PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T24	0-86	Sentence	denotes	A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection
T1	0-86	Sentence	denotes	A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection
T2	87-136	Sentence	denotes	Loss of A20 in Myeloid Cells Protects against IAV
T3	138-146	Sentence	denotes	Abstract
T25	147-291	Sentence	denotes	The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules.
T4	147-291	Sentence	denotes	The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules.
T26	292-550	Sentence	denotes	Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon.
T5	292-550	Sentence	denotes	Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon.
T27	551-704	Sentence	denotes	Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies.
T6	551-704	Sentence	denotes	Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies.
T28	705-865	Sentence	denotes	Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection.
T7	705-865	Sentence	denotes	Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection.
T29	866-1110	Sentence	denotes	A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon.
T8	866-1110	Sentence	denotes	A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon.
T30	1111-1238	Sentence	denotes	In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice.
T9	1111-1238	Sentence	denotes	In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice.
T31	1239-1336	Sentence	denotes	Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection.
T10	1239-1336	Sentence	denotes	Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection.
T32	1337-1587	Sentence	denotes	These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.
T11	1337-1587	Sentence	denotes	These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.
T12	1589-1603	Sentence	denotes	Author Summary
T13	1604-1806	Sentence	denotes	Influenza virus or flu epidemics represent a recurrent threat to the public health, especially for individuals which are part of a high-risk group such as children, elderly or immune-compromised people.
T14	1807-1930	Sentence	denotes	Sporadic pandemic flu outbreaks, such as the Spanish flu of 1918, may cause high grades of mortality among healthy persons.
T15	1931-2027	Sentence	denotes	A better understanding of how the immune system deals with these pathogens is of key importance.
T16	2028-2124	Sentence	denotes	The protein A20 is an important negative regulator of both innate and adaptive immune responses.
T17	2125-2292	Sentence	denotes	We show that the specific deletion of A20 in myeloid cells, such as macrophages and neutrophils, improves the resistance against otherwise lethal influenza infections.
T18	2293-2419	Sentence	denotes	This protective effect is mediated by an enhanced innate immune response following respiratory challenge with influenza virus.
T19	2420-2725	Sentence	denotes	Although exaggerated pulmonary immune responses are believed to be the primary cause of often life threatening influenza virus induced pneumonia, we demonstrate that boosting the innate immune response by selectively targeting the functionality of A20 in myeloid cells is beneficial for the host survival.
T20	2726-2856	Sentence	denotes	This finding provides us with a novel valuable approach for treating influenza and potentially other respiratory viral infections.
T21	2858-2870	Sentence	denotes	Introduction
T911	2871-2971	Sentence	denotes	Viruses are a class of highly diverse pathogens which depend on the host cell for their replication.
T22	2871-2971	Sentence	denotes	Viruses are a class of highly diverse pathogens which depend on the host cell for their replication.
T912	2972-3177	Sentence	denotes	The initiation of a protective innate antiviral immune response involves the action of specialized pattern recognition receptors (PRR), which detect conserved molecular structures of the invading pathogen.
T23	2972-3177	Sentence	denotes	The initiation of a protective innate antiviral immune response involves the action of specialized pattern recognition receptors (PRR), which detect conserved molecular structures of the invading pathogen.
T913	3178-3475	Sentence	denotes	Triggering of PRRs induces the production of host proinflammatory cytokines (e.g. TNF, IL-6 and IL-1) and type I interferons (interferon-α (IFN-α) and IFN-β) through activation of downstream signaling pathways that control various transcription factors such as NF-κB, AP-1, IRF3 and IRF7 [1], [2].
T24	3178-3475	Sentence	denotes	Triggering of PRRs induces the production of host proinflammatory cytokines (e.g. TNF, IL-6 and IL-1) and type I interferons (interferon-α (IFN-α) and IFN-β) through activation of downstream signaling pathways that control various transcription factors such as NF-κB, AP-1, IRF3 and IRF7 [1], [2].
T914	3476-3655	Sentence	denotes	The presence of viral nucleic acids, such as viral RNA and DNA, viral replication intermediates and viral transcription products, can be sensed by specific intracellular PRRs [3].
T25	3476-3655	Sentence	denotes	The presence of viral nucleic acids, such as viral RNA and DNA, viral replication intermediates and viral transcription products, can be sensed by specific intracellular PRRs [3].
T915	3656-3886	Sentence	denotes	Endosomal Toll-like receptors (TLRs) and cytoplasmic RNA helicase RIG-I-like receptors (RLRs) or Nod-like receptors (NLRs) detect the presence of viral single stranded (TLR7, TLR8, Nod2) or double stranded RNA (TLR3, RIG-I, MDA5).
T26	3656-3886	Sentence	denotes	Endosomal Toll-like receptors (TLRs) and cytoplasmic RNA helicase RIG-I-like receptors (RLRs) or Nod-like receptors (NLRs) detect the presence of viral single stranded (TLR7, TLR8, Nod2) or double stranded RNA (TLR3, RIG-I, MDA5).
T916	3887-4064	Sentence	denotes	Intracellular DNA sensors that mediate antiviral immune responses to DNA viruses include TLR9, DAI [4] and the PYHIN domain containing proteins AIM2 [5], [6], [7] and IFI16 [8].
T27	3887-4064	Sentence	denotes	Intracellular DNA sensors that mediate antiviral immune responses to DNA viruses include TLR9, DAI [4] and the PYHIN domain containing proteins AIM2 [5], [6], [7] and IFI16 [8].
T917	4065-4365	Sentence	denotes	TLR mediated antiviral responses are restricted to specialized type I IFN producing plasmacytoid dendritic cells (pDC), while most other cell types, including conventional DC (cDC), macrophages and fibroblasts, depend on the cytosolic RNA and DNA sensors for the production of antiviral proteins [9].
T28	4065-4365	Sentence	denotes	TLR mediated antiviral responses are restricted to specialized type I IFN producing plasmacytoid dendritic cells (pDC), while most other cell types, including conventional DC (cDC), macrophages and fibroblasts, depend on the cytosolic RNA and DNA sensors for the production of antiviral proteins [9].
T918	4366-4574	Sentence	denotes	Influenza A virus (IAV) is the etiological agent of a contagious acute respiratory disease that causes considerable mortality, which is generally believed to be due to an excessive host inflammatory response.
T29	4366-4574	Sentence	denotes	Influenza A virus (IAV) is the etiological agent of a contagious acute respiratory disease that causes considerable mortality, which is generally believed to be due to an excessive host inflammatory response.
T919	4575-4724	Sentence	denotes	Emergence of drug-resistant strains of influenza viruses with pandemic potential underscores the importance of developing novel antiviral strategies.
T30	4575-4724	Sentence	denotes	Emergence of drug-resistant strains of influenza viruses with pandemic potential underscores the importance of developing novel antiviral strategies.
T920	4725-4829	Sentence	denotes	In this context, understanding of the mechanisms that regulate IAV-induced immune responses is critical.
T31	4725-4829	Sentence	denotes	In this context, understanding of the mechanisms that regulate IAV-induced immune responses is critical.
T921	4830-4993	Sentence	denotes	IAV infection leads to the exposure in the host cell of single-stranded genomic RNA and double stranded RNA, the latter being an intermediate of viral replication.
T32	4830-4993	Sentence	denotes	IAV infection leads to the exposure in the host cell of single-stranded genomic RNA and double stranded RNA, the latter being an intermediate of viral replication.
T922	4994-5068	Sentence	denotes	TLR3 and RIG-I have been implicated as sensors of IAV infection [10]–[12].
T33	4994-5068	Sentence	denotes	TLR3 and RIG-I have been implicated as sensors of IAV infection [10]–[12].
T923	5069-5243	Sentence	denotes	Both receptors contribute to the proinflammatory response to IAV, but the initiation of the innate antiviral immune response largely depends on RIG-I mediated signaling [13].
T34	5069-5243	Sentence	denotes	Both receptors contribute to the proinflammatory response to IAV, but the initiation of the innate antiviral immune response largely depends on RIG-I mediated signaling [13].
T924	5244-5400	Sentence	denotes	Interestingly, RIG-I deficient mice are highly susceptible to IAV [14], [15], whereas TLR3 deficient mice have a survival advantage to acute infection [16].
T35	5244-5400	Sentence	denotes	Interestingly, RIG-I deficient mice are highly susceptible to IAV [14], [15], whereas TLR3 deficient mice have a survival advantage to acute infection [16].
T925	5401-5580	Sentence	denotes	These results indicate that an imbalance between the beneficial and harmful effects of mediators released by immune cells is likely to contribute to the pathogenesis of influenza.
T36	5401-5580	Sentence	denotes	These results indicate that an imbalance between the beneficial and harmful effects of mediators released by immune cells is likely to contribute to the pathogenesis of influenza.
T926	5581-5711	Sentence	denotes	RIG-I contains a C-terminal DExD/H box helicase domain, which is required for ligand recognition, and two N-terminal CARD domains.
T37	5581-5711	Sentence	denotes	RIG-I contains a C-terminal DExD/H box helicase domain, which is required for ligand recognition, and two N-terminal CARD domains.
T927	5712-5986	Sentence	denotes	Upon ligand binding, the CARD domains of RIG-I associate with the CARD domain of the MAVS (also termed IPS-1, VISA, Cardif) adaptor protein, which subsequently translocates to and inserts in the outer mitochondrial membrane via its C-terminal transmembrane domain [17]–[20].
T38	5712-5986	Sentence	denotes	Upon ligand binding, the CARD domains of RIG-I associate with the CARD domain of the MAVS (also termed IPS-1, VISA, Cardif) adaptor protein, which subsequently translocates to and inserts in the outer mitochondrial membrane via its C-terminal transmembrane domain [17]–[20].
T928	5987-6162	Sentence	denotes	Signaling downstream of MAVS requires the action of various ubiquitin modifying enzymes, which both positively and negatively regulate RIG-I mediated signal transduction [21].
T39	5987-6162	Sentence	denotes	Signaling downstream of MAVS requires the action of various ubiquitin modifying enzymes, which both positively and negatively regulate RIG-I mediated signal transduction [21].
T929	6163-6297	Sentence	denotes	K63-specific ubiquitin ligases (E3s), such as TRIM25 [22] and Riplet [23], [24], have been shown to directly promote RIG-I activation.
T40	6163-6297	Sentence	denotes	K63-specific ubiquitin ligases (E3s), such as TRIM25 [22] and Riplet [23], [24], have been shown to directly promote RIG-I activation.
T930	6298-6458	Sentence	denotes	In addition, well characterized ubiquitin ligases such as TRAF6 [25], [26] and TRAF3 [27] mediate respectively NF-κB and IRF3 activation upon RIG-I stimulation.
T41	6298-6458	Sentence	denotes	In addition, well characterized ubiquitin ligases such as TRAF6 [25], [26] and TRAF3 [27] mediate respectively NF-κB and IRF3 activation upon RIG-I stimulation.
T931	6459-6706	Sentence	denotes	On the other hand, deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30] and OTUB1/2 [31] have been shown to negatively regulate RLR signaling by specifically removing K63-linked polyubiquitin chains from several signaling molecules.
T42	6459-6706	Sentence	denotes	On the other hand, deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30] and OTUB1/2 [31] have been shown to negatively regulate RLR signaling by specifically removing K63-linked polyubiquitin chains from several signaling molecules.
T932	6707-6910	Sentence	denotes	Furthermore, various K48-specific ubiquitin ligases, such as AIP4 [32] and TRIAD3A [33] mark respectively MAVS and TRAF3 for proteasome mediated degradation, thus inhibiting further downstream signaling.
T43	6707-6910	Sentence	denotes	Furthermore, various K48-specific ubiquitin ligases, such as AIP4 [32] and TRIAD3A [33] mark respectively MAVS and TRAF3 for proteasome mediated degradation, thus inhibiting further downstream signaling.
T933	6911-7119	Sentence	denotes	Additionally, the attachment of K48-specific polyubiquitin chains to the IRF3 and IRF7 transcription factors by E3s such as RAUL [34], TRIM21 [35] and RBCK1 [36] further dampens antiviral signal transduction.
T44	6911-7119	Sentence	denotes	Additionally, the attachment of K48-specific polyubiquitin chains to the IRF3 and IRF7 transcription factors by E3s such as RAUL [34], TRIM21 [35] and RBCK1 [36] further dampens antiviral signal transduction.
T934	7120-7198	Sentence	denotes	A20 is an ubiquitin-editing enzyme belonging to the OTU-domain family of DUBs.
T45	7120-7198	Sentence	denotes	A20 is an ubiquitin-editing enzyme belonging to the OTU-domain family of DUBs.
T935	7199-7304	Sentence	denotes	Interestingly, A20 also harbors atypical zinc finger dependent K48-specific E3 ubiquitin ligase activity.
T46	7199-7304	Sentence	denotes	Interestingly, A20 also harbors atypical zinc finger dependent K48-specific E3 ubiquitin ligase activity.
T936	7305-7542	Sentence	denotes	Both catalytic and noncatalytic mechanisms were previously shown to be involved in the negative regulation of proinflammatory signaling by A20 in response to multiple receptors such as TNF receptor I [37]–[39], TLR4 [40], and IL-1R [41].
T47	7305-7542	Sentence	denotes	Both catalytic and noncatalytic mechanisms were previously shown to be involved in the negative regulation of proinflammatory signaling by A20 in response to multiple receptors such as TNF receptor I [37]–[39], TLR4 [40], and IL-1R [41].
T937	7543-7716	Sentence	denotes	The anti-inflammatory role of A20 is clearly demonstrated by the fact that A20 deficient mice die early after birth due to severe multi-organ inflammation and cachexia [37].
T48	7543-7716	Sentence	denotes	The anti-inflammatory role of A20 is clearly demonstrated by the fact that A20 deficient mice die early after birth due to severe multi-organ inflammation and cachexia [37].
T938	7717-7939	Sentence	denotes	More recently, gene targeting of A20 in specific cell types was shown to be associated with autoimmunity and chronic inflammation [42]–[48], further illustrating that A20 is an important brake on the inflammatory response.
T49	7717-7939	Sentence	denotes	More recently, gene targeting of A20 in specific cell types was shown to be associated with autoimmunity and chronic inflammation [42]–[48], further illustrating that A20 is an important brake on the inflammatory response.
T939	7940-8164	Sentence	denotes	The relevance of these findings for human disease has recently been illustrated through the identification of polymorphisms in the A20 locus that are associated with several autoimmune diseases and chronic inflammation [45].
T50	7940-8164	Sentence	denotes	The relevance of these findings for human disease has recently been illustrated through the identification of polymorphisms in the A20 locus that are associated with several autoimmune diseases and chronic inflammation [45].
T940	8165-8543	Sentence	denotes	In contrast to its well established function in the regulation of proinflammatory responses, the role of A20 in the regulation of antiviral immune responses is less well described and limited to a number of in vitro studies using overexpression or silencing in specific cell lines, indicating that A20 may regulate RIG-I- and TLR3-induced signaling to NF-κB and IRF-3 [49]–[52].
T51	8165-8543	Sentence	denotes	In contrast to its well established function in the regulation of proinflammatory responses, the role of A20 in the regulation of antiviral immune responses is less well described and limited to a number of in vitro studies using overexpression or silencing in specific cell lines, indicating that A20 may regulate RIG-I- and TLR3-induced signaling to NF-κB and IRF-3 [49]–[52].
T941	8544-8644	Sentence	denotes	However, the precise in vivo role of A20 in the response to viral infection remains to be clarified.
T52	8544-8644	Sentence	denotes	However, the precise in vivo role of A20 in the response to viral infection remains to be clarified.
T942	8645-8921	Sentence	denotes	Using myeloid cell specific A20 knockout mice (A20myel-KO) that were recently generated in our lab and primary cells derived of these mice, we here provide evidence that A20 is a crucial negative regulator of IAV-induced proinflammatory and antiviral signaling in macrophages.
T53	8645-8921	Sentence	denotes	Using myeloid cell specific A20 knockout mice (A20myel-KO) that were recently generated in our lab and primary cells derived of these mice, we here provide evidence that A20 is a crucial negative regulator of IAV-induced proinflammatory and antiviral signaling in macrophages.
T943	8922-9059	Sentence	denotes	Interestingly, A20myel-KO mice show enhanced survival and reduced morbidity in response to IAV lung infection compared to wild type mice.
T54	8922-9059	Sentence	denotes	Interestingly, A20myel-KO mice show enhanced survival and reduced morbidity in response to IAV lung infection compared to wild type mice.
T944	9060-9285	Sentence	denotes	Protection against IAV in A20myel-KO mice is associated with increased cytokine and chemokine production, augmented recruitment of innate immune cells such as neutophils and alveolar macrophages, and enhanced viral clearance.
T55	9060-9285	Sentence	denotes	Protection against IAV in A20myel-KO mice is associated with increased cytokine and chemokine production, augmented recruitment of innate immune cells such as neutophils and alveolar macrophages, and enhanced viral clearance.
T945	9286-9432	Sentence	denotes	These results suggest that boosting the innate immune response to IAV by targeting A20 activity in myeloid cells might have therapeutic potential.
T56	9286-9432	Sentence	denotes	These results suggest that boosting the innate immune response to IAV by targeting A20 activity in myeloid cells might have therapeutic potential.
T57	9434-9441	Sentence	denotes	Results
T4724	9443-9495	Sentence	denotes	A20 inhibits RIG-I-induced NF-κB and IRF3 activation
T58	9443-9495	Sentence	denotes	A20 inhibits RIG-I-induced NF-κB and IRF3 activation
T4725	9496-9696	Sentence	denotes	RIG-I signaling induces the activation of NF-κB, IRF3 and IRF7 transcription factors, which promote the expression of proinflammatory cytokines and type I IFNs that restrict further viral propagation.
T59	9496-9696	Sentence	denotes	RIG-I signaling induces the activation of NF-κB, IRF3 and IRF7 transcription factors, which promote the expression of proinflammatory cytokines and type I IFNs that restrict further viral propagation.
T4726	9697-9836	Sentence	denotes	Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52].
T60	9697-9836	Sentence	denotes	Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52].
T4727	9837-10161	Sentence	denotes	Similarly, we show that A20 overexpression in HEK293T cells prevents NF-κB- and IRF3-dependent luciferase reporter gene activation induced by transfection of a truncated constitutive active form of RIG-I [53], corresponding to only the two N-terminal CARD domains of RIG-I [RIG-I (2CARD)] (figure 1A, left and middle graph).
T61	9837-10161	Sentence	denotes	Similarly, we show that A20 overexpression in HEK293T cells prevents NF-κB- and IRF3-dependent luciferase reporter gene activation induced by transfection of a truncated constitutive active form of RIG-I [53], corresponding to only the two N-terminal CARD domains of RIG-I [RIG-I (2CARD)] (figure 1A, left and middle graph).
T4728	10162-10225	Sentence	denotes	We next investigated whether A20 also inhibits IRF7 activation.
T62	10162-10225	Sentence	denotes	We next investigated whether A20 also inhibits IRF7 activation.
T4729	10226-10388	Sentence	denotes	Unlike IRF3, IRF7 is not or weakly expressed under naïve conditions and IRF7 protein levels are rapidly upregulated upon virus-induced IRF3 activation [54], [55].
T63	10226-10388	Sentence	denotes	Unlike IRF3, IRF7 is not or weakly expressed under naïve conditions and IRF7 protein levels are rapidly upregulated upon virus-induced IRF3 activation [54], [55].
T4730	10389-10617	Sentence	denotes	To determine the effect of A20 on IRF7 activation, we therefore transfected minor amounts of an IRF7 expression plasmid together with plasmids encoding RIG-I (2CARD), A20 and an IRF7-specific IFNα4 luciferase reporter construct.
T64	10389-10617	Sentence	denotes	To determine the effect of A20 on IRF7 activation, we therefore transfected minor amounts of an IRF7 expression plasmid together with plasmids encoding RIG-I (2CARD), A20 and an IRF7-specific IFNα4 luciferase reporter construct.
T4731	10618-10842	Sentence	denotes	RIG-I (2CARD) expression in the absence of IRF7 co-expression showed negligible IFNα4 promoter activation (grey bar, figure 1A, right graph), whereas significant reporter gene expression was observed in the presence of IRF7.
T65	10618-10842	Sentence	denotes	RIG-I (2CARD) expression in the absence of IRF7 co-expression showed negligible IFNα4 promoter activation (grey bar, figure 1A, right graph), whereas significant reporter gene expression was observed in the presence of IRF7.
T4732	10843-10980	Sentence	denotes	Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph).
T66	10843-10980	Sentence	denotes	Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph).
T4733	10981-11081	Sentence	denotes	These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.
T67	10981-11081	Sentence	denotes	These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.
T68	11082-11198	Sentence	denotes	10.1371/journal.ppat.1002570.g001 Figure 1 A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.
T19347	11126-11198	Sentence	denotes	A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.
T19348	11199-11415	Sentence	denotes	(A) HEK293T cells were transfected with NF-κB (left), IRF3 (middle) or IRF7 dependent luciferase reporter (right) plasmids, together with plasmids expressing RIG-I (2CARD), IRF7 (right) and increasing amounts of A20.
T69	11199-11415	Sentence	denotes	(A) HEK293T cells were transfected with NF-κB (left), IRF3 (middle) or IRF7 dependent luciferase reporter (right) plasmids, together with plasmids expressing RIG-I (2CARD), IRF7 (right) and increasing amounts of A20.
T19349	11416-11483	Sentence	denotes	Luciferase activity in cell extracts was analyzed in a luminometer.
T70	11416-11483	Sentence	denotes	Luciferase activity in cell extracts was analyzed in a luminometer.
T19350	11484-11814	Sentence	denotes	Numbers are averages +/− SD of 3 samples per set-up. (B–D) A20myel-KO and wild type (A20myel-WT) BMDM were transfected with LMW poly(I:C) and analyzed at different time points (minutes) after the start of transfection for IκBα phosphorylation and degradation, and IRF3 phosphorylation by immunoblotting of total cell extracts (B).
T71	11484-11814	Sentence	denotes	Numbers are averages +/− SD of 3 samples per set-up. (B–D) A20myel-KO and wild type (A20myel-WT) BMDM were transfected with LMW poly(I:C) and analyzed at different time points (minutes) after the start of transfection for IκBα phosphorylation and degradation, and IRF3 phosphorylation by immunoblotting of total cell extracts (B).
T19351	11815-11946	Sentence	denotes	The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control.
T72	11815-11946	Sentence	denotes	The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control.
T19352	11947-12064	Sentence	denotes	Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C).
T73	11947-12064	Sentence	denotes	Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C).
T19353	12065-12214	Sentence	denotes	Quantification of band density for nuclear p65 and IRF3 is shown in bar graphs (expressed as relative density compared to the first lane; = time 0).
T74	12065-12214	Sentence	denotes	Quantification of band density for nuclear p65 and IRF3 is shown in bar graphs (expressed as relative density compared to the first lane; = time 0).
T19354	12215-12397	Sentence	denotes	IL-6, TNF and IFNβ levels in the supernatant of cells used in (B) were determined by bioassay (IL-6) and ELISA (TNF and IFNβ) as described in the ‘materials and methods’ section (D).
T75	12215-12397	Sentence	denotes	IL-6, TNF and IFNβ levels in the supernatant of cells used in (B) were determined by bioassay (IL-6) and ELISA (TNF and IFNβ) as described in the ‘materials and methods’ section (D).
T19355	12398-12451	Sentence	denotes	Data are representative of 2 independent experiments.
T76	12398-12451	Sentence	denotes	Data are representative of 2 independent experiments.
T4734	12452-12641	Sentence	denotes	To study the effect of endogenously expressed A20 on RIG-I-induced signaling in a more immunological relevant context, we performed further experiments in A20 deficient primary macrophages.
T77	12452-12641	Sentence	denotes	To study the effect of endogenously expressed A20 on RIG-I-induced signaling in a more immunological relevant context, we performed further experiments in A20 deficient primary macrophages.
T4735	12642-12856	Sentence	denotes	Since A20 full knockout mice die prematurely as a result of severe multi-organ inflammation [37], we generated mice carrying a conditional A20 allele in which exon IV and exon V were flanked by two loxP sites [56].
T78	12642-12856	Sentence	denotes	Since A20 full knockout mice die prematurely as a result of severe multi-organ inflammation [37], we generated mice carrying a conditional A20 allele in which exon IV and exon V were flanked by two loxP sites [56].
T4736	12857-13082	Sentence	denotes	Crossing these mice with transgenic mice expressing Cre recombinase under control of the lysozyme M promoter leads to specific deletion in myeloid cells and allowed us to generate myeloid cell specific A20 knockout mice [48].
T79	12857-13082	Sentence	denotes	Crossing these mice with transgenic mice expressing Cre recombinase under control of the lysozyme M promoter leads to specific deletion in myeloid cells and allowed us to generate myeloid cell specific A20 knockout mice [48].
T4737	13083-13316	Sentence	denotes	To stimulate the RIG-I receptor, we transfected A20myel-KO BMDM and wild type control cells with minimal amounts of low molecular weight (LMW) poly(I:C), which is known to preferentially bind and activate RIG-I rather than MDA5 [57].
T80	13083-13316	Sentence	denotes	To stimulate the RIG-I receptor, we transfected A20myel-KO BMDM and wild type control cells with minimal amounts of low molecular weight (LMW) poly(I:C), which is known to preferentially bind and activate RIG-I rather than MDA5 [57].
T4738	13317-13472	Sentence	denotes	Of note, this concentration of poly(I:C) was unable to induce significant TLR3 dependent NF-κB and IRF3 activation or cytokine production (data not shown).
T81	13317-13472	Sentence	denotes	Of note, this concentration of poly(I:C) was unable to induce significant TLR3 dependent NF-κB and IRF3 activation or cytokine production (data not shown).
T4739	13473-13611	Sentence	denotes	As expected, poly(I:C) transfection induced the rapid expression of A20 in wild type, but not in A20myel-KO BMDM (figure 1B, upper panel).
T82	13473-13611	Sentence	denotes	As expected, poly(I:C) transfection induced the rapid expression of A20 in wild type, but not in A20myel-KO BMDM (figure 1B, upper panel).
T4740	13612-13773	Sentence	denotes	At early time points, slightly slower migrating forms of A20 were observed, indicating that A20 undergoes a yet unknown modification upon poly(I:C) transfection.
T83	13612-13773	Sentence	denotes	At early time points, slightly slower migrating forms of A20 were observed, indicating that A20 undergoes a yet unknown modification upon poly(I:C) transfection.
T4741	13774-13943	Sentence	denotes	Compared to wild type BMBM, A20 deficient cells showed enhanced NF-κB activation as indicated by increased phosphorylation and sustained degradation of IκBα (figure 1B).
T84	13774-13943	Sentence	denotes	Compared to wild type BMBM, A20 deficient cells showed enhanced NF-κB activation as indicated by increased phosphorylation and sustained degradation of IκBα (figure 1B).
T4742	13944-14141	Sentence	denotes	Furthermore, nuclear translocation of the p65 NF-κB subunit was enhanced in poly(I:C) transfected A20myel-KO BMDM, reaching a maximum at earlier time points compared to wild type cells (figure 1C).
T85	13944-14141	Sentence	denotes	Furthermore, nuclear translocation of the p65 NF-κB subunit was enhanced in poly(I:C) transfected A20myel-KO BMDM, reaching a maximum at earlier time points compared to wild type cells (figure 1C).
T4743	14142-14363	Sentence	denotes	IRF3 is known to be activated upon phosphorylation of a series of carboxyl terminal serine residues by the IKK-like kinases TBK1 and IKKε [58], leading to its dimerization and subsequent translocation to the nucleus [59].
T86	14142-14363	Sentence	denotes	IRF3 is known to be activated upon phosphorylation of a series of carboxyl terminal serine residues by the IKK-like kinases TBK1 and IKKε [58], leading to its dimerization and subsequent translocation to the nucleus [59].
T4744	14364-14565	Sentence	denotes	Using immunoblotting with an antibody directed against phosphorylated Ser396, maximum IRF3 phosphorylation was detected at earlier time points in A20myel-KO BMDM compared to wild type BMDM (figure 1B).
T87	14364-14565	Sentence	denotes	Using immunoblotting with an antibody directed against phosphorylated Ser396, maximum IRF3 phosphorylation was detected at earlier time points in A20myel-KO BMDM compared to wild type BMDM (figure 1B).
T4745	14566-14716	Sentence	denotes	Similar to p65, IRF3 nuclear translocation reached its maximum at an earlier time point in A20 deficient BMDM compared to wild type cells (figure 1C).
T88	14566-14716	Sentence	denotes	Similar to p65, IRF3 nuclear translocation reached its maximum at an earlier time point in A20 deficient BMDM compared to wild type cells (figure 1C).
T4746	14717-14814	Sentence	denotes	NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ.
T89	14717-14814	Sentence	denotes	NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ.
T4747	14815-14967	Sentence	denotes	In line with the enhanced activation of NF-κB and IRF3 as described above, A20myel-KO BMDM secreted increased amounts of IL-6, TNF and IFNβ (figure 1D).
T90	14815-14967	Sentence	denotes	In line with the enhanced activation of NF-κB and IRF3 as described above, A20myel-KO BMDM secreted increased amounts of IL-6, TNF and IFNβ (figure 1D).
T4748	14968-15044	Sentence	denotes	Similar results were obtained using peritoneal macrophages (data not shown).
T91	14968-15044	Sentence	denotes	Similar results were obtained using peritoneal macrophages (data not shown).
T4749	15045-15207	Sentence	denotes	Together, these results demonstrate that A20 plays an important role in the negative regulation of RIG-I-induced NF-κB and IRF3 activation in primary macrophages.
T92	15045-15207	Sentence	denotes	Together, these results demonstrate that A20 plays an important role in the negative regulation of RIG-I-induced NF-κB and IRF3 activation in primary macrophages.
T7698	15209-15269	Sentence	denotes	A20 negatively regulates IAV-induced gene expression in BMDM
T93	15209-15269	Sentence	denotes	A20 negatively regulates IAV-induced gene expression in BMDM
T7699	15270-15439	Sentence	denotes	To investigate the role of A20 in the IAV-induced proinflammatory and antiviral innate immune responses, we infected A20 deficient and control BMDM with IAV X-47 (H3N2).
T94	15270-15439	Sentence	denotes	To investigate the role of A20 in the IAV-induced proinflammatory and antiviral innate immune responses, we infected A20 deficient and control BMDM with IAV X-47 (H3N2).
T7700	15440-15560	Sentence	denotes	A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A).
T95	15440-15560	Sentence	denotes	A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A).
T7701	15561-15695	Sentence	denotes	Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A).
T96	15561-15695	Sentence	denotes	Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A).
T7702	15696-15831	Sentence	denotes	In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).
T97	15696-15831	Sentence	denotes	In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).
T98	15832-15962	Sentence	denotes	10.1371/journal.ppat.1002570.g002 Figure 2 A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.
T20279	15876-15962	Sentence	denotes	A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.
T20280	15963-16053	Sentence	denotes	(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1).
T99	15963-16053	Sentence	denotes	(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1).
T20281	16054-16182	Sentence	denotes	At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A).
T100	16054-16182	Sentence	denotes	At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A).
T20282	16183-16408	Sentence	denotes	18 hours post infection cell culture supernatant was analyzed for TNF and IFNβ protein levels by ELISA and multiplexing technologies (B). (C–D) Alveolar macrophages were mock treated or infected with IAV (moi 1) for 18 hours.
T101	16183-16408	Sentence	denotes	18 hours post infection cell culture supernatant was analyzed for TNF and IFNβ protein levels by ELISA and multiplexing technologies (B). (C–D) Alveolar macrophages were mock treated or infected with IAV (moi 1) for 18 hours.
T20283	16409-16466	Sentence	denotes	IL-6 and IFNβ mRNA expression was determined by qPCR (C).
T102	16409-16466	Sentence	denotes	IL-6 and IFNβ mRNA expression was determined by qPCR (C).
T20284	16467-16581	Sentence	denotes	Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D).
T103	16467-16581	Sentence	denotes	Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D).
T20285	16582-16640	Sentence	denotes	Error bars represent mean values (+/− SEM) of 2–3 samples.
T104	16582-16640	Sentence	denotes	Error bars represent mean values (+/− SEM) of 2–3 samples.
T20286	16641-16710	Sentence	denotes	Results are representative for 2 independent experiments. ***p<0,001.
T105	16641-16710	Sentence	denotes	Results are representative for 2 independent experiments. ***p<0,001.
T7703	16711-16896	Sentence	denotes	Upon host infection with IAV, alveolar macrophages are an important source of cytokines and chemokines, attracting innate immune cells to the lung during the primary phase of infection.
T106	16711-16896	Sentence	denotes	Upon host infection with IAV, alveolar macrophages are an important source of cytokines and chemokines, attracting innate immune cells to the lung during the primary phase of infection.
T7704	16897-17102	Sentence	denotes	To test whether A20 directly controls IAV-induced gene expression in alveolar macrophages, we isolated these cells from lungs of A20myel-KO and control littermates and infected them in vitro with IAV X-47.
T107	16897-17102	Sentence	denotes	To test whether A20 directly controls IAV-induced gene expression in alveolar macrophages, we isolated these cells from lungs of A20myel-KO and control littermates and infected them in vitro with IAV X-47.
T7705	17103-17374	Sentence	denotes	Expression and secretion of the proinflammatory cytokines IL-6 and TNF, the type-I IFN IFNβ and IFNα4 and the chemokines MCP-1 (ccl2) and KC (cxcl1) was significantly higher in IAV infected cells lacking A20 compared to infected wild type cells (figure 2C and figure S1).
T108	17103-17374	Sentence	denotes	Expression and secretion of the proinflammatory cytokines IL-6 and TNF, the type-I IFN IFNβ and IFNα4 and the chemokines MCP-1 (ccl2) and KC (cxcl1) was significantly higher in IAV infected cells lacking A20 compared to infected wild type cells (figure 2C and figure S1).
T7706	17375-17622	Sentence	denotes	Taken together these results demonstrate that A20 negatively regulates IAV-induced proinflammatory and antiviral gene expression in alveolar macrophages, consistent with the inhibitory effect of A20 seen on RIG-I-induced NF-κB and IRF3 activation.
T109	17375-17622	Sentence	denotes	Taken together these results demonstrate that A20 negatively regulates IAV-induced proinflammatory and antiviral gene expression in alveolar macrophages, consistent with the inhibitory effect of A20 seen on RIG-I-induced NF-κB and IRF3 activation.
T8720	17624-17691	Sentence	denotes	A20 deficiency in myeloid cells protects against IAV lung infection
T110	17624-17691	Sentence	denotes	A20 deficiency in myeloid cells protects against IAV lung infection
T8721	17692-17968	Sentence	denotes	To determine the role of A20 expression in myeloid cells during an IAV infection in vivo, we intranasally inoculated both A20myel-KO mice and control littermates with a sublethal dose of the mouse adapted IAV X-47 (H3N2) strain and monitored morbidity in terms of weight loss.
T111	17692-17968	Sentence	denotes	To determine the role of A20 expression in myeloid cells during an IAV infection in vivo, we intranasally inoculated both A20myel-KO mice and control littermates with a sublethal dose of the mouse adapted IAV X-47 (H3N2) strain and monitored morbidity in terms of weight loss.
T8722	17969-18112	Sentence	denotes	A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A).
T112	17969-18112	Sentence	denotes	A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A).
T8723	18113-18368	Sentence	denotes	Also, total protein concentration in BAL fluid, which reflects lung damage and vascular leakage, was increased significantly at 7 and 10 days post infection in both wild type and A20myel-KO mice, and was slightly lower in A20myel-KO mice (data not shown).
T113	18113-18368	Sentence	denotes	Also, total protein concentration in BAL fluid, which reflects lung damage and vascular leakage, was increased significantly at 7 and 10 days post infection in both wild type and A20myel-KO mice, and was slightly lower in A20myel-KO mice (data not shown).
T8724	18369-18445	Sentence	denotes	Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection.
T114	18369-18445	Sentence	denotes	Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection.
T8725	18446-18562	Sentence	denotes	No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection.
T115	18446-18562	Sentence	denotes	No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection.
T8726	18563-18764	Sentence	denotes	However, after 10 days, almost no virus could be detected in the lungs of A20myel-KO mice while abundant infectious viral particles could still be isolated from lungs of all wild type mice (figure 3B).
T116	18563-18764	Sentence	denotes	However, after 10 days, almost no virus could be detected in the lungs of A20myel-KO mice while abundant infectious viral particles could still be isolated from lungs of all wild type mice (figure 3B).
T8727	18765-18922	Sentence	denotes	This indicates that loss of A20 in myeloid cells does not affect early viral replication but contributes to viral clearance at later stages during infection.
T117	18765-18922	Sentence	denotes	This indicates that loss of A20 in myeloid cells does not affect early viral replication but contributes to viral clearance at later stages during infection.
T118	18923-19035	Sentence	denotes	10.1371/journal.ppat.1002570.g003 Figure 3 A20 deficiency in myeloid cells protects against IAV lung infection.
T20779	18967-19035	Sentence	denotes	A20 deficiency in myeloid cells protects against IAV lung infection.
T20780	19036-19157	Sentence	denotes	A20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A).
T119	19036-19157	Sentence	denotes	A20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A).
T20781	19158-19271	Sentence	denotes	At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B).
T120	19158-19271	Sentence	denotes	At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B).
T20782	19272-19319	Sentence	denotes	The dashed line represents the detection limit.
T121	19272-19319	Sentence	denotes	The dashed line represents the detection limit.
T20783	19320-19339	Sentence	denotes	ND, not detectable.
T122	19320-19339	Sentence	denotes	ND, not detectable.
T20784	19340-19513	Sentence	denotes	BAL was isolated from IAV infected mice at 4, 7 and 10 days p.i. and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were analyzed by ELISA and multiplexing technologies (C).
T123	19340-19513	Sentence	denotes	BAL was isolated from IAV infected mice at 4, 7 and 10 days p.i. and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were analyzed by ELISA and multiplexing technologies (C).
T20785	19514-19659	Sentence	denotes	Absolute numbers of resident alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 4, 7 and 10 days p.i. (D).
T124	19514-19659	Sentence	denotes	Absolute numbers of resident alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 4, 7 and 10 days p.i. (D).
T20786	19660-19789	Sentence	denotes	Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. p<0.05; *p<0.01.
T125	19660-19789	Sentence	denotes	Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. p<0.05; *p<0.01.
T8728	19790-20022	Sentence	denotes	To verify if A20 deficiency in myeloid cells affects IAV-induced gene expression in the lung, we analyzed the levels of several chemokines and cytokines in the bronchoalveolar lavage (BAL) at day 4, 7 and day 10 following infection.
T126	19790-20022	Sentence	denotes	To verify if A20 deficiency in myeloid cells affects IAV-induced gene expression in the lung, we analyzed the levels of several chemokines and cytokines in the bronchoalveolar lavage (BAL) at day 4, 7 and day 10 following infection.
T8729	20023-20207	Sentence	denotes	Levels of KC and MIP-2 chemokines, as well as IL-6 were significantly higher at day 7 p.i. in BAL from IAV infected A20myel-KO mice compared to IAV infected wild type mice (figure 3C).
T127	20023-20207	Sentence	denotes	Levels of KC and MIP-2 chemokines, as well as IL-6 were significantly higher at day 7 p.i. in BAL from IAV infected A20myel-KO mice compared to IAV infected wild type mice (figure 3C).
T8730	20208-20395	Sentence	denotes	Unlike our observations with in vitro stimulated primary macrophages we could not detect a significant increase in MCP-1 or IFNα production in the lungs of A20myel-KO animals (figure 3C).
T128	20208-20395	Sentence	denotes	Unlike our observations with in vitro stimulated primary macrophages we could not detect a significant increase in MCP-1 or IFNα production in the lungs of A20myel-KO animals (figure 3C).
T8731	20396-20645	Sentence	denotes	KC is the murine orthologue of IL-8 and serves together with MIP-2 as a chemoattractant for neutrophils, while MCP-1 is mainly known as a chemoattractant for monocytes, which eventually develop into macrophages upon entering the alveolar lumen [60].
T129	20396-20645	Sentence	denotes	KC is the murine orthologue of IL-8 and serves together with MIP-2 as a chemoattractant for neutrophils, while MCP-1 is mainly known as a chemoattractant for monocytes, which eventually develop into macrophages upon entering the alveolar lumen [60].
T8732	20646-20935	Sentence	denotes	Consistent with the higher KC and MIP-2 levels in A20myel-KO mice, the number of neutrophils (CD11b+ Ly6C+ Ly6G+ F4/80−) that were recruited in the bronchoalveolar spaces upon IAV infection was clearly higher throughout infection in A20myel-KO mice compared to control animals (figure 3D).
T130	20646-20935	Sentence	denotes	Consistent with the higher KC and MIP-2 levels in A20myel-KO mice, the number of neutrophils (CD11b+ Ly6C+ Ly6G+ F4/80−) that were recruited in the bronchoalveolar spaces upon IAV infection was clearly higher throughout infection in A20myel-KO mice compared to control animals (figure 3D).
T8733	20936-21197	Sentence	denotes	Although we could detect a significant increase in monocyte (CD11b+ Ly6C+) recruitment at day 4 post infection, this was not evident at later time points after infection (figure 3D), which is consistent with the equal expression of MCP-1 in both groups of mice.
T131	20936-21197	Sentence	denotes	Although we could detect a significant increase in monocyte (CD11b+ Ly6C+) recruitment at day 4 post infection, this was not evident at later time points after infection (figure 3D), which is consistent with the equal expression of MCP-1 in both groups of mice.
T8734	21198-21407	Sentence	denotes	The number of resident alveolar macrophages (autofluorescent+ CD11c+ F4/80+ CD11b−) was also elevated in A20myel-KO mice but did not differ significantly between IAV infected or mock infected mice (figure 3D).
T132	21198-21407	Sentence	denotes	The number of resident alveolar macrophages (autofluorescent+ CD11c+ F4/80+ CD11b−) was also elevated in A20myel-KO mice but did not differ significantly between IAV infected or mock infected mice (figure 3D).
T8735	21408-21538	Sentence	denotes	Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63].
T133	21408-21538	Sentence	denotes	Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63].
T8736	21539-21787	Sentence	denotes	To test whether A20 expression in myeloid cells regulates the antiviral CTL response, total CD8+ T cells and virus specific Granzyme B (GrB) and IFNγ expressing CD8+ T cells were measured in BAL and lung parenchyma of wild type and A20myel-KO mice.
T134	21539-21787	Sentence	denotes	To test whether A20 expression in myeloid cells regulates the antiviral CTL response, total CD8+ T cells and virus specific Granzyme B (GrB) and IFNγ expressing CD8+ T cells were measured in BAL and lung parenchyma of wild type and A20myel-KO mice.
T8737	21788-21970	Sentence	denotes	A clear increase in CD8+ T cells could be detected at day 7 and 10 post infection, but no differences were observed between A20myel-KO and wild type mice (figure S2A and figure S2C).
T135	21788-21970	Sentence	denotes	A clear increase in CD8+ T cells could be detected at day 7 and 10 post infection, but no differences were observed between A20myel-KO and wild type mice (figure S2A and figure S2C).
T8738	21971-22135	Sentence	denotes	Also, the number of GrB and IFNγ CD8+ T cells as well as IFNγ expression levels in the lungs were not altered by the absence of A20 in myeloid cells (figure S2A–C).
T136	21971-22135	Sentence	denotes	Also, the number of GrB and IFNγ CD8+ T cells as well as IFNγ expression levels in the lungs were not altered by the absence of A20 in myeloid cells (figure S2A–C).
T8739	22136-22217	Sentence	denotes	Protection against IAV infection is also provided by the humoral immune response.
T137	22136-22217	Sentence	denotes	Protection against IAV infection is also provided by the humoral immune response.
T8740	22218-22399	Sentence	denotes	To test whether loss of A20 in myeloid cells affects B cell mediated immunity, we determined hemagglutinin (HA) antibody titers in the serum of A20myel-KO and wild type littermates.
T138	22218-22399	Sentence	denotes	To test whether loss of A20 in myeloid cells affects B cell mediated immunity, we determined hemagglutinin (HA) antibody titers in the serum of A20myel-KO and wild type littermates.
T8741	22400-22581	Sentence	denotes	However, no differences could be detected between wild type and A20myel-KO animals (figure S2D), indicating that humoral immunity is not affected by A20 expression in myeloid cells.
T139	22400-22581	Sentence	denotes	However, no differences could be detected between wild type and A20myel-KO animals (figure S2D), indicating that humoral immunity is not affected by A20 expression in myeloid cells.
T8742	22582-22861	Sentence	denotes	Together, these data suggest that mechanisms other than adaptive immunity, such as an increased innate immune response, characterized by an increased influx of neutrophils and increased numbers of alveolar macrophages, contribute to the better viral clearance in A20myel-KO mice.
T140	22582-22861	Sentence	denotes	Together, these data suggest that mechanisms other than adaptive immunity, such as an increased innate immune response, characterized by an increased influx of neutrophils and increased numbers of alveolar macrophages, contribute to the better viral clearance in A20myel-KO mice.
T8743	22862-22974	Sentence	denotes	It is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung.
T141	22862-22974	Sentence	denotes	It is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung.
T8744	22975-23197	Sentence	denotes	We therefore analyzed whether the increased proinflammatory cytokine production and infiltration of proinflammatory cells in A20myel-KO mice affects mortality induced by intranasal infection with a lethal dose of IAV X-47.
T142	22975-23197	Sentence	denotes	We therefore analyzed whether the increased proinflammatory cytokine production and infiltration of proinflammatory cells in A20myel-KO mice affects mortality induced by intranasal infection with a lethal dose of IAV X-47.
T8745	23198-23340	Sentence	denotes	Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A).
T143	23198-23340	Sentence	denotes	Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A).
T8746	23341-23607	Sentence	denotes	A20myel-KO mice still showed significant weight loss and lung damage (as reflected by increased total protein concentration in the BAL; data not shown) during the course of infection but were able to recover, in contrast to wild type mice that succumbed (figure 4B).
T144	23341-23607	Sentence	denotes	A20myel-KO mice still showed significant weight loss and lung damage (as reflected by increased total protein concentration in the BAL; data not shown) during the course of infection but were able to recover, in contrast to wild type mice that succumbed (figure 4B).
T8747	23608-23904	Sentence	denotes	Similar to our observations with sublethal IAV infection, pulmonary KC and MIP-2 production was stronger in A20myel-KO animals compared to wild type mice following lethal IAV infection (figure 4C), which correlates with the increased numbers of neutrophils in the lungs of these mice (figure 4C).
T145	23608-23904	Sentence	denotes	Similar to our observations with sublethal IAV infection, pulmonary KC and MIP-2 production was stronger in A20myel-KO animals compared to wild type mice following lethal IAV infection (figure 4C), which correlates with the increased numbers of neutrophils in the lungs of these mice (figure 4C).
T8748	23905-24140	Sentence	denotes	Also levels of the proinflammatory cytokines IL-6, TNF and IL-1β, which are often associated with immunopathogenesis in humans [64], were increased in the lungs of A20myel-KO mice compared to control animals (figure 4C and figure S3A).
T146	23905-24140	Sentence	denotes	Also levels of the proinflammatory cytokines IL-6, TNF and IL-1β, which are often associated with immunopathogenesis in humans [64], were increased in the lungs of A20myel-KO mice compared to control animals (figure 4C and figure S3A).
T8749	24141-24306	Sentence	denotes	Again, MCP-1 production was not increased and even lower in A20myel-KO mice (figure 4C), and also monocyte recruitment was not different between both groups of mice.
T147	24141-24306	Sentence	denotes	Again, MCP-1 production was not increased and even lower in A20myel-KO mice (figure 4C), and also monocyte recruitment was not different between both groups of mice.
T8750	24307-24435	Sentence	denotes	We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F).
T148	24307-24435	Sentence	denotes	We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F).
T8751	24436-24607	Sentence	denotes	Collectively these data indicate that A20 deficiency in myeloid cells is associated with an increased innate immune response and protection against a lethal IAV infection.
T149	24436-24607	Sentence	denotes	Collectively these data indicate that A20 deficiency in myeloid cells is associated with an increased innate immune response and protection against a lethal IAV infection.
T150	24608-24722	Sentence	denotes	10.1371/journal.ppat.1002570.g004 Figure 4 A20 deficiency in myeloid cells protects against lethal IAV infection.
T21219	24652-24722	Sentence	denotes	A20 deficiency in myeloid cells protects against lethal IAV infection.
T21220	24723-24930	Sentence	denotes	A20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p<0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i.
T151	24723-24930	Sentence	denotes	A20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p<0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i.
T21221	24931-25124	Sentence	denotes	BAL fluid was collected from wild type (A20myel-WT) and A20myel-KO mice 6 days following infection with a lethal dose IAV and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were determined (C).
T152	24931-25124	Sentence	denotes	BAL fluid was collected from wild type (A20myel-WT) and A20myel-KO mice 6 days following infection with a lethal dose IAV and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were determined (C).
T21222	25125-25251	Sentence	denotes	Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D).
T153	25125-25251	Sentence	denotes	Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D).
T21223	25252-25310	Sentence	denotes	Numbers are averages +/− SEM of at least 5 mice per group.
T154	25252-25310	Sentence	denotes	Numbers are averages +/− SEM of at least 5 mice per group.
T21224	25311-25395	Sentence	denotes	Data are representative of 2 independent experiments. p<0.05; p<0.01, **p<0.001.
T155	25311-25395	Sentence	denotes	Data are representative of 2 independent experiments. p<0.05; p<0.01, **p<0.001.
T156	25397-25407	Sentence	denotes	Discussion
T11703	25408-25554	Sentence	denotes	In the present study we have investigated the contribution of A20 expression in myeloid cells in the innate immune response to IAV lung infection.
T157	25408-25554	Sentence	denotes	In the present study we have investigated the contribution of A20 expression in myeloid cells in the innate immune response to IAV lung infection.
T11704	25555-25701	Sentence	denotes	In the pulmonary environment, macrophages populate both lung parenchyma and the alveolar lumen where they are referred to as alveolar macrophages.
T158	25555-25701	Sentence	denotes	In the pulmonary environment, macrophages populate both lung parenchyma and the alveolar lumen where they are referred to as alveolar macrophages.
T11705	25702-25801	Sentence	denotes	Under naïve conditions, alveolar macrophages exert important immunomodulatory functions [65], [66].
T159	25702-25801	Sentence	denotes	Under naïve conditions, alveolar macrophages exert important immunomodulatory functions [65], [66].
T11706	25802-25977	Sentence	denotes	However, alveolar macrophages are also crucial in the innate immune response against IAV as they are one of the first cells that encounter infectious IAV particles [67], [68].
T160	25802-25977	Sentence	denotes	However, alveolar macrophages are also crucial in the innate immune response against IAV as they are one of the first cells that encounter infectious IAV particles [67], [68].
T11707	25978-26195	Sentence	denotes	They are an important source of proinflammatory cytokines and chemokines that attract innate immune cells to the lung during the primary phase of infection [69], and they are the primary producers of type I IFNs [70].
T161	25978-26195	Sentence	denotes	They are an important source of proinflammatory cytokines and chemokines that attract innate immune cells to the lung during the primary phase of infection [69], and they are the primary producers of type I IFNs [70].
T11708	26196-26355	Sentence	denotes	Alveolar macrophages are also known to phagocytose virus infected apoptotic cells and antibody coated viral particles, further contributing to viral clearance.
T162	26196-26355	Sentence	denotes	Alveolar macrophages are also known to phagocytose virus infected apoptotic cells and antibody coated viral particles, further contributing to viral clearance.
T11709	26356-26558	Sentence	denotes	We could show that BMDM or alveolar macrophages derived from A20myel-KO mice express higher amounts of chemokines, cytokines and type I IFNs compared to wild type mice in response to in vitro infection.
T163	26356-26558	Sentence	denotes	We could show that BMDM or alveolar macrophages derived from A20myel-KO mice express higher amounts of chemokines, cytokines and type I IFNs compared to wild type mice in response to in vitro infection.
T11710	26559-26815	Sentence	denotes	Similarly, in vivo infection with IAV resulted in higher levels of primarily neutrophil attracting chemokines such as KC and MIP-2 and several proinflammatory cytokines such as IL-6, TNF and IL-1β in the lungs of A20myel-KO mice compared to wild type mice.
T164	26559-26815	Sentence	denotes	Similarly, in vivo infection with IAV resulted in higher levels of primarily neutrophil attracting chemokines such as KC and MIP-2 and several proinflammatory cytokines such as IL-6, TNF and IL-1β in the lungs of A20myel-KO mice compared to wild type mice.
T11711	26816-26994	Sentence	denotes	This was associated with a selective enhancement of neutrophil recruitment to the bronchoalveolar compartment, and resulted in improved viral clearance later on during infection.
T165	26816-26994	Sentence	denotes	This was associated with a selective enhancement of neutrophil recruitment to the bronchoalveolar compartment, and resulted in improved viral clearance later on during infection.
T11712	26995-27168	Sentence	denotes	Although the role of neutrophils during viral infection is still under debate, recent evidence supports a protective function of these cells during IAV infection [71], [72].
T166	26995-27168	Sentence	denotes	Although the role of neutrophils during viral infection is still under debate, recent evidence supports a protective function of these cells during IAV infection [71], [72].
T11713	27169-27374	Sentence	denotes	MCP-1 levels were not affected by the absence of A20 in myeloid cells, which is consistent with the notion that airway epithelial cells are the primary source of MCP-1 production during IAV infection [73].
T167	27169-27374	Sentence	denotes	MCP-1 levels were not affected by the absence of A20 in myeloid cells, which is consistent with the notion that airway epithelial cells are the primary source of MCP-1 production during IAV infection [73].
T11714	27375-27567	Sentence	denotes	Mice deficient for the MCP-1 receptor CCR2, which is expressed on a subset of circulating monocytes, are protected against IAV infection and display reduced signs of immunopathology [74]–[76].
T168	27375-27567	Sentence	denotes	Mice deficient for the MCP-1 receptor CCR2, which is expressed on a subset of circulating monocytes, are protected against IAV infection and display reduced signs of immunopathology [74]–[76].
T11715	27568-27757	Sentence	denotes	During IAV infection these monocytes develop into inflammatory dendritic cells or proinflammatory macrophages [77] and are considered major contributors to IAV-induced immunopathology [78].
T169	27568-27757	Sentence	denotes	During IAV infection these monocytes develop into inflammatory dendritic cells or proinflammatory macrophages [77] and are considered major contributors to IAV-induced immunopathology [78].
T11716	27758-28022	Sentence	denotes	A20myel-KO mice were protected against a lethal IAV infection, which is rather surprising since an excessive proinflammatory cytokine response and an excessive influx of inflammatory cells is generally believed to be associated with increased lethality [64], [79].
T170	27758-28022	Sentence	denotes	A20myel-KO mice were protected against a lethal IAV infection, which is rather surprising since an excessive proinflammatory cytokine response and an excessive influx of inflammatory cells is generally believed to be associated with increased lethality [64], [79].
T11717	28023-28301	Sentence	denotes	However, the selective effect of A20 deficiency on neutrophil recruitment, without altering inflammatory monocyte (Ly6C+ CD11b+) recruitment, further support the idea that monocytes and not neutrophils are major contributors to IAV-associated immunopathology and lethality [78].
T171	28023-28301	Sentence	denotes	However, the selective effect of A20 deficiency on neutrophil recruitment, without altering inflammatory monocyte (Ly6C+ CD11b+) recruitment, further support the idea that monocytes and not neutrophils are major contributors to IAV-associated immunopathology and lethality [78].
T11718	28302-28447	Sentence	denotes	We show that A20 deficient BMDM display enhanced NF-κB and IRF3 activation in response to RIG-I stimulation by synthetic LMW double stranded RNA.
T172	28302-28447	Sentence	denotes	We show that A20 deficient BMDM display enhanced NF-κB and IRF3 activation in response to RIG-I stimulation by synthetic LMW double stranded RNA.
T11719	28448-28663	Sentence	denotes	RIG-I has previously been shown to play a key role in the innate immune response to IAV [13], suggesting that the increased immune response of A20myel-KO mice to IAV lung infection reflects enhanced RIG-I signaling.
T173	28448-28663	Sentence	denotes	RIG-I has previously been shown to play a key role in the innate immune response to IAV [13], suggesting that the increased immune response of A20myel-KO mice to IAV lung infection reflects enhanced RIG-I signaling.
T11720	28664-28906	Sentence	denotes	We propose that A20 inhibits IAV-induced proinflammatory gene expression (as shown in our manuscript for TNF, IL-6, KC, MIP-2, and IFNβ) by negatively regulating NF-κB and IRF3 activation, which are the major pathways controlling these genes.
T174	28664-28906	Sentence	denotes	We propose that A20 inhibits IAV-induced proinflammatory gene expression (as shown in our manuscript for TNF, IL-6, KC, MIP-2, and IFNβ) by negatively regulating NF-κB and IRF3 activation, which are the major pathways controlling these genes.
T11721	28907-29049	Sentence	denotes	However, this does not exclude an additional effect of A20 on other signaling pathways that may contribute to proinflammatory gene expression.
T175	28907-29049	Sentence	denotes	However, this does not exclude an additional effect of A20 on other signaling pathways that may contribute to proinflammatory gene expression.
T11722	29050-29192	Sentence	denotes	A20 is believed to exert its NF-κB and IRF3 inhibitory functions by modulating the ubiquitination status of different signaling proteins [80].
T176	29050-29192	Sentence	denotes	A20 is believed to exert its NF-κB and IRF3 inhibitory functions by modulating the ubiquitination status of different signaling proteins [80].
T11723	29193-29465	Sentence	denotes	In this context, it was recently shown that A20 cooperates with the ubiquitin-binding proteins TAX1BP1 and ABIN1 to to disrupt the TRAF3-TBK1-IKKε complex, thereby negatively affecting K63-polyubiquitination of TBK1 and IKKε, and their ability to activity IRF3 [81], [82].
T177	29193-29465	Sentence	denotes	In this context, it was recently shown that A20 cooperates with the ubiquitin-binding proteins TAX1BP1 and ABIN1 to to disrupt the TRAF3-TBK1-IKKε complex, thereby negatively affecting K63-polyubiquitination of TBK1 and IKKε, and their ability to activity IRF3 [81], [82].
T11724	29466-29575	Sentence	denotes	Whether similar mechanisms are involved in the regulation of RIG-I induced NF-κB activation is still unclear.
T178	29466-29575	Sentence	denotes	Whether similar mechanisms are involved in the regulation of RIG-I induced NF-κB activation is still unclear.
T11725	29576-29760	Sentence	denotes	So far we were unable to clearly detect ubiquitination of TBK1 and IKKε in primary macrophages, preventing us from studying the effect of A20 deficiency on their ubiquitination status.
T179	29576-29760	Sentence	denotes	So far we were unable to clearly detect ubiquitination of TBK1 and IKKε in primary macrophages, preventing us from studying the effect of A20 deficiency on their ubiquitination status.
T11726	29761-29886	Sentence	denotes	It cannot be excluded however that A20 also targets other substrates that mediate NF-κB and IRF3 activation in myeloid cells.
T180	29761-29886	Sentence	denotes	It cannot be excluded however that A20 also targets other substrates that mediate NF-κB and IRF3 activation in myeloid cells.
T11727	29887-29987	Sentence	denotes	The identification of these substrates will be the topic of future investigations in our laboratory.
T181	29887-29987	Sentence	denotes	The identification of these substrates will be the topic of future investigations in our laboratory.
T11728	29988-30211	Sentence	denotes	Multiple other deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30], OTUB1/2 [31], and A20 [49]–[52] have been shown to negatively regulate RIG-I signaling to NF-κB and IRF-3, implicating possible redundancy.
T182	29988-30211	Sentence	denotes	Multiple other deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30], OTUB1/2 [31], and A20 [49]–[52] have been shown to negatively regulate RIG-I signaling to NF-κB and IRF-3, implicating possible redundancy.
T11729	30212-30318	Sentence	denotes	However, evidence so far was limited to in vitro data and was obtained under non-physiological conditions.
T183	30212-30318	Sentence	denotes	However, evidence so far was limited to in vitro data and was obtained under non-physiological conditions.
T11730	30319-30606	Sentence	denotes	The clear protective phenotype of A20myel-KO mice that we here describe indicates that A20 expression in myeloid cells is a central gatekeeper of RIG-I induced signaling in response to IAV infection and that other DUBs cannot substitute for A20 deficiency under physiological conditions.
T184	30319-30606	Sentence	denotes	The clear protective phenotype of A20myel-KO mice that we here describe indicates that A20 expression in myeloid cells is a central gatekeeper of RIG-I induced signaling in response to IAV infection and that other DUBs cannot substitute for A20 deficiency under physiological conditions.
T11731	30607-30769	Sentence	denotes	If A20 has a similar non-redundant role in other cell types that are implicated in the response to IAV, such as lung epithelial cells, remains to be investigated.
T185	30607-30769	Sentence	denotes	If A20 has a similar non-redundant role in other cell types that are implicated in the response to IAV, such as lung epithelial cells, remains to be investigated.
T11732	30770-30896	Sentence	denotes	Understanding and controlling the activation of innate antiviral immune responses is an important step toward novel therapies.
T186	30770-30896	Sentence	denotes	Understanding and controlling the activation of innate antiviral immune responses is an important step toward novel therapies.
T11733	30897-31066	Sentence	denotes	About a fifth of world's population is infected by IAV annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised.
T187	30897-31066	Sentence	denotes	About a fifth of world's population is infected by IAV annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised.
T11734	31067-31175	Sentence	denotes	The high mutation rate of IAV turns all attempts of vaccine and antiviral design into a never ending battle.
T188	31067-31175	Sentence	denotes	The high mutation rate of IAV turns all attempts of vaccine and antiviral design into a never ending battle.
T11735	31176-31315	Sentence	denotes	In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen.
T189	31176-31315	Sentence	denotes	In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen.
T11736	31316-31421	Sentence	denotes	Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication.
T190	31316-31421	Sentence	denotes	Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication.
T11737	31422-31698	Sentence	denotes	Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism [83].
T191	31422-31698	Sentence	denotes	Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism [83].
T11738	31699-31896	Sentence	denotes	Our results suggest that not only siRNA targeting IAV components, but boosting the antiviral immune response by intranasal administration of siRNA against A20 might be a valid therapeutic approach.
T192	31699-31896	Sentence	denotes	Our results suggest that not only siRNA targeting IAV components, but boosting the antiviral immune response by intranasal administration of siRNA against A20 might be a valid therapeutic approach.
T11739	31897-31971	Sentence	denotes	Also small compound inhibitors of A20 might be an interesting alternative.
T193	31897-31971	Sentence	denotes	Also small compound inhibitors of A20 might be an interesting alternative.
T11740	31972-32107	Sentence	denotes	Finally, similar targeting of A20 might be of interest in the battle against other viral infections including RSV and SARS coronavirus.
T194	31972-32107	Sentence	denotes	Finally, similar targeting of A20 might be of interest in the battle against other viral infections including RSV and SARS coronavirus.
T195	32109-32130	Sentence	denotes	Materials and Methods
T14796	32132-32148	Sentence	denotes	Ethics statement
T196	32132-32148	Sentence	denotes	Ethics statement
T14797	32149-32358	Sentence	denotes	All experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations.
T197	32149-32358	Sentence	denotes	All experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations.
T14798	32359-32482	Sentence	denotes	Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001).
T198	32359-32482	Sentence	denotes	Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001).
T14799	32483-32540	Sentence	denotes	All efforts were made to ameliorate suffering of animals.
T199	32483-32540	Sentence	denotes	All efforts were made to ameliorate suffering of animals.
T14800	32541-32660	Sentence	denotes	Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).
T200	32541-32660	Sentence	denotes	Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).
T14992	32662-32666	Sentence	denotes	Mice
T201	32662-32666	Sentence	denotes	Mice
T14993	32667-32725	Sentence	denotes	A20fl/fl mice were generated as previously described [56].
T202	32667-32725	Sentence	denotes	A20fl/fl mice were generated as previously described [56].
T203	32726-32792	Sentence	denotes	A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I.
T14994	32726-32940	Sentence	denotes	A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I. Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48].
T204	32793-32940	Sentence	denotes	Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48].
T14995	32941-33090	Sentence	denotes	Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities.
T205	32941-33090	Sentence	denotes	Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities.
T14996	33091-33191	Sentence	denotes	Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates.
T206	33091-33191	Sentence	denotes	Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates.
T14997	33192-33273	Sentence	denotes	A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background.
T207	33192-33273	Sentence	denotes	A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background.
T14998	33274-33396	Sentence	denotes	A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.
T208	33274-33396	Sentence	denotes	A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.
T15282	33398-33447	Sentence	denotes	Viral infection and determination of viral titers
T209	33398-33447	Sentence	denotes	Viral infection and determination of viral titers
T15283	33448-33528	Sentence	denotes	Mouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells.
T210	33448-33528	Sentence	denotes	Mouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells.
T15284	33529-33702	Sentence	denotes	For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally.
T211	33529-33702	Sentence	denotes	For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally.
T15285	33703-33791	Sentence	denotes	For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47.
T212	33703-33791	Sentence	denotes	For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47.
T15286	33792-33972	Sentence	denotes	To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS.
T213	33792-33972	Sentence	denotes	To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS.
T15287	33973-34163	Sentence	denotes	Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
T214	33973-34163	Sentence	denotes	Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
T15288	34164-34281	Sentence	denotes	For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored.
T215	34164-34281	Sentence	denotes	For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored.
T15289	34282-34365	Sentence	denotes	TCID50 titers were then calculated according to the method of Reed and Muench [85].
T216	34282-34365	Sentence	denotes	TCID50 titers were then calculated according to the method of Reed and Muench [85].
T15660	34367-34424	Sentence	denotes	Determination of HAI (hemagglutination inhibition) titers
T217	34367-34424	Sentence	denotes	Determination of HAI (hemagglutination inhibition) titers
T15661	34425-34652	Sentence	denotes	To determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination.
T218	34425-34652	Sentence	denotes	To determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination.
T15662	34653-34787	Sentence	denotes	After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min.
T219	34653-34787	Sentence	denotes	After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min.
T15663	34788-34879	Sentence	denotes	To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC.
T220	34788-34879	Sentence	denotes	To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC.
T15664	34880-35036	Sentence	denotes	Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates.
T221	34880-35036	Sentence	denotes	Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates.
T15665	35037-35126	Sentence	denotes	Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later.
T222	35037-35126	Sentence	denotes	Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later.
T15666	35127-35373	Sentence	denotes	Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.
T223	35127-35373	Sentence	denotes	Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.
T16109	35375-35444	Sentence	denotes	In vivo intracellular GrB and IFNγ staining of activated CD8+ T cells
T224	35375-35444	Sentence	denotes	In vivo intracellular GrB and IFNγ staining of activated CD8+ T cells
T16110	35445-35605	Sentence	denotes	Granzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86].
T225	35445-35605	Sentence	denotes	Granzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86].
T226	35606-35740	Sentence	denotes	6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A.
T16111	35606-35865	Sentence	denotes	6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A. Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions.
T227	35741-35865	Sentence	denotes	Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions.
T16112	35866-35962	Sentence	denotes	Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression.
T228	35866-35962	Sentence	denotes	Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression.
T16113	35963-36071	Sentence	denotes	Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.
T229	35963-36071	Sentence	denotes	Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.
T16543	36073-36095	Sentence	denotes	Cells and transfection
T230	36073-36095	Sentence	denotes	Cells and transfection
T16544	36096-36230	Sentence	denotes	HEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
T231	36096-36230	Sentence	denotes	HEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
T232	36231-36451	Sentence	denotes	HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T.
T16545	36231-36793	Sentence	denotes	HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T. Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J. Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization.
T233	36452-36668	Sentence	denotes	Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J.
T234	36669-36793	Sentence	denotes	Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization.
T16546	36794-36928	Sentence	denotes	Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.
T235	36794-36928	Sentence	denotes	Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.
T16547	36929-37129	Sentence	denotes	For the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF.
T236	36929-37129	Sentence	denotes	For the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF.
T16548	37130-37227	Sentence	denotes	BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies.
T237	37130-37227	Sentence	denotes	BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies.
T16549	37228-37361	Sentence	denotes	For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA.
T238	37228-37361	Sentence	denotes	For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA.
T16550	37362-37501	Sentence	denotes	Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
T239	37362-37501	Sentence	denotes	Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.
T17160	37503-37519	Sentence	denotes	Western blotting
T240	37503-37519	Sentence	denotes	Western blotting
T17161	37520-37712	Sentence	denotes	For total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors.
T241	37520-37712	Sentence	denotes	For total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors.
T242	37713-37945	Sentence	denotes	Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C.
T17162	37713-38058	Sentence	denotes	Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min.
T243	37946-38058	Sentence	denotes	Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min.
T244	38059-38296	Sentence	denotes	The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C.
T17163	38059-38429	Sentence	denotes	The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences).
T245	38297-38429	Sentence	denotes	The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences).
T17164	38430-38638	Sentence	denotes	Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals).
T246	38430-38638	Sentence	denotes	Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals).
T17165	38639-38757	Sentence	denotes	The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool.
T247	38639-38757	Sentence	denotes	The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool.
T17166	38758-38829	Sentence	denotes	All intensities were calculated relative to the first lane ( = time 0).
T248	38758-38829	Sentence	denotes	All intensities were calculated relative to the first lane ( = time 0).
T17826	38831-38845	Sentence	denotes	Flow cytometry
T249	38831-38845	Sentence	denotes	Flow cytometry
T17827	38846-38970	Sentence	denotes	Lungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min.
T250	38846-38970	Sentence	denotes	Lungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min.
T17828	38971-39044	Sentence	denotes	Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh.
T251	38971-39044	Sentence	denotes	Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh.
T17829	39045-39158	Sentence	denotes	For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA.
T252	39045-39158	Sentence	denotes	For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA.
T253	39159-39558	Sentence	denotes	Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen.
T17830	39159-39656	Sentence	denotes	Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen. Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences).
T254	39559-39656	Sentence	denotes	Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences).
T17831	39657-39727	Sentence	denotes	Propidium iodide was used to discriminate between live and dead cells.
T255	39657-39727	Sentence	denotes	Propidium iodide was used to discriminate between live and dead cells.
T18266	39729-39752	Sentence	denotes	Cytokine quantification
T256	39729-39752	Sentence	denotes	Cytokine quantification
T18267	39753-39881	Sentence	denotes	For TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies.
T257	39753-39881	Sentence	denotes	For TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies.
T18268	39882-39975	Sentence	denotes	IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories).
T258	39882-39975	Sentence	denotes	IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories).
T18269	39976-40095	Sentence	denotes	For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences).
T259	39976-40095	Sentence	denotes	For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences).
T18270	40096-40246	Sentence	denotes	Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions.
T260	40096-40246	Sentence	denotes	Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions.
T18271	40247-40329	Sentence	denotes	Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.
T261	40247-40329	Sentence	denotes	Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.

T24

0-86

Sentence

denotes

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

T1

0-86

Sentence

denotes

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

T2

87-136

Sentence

denotes

Loss of A20 in Myeloid Cells Protects against IAV

T3

138-146

Sentence

denotes

Abstract

T25

147-291

Sentence

denotes

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules.

T4

147-291

Sentence

denotes

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules.

T26

292-550

Sentence

denotes

Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon.

T5

292-550

Sentence

denotes

Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon.

T27

551-704

Sentence

denotes

Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies.

T6

551-704

Sentence

denotes

Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies.

T28

705-865

Sentence

denotes

Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection.

T7

705-865

Sentence

denotes

Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection.

T29

866-1110

Sentence

denotes

A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon.

T8

866-1110

Sentence

denotes

A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon.

T30

1111-1238

Sentence

denotes

In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice.

T9

1111-1238

Sentence

denotes

In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice.

T31

1239-1336

Sentence

denotes

Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection.

T10

1239-1336

Sentence

denotes

Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection.

T32

1337-1587

Sentence

denotes

These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.

T11

1337-1587

Sentence

denotes

These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.

T12

1589-1603

Sentence

denotes

Author Summary

T13

1604-1806

Sentence

denotes

Influenza virus or flu epidemics represent a recurrent threat to the public health, especially for individuals which are part of a high-risk group such as children, elderly or immune-compromised people.

T14

1807-1930

Sentence

denotes

Sporadic pandemic flu outbreaks, such as the Spanish flu of 1918, may cause high grades of mortality among healthy persons.

T15

1931-2027

Sentence

denotes

A better understanding of how the immune system deals with these pathogens is of key importance.

T16

2028-2124

Sentence

denotes

The protein A20 is an important negative regulator of both innate and adaptive immune responses.

T17

2125-2292

Sentence

denotes

We show that the specific deletion of A20 in myeloid cells, such as macrophages and neutrophils, improves the resistance against otherwise lethal influenza infections.

T18

2293-2419

Sentence

denotes

This protective effect is mediated by an enhanced innate immune response following respiratory challenge with influenza virus.

T19

2420-2725

Sentence

denotes

Although exaggerated pulmonary immune responses are believed to be the primary cause of often life threatening influenza virus induced pneumonia, we demonstrate that boosting the innate immune response by selectively targeting the functionality of A20 in myeloid cells is beneficial for the host survival.

T20

2726-2856

Sentence

denotes

This finding provides us with a novel valuable approach for treating influenza and potentially other respiratory viral infections.

T21

2858-2870

Sentence

denotes

Introduction

T911

2871-2971

Sentence

denotes

Viruses are a class of highly diverse pathogens which depend on the host cell for their replication.

T22

2871-2971

Sentence

denotes

Viruses are a class of highly diverse pathogens which depend on the host cell for their replication.

T912

2972-3177

Sentence

denotes

The initiation of a protective innate antiviral immune response involves the action of specialized pattern recognition receptors (PRR), which detect conserved molecular structures of the invading pathogen.

T23

2972-3177

Sentence

denotes

The initiation of a protective innate antiviral immune response involves the action of specialized pattern recognition receptors (PRR), which detect conserved molecular structures of the invading pathogen.

T913

3178-3475

Sentence

denotes

Triggering of PRRs induces the production of host proinflammatory cytokines (e.g. TNF, IL-6 and IL-1) and type I interferons (interferon-α (IFN-α) and IFN-β) through activation of downstream signaling pathways that control various transcription factors such as NF-κB, AP-1, IRF3 and IRF7 [1], [2].

T24

3178-3475

Sentence

denotes

Triggering of PRRs induces the production of host proinflammatory cytokines (e.g. TNF, IL-6 and IL-1) and type I interferons (interferon-α (IFN-α) and IFN-β) through activation of downstream signaling pathways that control various transcription factors such as NF-κB, AP-1, IRF3 and IRF7 [1], [2].

T914

3476-3655

Sentence

denotes

The presence of viral nucleic acids, such as viral RNA and DNA, viral replication intermediates and viral transcription products, can be sensed by specific intracellular PRRs [3].

T25

3476-3655

Sentence

denotes

The presence of viral nucleic acids, such as viral RNA and DNA, viral replication intermediates and viral transcription products, can be sensed by specific intracellular PRRs [3].

T915

3656-3886

Sentence

denotes

Endosomal Toll-like receptors (TLRs) and cytoplasmic RNA helicase RIG-I-like receptors (RLRs) or Nod-like receptors (NLRs) detect the presence of viral single stranded (TLR7, TLR8, Nod2) or double stranded RNA (TLR3, RIG-I, MDA5).

T26

3656-3886

Sentence

denotes

Endosomal Toll-like receptors (TLRs) and cytoplasmic RNA helicase RIG-I-like receptors (RLRs) or Nod-like receptors (NLRs) detect the presence of viral single stranded (TLR7, TLR8, Nod2) or double stranded RNA (TLR3, RIG-I, MDA5).

T916

3887-4064

Sentence

denotes

Intracellular DNA sensors that mediate antiviral immune responses to DNA viruses include TLR9, DAI [4] and the PYHIN domain containing proteins AIM2 [5], [6], [7] and IFI16 [8].

T27

3887-4064

Sentence

denotes

Intracellular DNA sensors that mediate antiviral immune responses to DNA viruses include TLR9, DAI [4] and the PYHIN domain containing proteins AIM2 [5], [6], [7] and IFI16 [8].

T917

4065-4365

Sentence

denotes

TLR mediated antiviral responses are restricted to specialized type I IFN producing plasmacytoid dendritic cells (pDC), while most other cell types, including conventional DC (cDC), macrophages and fibroblasts, depend on the cytosolic RNA and DNA sensors for the production of antiviral proteins [9].

T28

4065-4365

Sentence

denotes

TLR mediated antiviral responses are restricted to specialized type I IFN producing plasmacytoid dendritic cells (pDC), while most other cell types, including conventional DC (cDC), macrophages and fibroblasts, depend on the cytosolic RNA and DNA sensors for the production of antiviral proteins [9].

T918

4366-4574

Sentence

denotes

Influenza A virus (IAV) is the etiological agent of a contagious acute respiratory disease that causes considerable mortality, which is generally believed to be due to an excessive host inflammatory response.

T29

4366-4574

Sentence

denotes

Influenza A virus (IAV) is the etiological agent of a contagious acute respiratory disease that causes considerable mortality, which is generally believed to be due to an excessive host inflammatory response.

T919

4575-4724

Sentence

denotes

Emergence of drug-resistant strains of influenza viruses with pandemic potential underscores the importance of developing novel antiviral strategies.

T30

4575-4724

Sentence

denotes

Emergence of drug-resistant strains of influenza viruses with pandemic potential underscores the importance of developing novel antiviral strategies.

T920

4725-4829

Sentence

denotes

In this context, understanding of the mechanisms that regulate IAV-induced immune responses is critical.

T31

4725-4829

Sentence

denotes

In this context, understanding of the mechanisms that regulate IAV-induced immune responses is critical.

T921

4830-4993

Sentence

denotes

IAV infection leads to the exposure in the host cell of single-stranded genomic RNA and double stranded RNA, the latter being an intermediate of viral replication.

T32

4830-4993

Sentence

denotes

IAV infection leads to the exposure in the host cell of single-stranded genomic RNA and double stranded RNA, the latter being an intermediate of viral replication.

T922

4994-5068

Sentence

denotes

TLR3 and RIG-I have been implicated as sensors of IAV infection [10]–[12].

T33

4994-5068

Sentence

denotes

TLR3 and RIG-I have been implicated as sensors of IAV infection [10]–[12].

T923

5069-5243

Sentence

denotes

Both receptors contribute to the proinflammatory response to IAV, but the initiation of the innate antiviral immune response largely depends on RIG-I mediated signaling [13].

T34

5069-5243

Sentence

denotes

Both receptors contribute to the proinflammatory response to IAV, but the initiation of the innate antiviral immune response largely depends on RIG-I mediated signaling [13].

T924

5244-5400

Sentence

denotes

Interestingly, RIG-I deficient mice are highly susceptible to IAV [14], [15], whereas TLR3 deficient mice have a survival advantage to acute infection [16].

T35

5244-5400

Sentence

denotes

Interestingly, RIG-I deficient mice are highly susceptible to IAV [14], [15], whereas TLR3 deficient mice have a survival advantage to acute infection [16].

T925

5401-5580

Sentence

denotes

These results indicate that an imbalance between the beneficial and harmful effects of mediators released by immune cells is likely to contribute to the pathogenesis of influenza.

T36

5401-5580

Sentence

denotes

These results indicate that an imbalance between the beneficial and harmful effects of mediators released by immune cells is likely to contribute to the pathogenesis of influenza.

T926

5581-5711

Sentence

denotes

RIG-I contains a C-terminal DExD/H box helicase domain, which is required for ligand recognition, and two N-terminal CARD domains.

T37

5581-5711

Sentence

denotes

RIG-I contains a C-terminal DExD/H box helicase domain, which is required for ligand recognition, and two N-terminal CARD domains.

T927

5712-5986

Sentence

denotes

Upon ligand binding, the CARD domains of RIG-I associate with the CARD domain of the MAVS (also termed IPS-1, VISA, Cardif) adaptor protein, which subsequently translocates to and inserts in the outer mitochondrial membrane via its C-terminal transmembrane domain [17]–[20].

T38

5712-5986

Sentence

denotes

Upon ligand binding, the CARD domains of RIG-I associate with the CARD domain of the MAVS (also termed IPS-1, VISA, Cardif) adaptor protein, which subsequently translocates to and inserts in the outer mitochondrial membrane via its C-terminal transmembrane domain [17]–[20].

T928

5987-6162

Sentence

denotes

Signaling downstream of MAVS requires the action of various ubiquitin modifying enzymes, which both positively and negatively regulate RIG-I mediated signal transduction [21].

T39

5987-6162

Sentence

denotes

Signaling downstream of MAVS requires the action of various ubiquitin modifying enzymes, which both positively and negatively regulate RIG-I mediated signal transduction [21].

T929

6163-6297

Sentence

denotes

K63-specific ubiquitin ligases (E3s), such as TRIM25 [22] and Riplet [23], [24], have been shown to directly promote RIG-I activation.

T40

6163-6297

Sentence

denotes

K63-specific ubiquitin ligases (E3s), such as TRIM25 [22] and Riplet [23], [24], have been shown to directly promote RIG-I activation.

T930

6298-6458

Sentence

denotes

In addition, well characterized ubiquitin ligases such as TRAF6 [25], [26] and TRAF3 [27] mediate respectively NF-κB and IRF3 activation upon RIG-I stimulation.

T41

6298-6458

Sentence

denotes

In addition, well characterized ubiquitin ligases such as TRAF6 [25], [26] and TRAF3 [27] mediate respectively NF-κB and IRF3 activation upon RIG-I stimulation.

T931

6459-6706

Sentence

denotes

On the other hand, deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30] and OTUB1/2 [31] have been shown to negatively regulate RLR signaling by specifically removing K63-linked polyubiquitin chains from several signaling molecules.

T42

6459-6706

Sentence

denotes

On the other hand, deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30] and OTUB1/2 [31] have been shown to negatively regulate RLR signaling by specifically removing K63-linked polyubiquitin chains from several signaling molecules.

T932

6707-6910

Sentence

denotes

Furthermore, various K48-specific ubiquitin ligases, such as AIP4 [32] and TRIAD3A [33] mark respectively MAVS and TRAF3 for proteasome mediated degradation, thus inhibiting further downstream signaling.

T43

6707-6910

Sentence

denotes

Furthermore, various K48-specific ubiquitin ligases, such as AIP4 [32] and TRIAD3A [33] mark respectively MAVS and TRAF3 for proteasome mediated degradation, thus inhibiting further downstream signaling.

T933

6911-7119

Sentence

denotes

Additionally, the attachment of K48-specific polyubiquitin chains to the IRF3 and IRF7 transcription factors by E3s such as RAUL [34], TRIM21 [35] and RBCK1 [36] further dampens antiviral signal transduction.

T44

6911-7119

Sentence

denotes

Additionally, the attachment of K48-specific polyubiquitin chains to the IRF3 and IRF7 transcription factors by E3s such as RAUL [34], TRIM21 [35] and RBCK1 [36] further dampens antiviral signal transduction.

T934

7120-7198

Sentence

denotes

A20 is an ubiquitin-editing enzyme belonging to the OTU-domain family of DUBs.

T45

7120-7198

Sentence

denotes

A20 is an ubiquitin-editing enzyme belonging to the OTU-domain family of DUBs.

T935

7199-7304

Sentence

denotes

Interestingly, A20 also harbors atypical zinc finger dependent K48-specific E3 ubiquitin ligase activity.

T46

7199-7304

Sentence

denotes

Interestingly, A20 also harbors atypical zinc finger dependent K48-specific E3 ubiquitin ligase activity.

T936

7305-7542

Sentence

denotes

Both catalytic and noncatalytic mechanisms were previously shown to be involved in the negative regulation of proinflammatory signaling by A20 in response to multiple receptors such as TNF receptor I [37]–[39], TLR4 [40], and IL-1R [41].

T47

7305-7542

Sentence

denotes

Both catalytic and noncatalytic mechanisms were previously shown to be involved in the negative regulation of proinflammatory signaling by A20 in response to multiple receptors such as TNF receptor I [37]–[39], TLR4 [40], and IL-1R [41].

T937

7543-7716

Sentence

denotes

The anti-inflammatory role of A20 is clearly demonstrated by the fact that A20 deficient mice die early after birth due to severe multi-organ inflammation and cachexia [37].

T48

7543-7716

Sentence

denotes

The anti-inflammatory role of A20 is clearly demonstrated by the fact that A20 deficient mice die early after birth due to severe multi-organ inflammation and cachexia [37].

T938

7717-7939

Sentence

denotes

More recently, gene targeting of A20 in specific cell types was shown to be associated with autoimmunity and chronic inflammation [42]–[48], further illustrating that A20 is an important brake on the inflammatory response.

T49

7717-7939

Sentence

denotes

More recently, gene targeting of A20 in specific cell types was shown to be associated with autoimmunity and chronic inflammation [42]–[48], further illustrating that A20 is an important brake on the inflammatory response.

T939

7940-8164

Sentence

denotes

The relevance of these findings for human disease has recently been illustrated through the identification of polymorphisms in the A20 locus that are associated with several autoimmune diseases and chronic inflammation [45].

T50

7940-8164

Sentence

denotes

The relevance of these findings for human disease has recently been illustrated through the identification of polymorphisms in the A20 locus that are associated with several autoimmune diseases and chronic inflammation [45].

T940

8165-8543

Sentence

denotes

In contrast to its well established function in the regulation of proinflammatory responses, the role of A20 in the regulation of antiviral immune responses is less well described and limited to a number of in vitro studies using overexpression or silencing in specific cell lines, indicating that A20 may regulate RIG-I- and TLR3-induced signaling to NF-κB and IRF-3 [49]–[52].

T51

8165-8543

Sentence

denotes

In contrast to its well established function in the regulation of proinflammatory responses, the role of A20 in the regulation of antiviral immune responses is less well described and limited to a number of in vitro studies using overexpression or silencing in specific cell lines, indicating that A20 may regulate RIG-I- and TLR3-induced signaling to NF-κB and IRF-3 [49]–[52].

T941

8544-8644

Sentence

denotes

However, the precise in vivo role of A20 in the response to viral infection remains to be clarified.

T52

8544-8644

Sentence

denotes

However, the precise in vivo role of A20 in the response to viral infection remains to be clarified.

T942

8645-8921

Sentence

denotes

Using myeloid cell specific A20 knockout mice (A20myel-KO) that were recently generated in our lab and primary cells derived of these mice, we here provide evidence that A20 is a crucial negative regulator of IAV-induced proinflammatory and antiviral signaling in macrophages.

T53

8645-8921

Sentence

denotes

Using myeloid cell specific A20 knockout mice (A20myel-KO) that were recently generated in our lab and primary cells derived of these mice, we here provide evidence that A20 is a crucial negative regulator of IAV-induced proinflammatory and antiviral signaling in macrophages.

T943

8922-9059

Sentence

denotes

Interestingly, A20myel-KO mice show enhanced survival and reduced morbidity in response to IAV lung infection compared to wild type mice.

T54

8922-9059

Sentence

denotes

Interestingly, A20myel-KO mice show enhanced survival and reduced morbidity in response to IAV lung infection compared to wild type mice.

T944

9060-9285

Sentence

denotes

Protection against IAV in A20myel-KO mice is associated with increased cytokine and chemokine production, augmented recruitment of innate immune cells such as neutophils and alveolar macrophages, and enhanced viral clearance.

T55

9060-9285

Sentence

denotes

Protection against IAV in A20myel-KO mice is associated with increased cytokine and chemokine production, augmented recruitment of innate immune cells such as neutophils and alveolar macrophages, and enhanced viral clearance.

T945

9286-9432

Sentence

denotes

These results suggest that boosting the innate immune response to IAV by targeting A20 activity in myeloid cells might have therapeutic potential.

T56

9286-9432

Sentence

denotes

These results suggest that boosting the innate immune response to IAV by targeting A20 activity in myeloid cells might have therapeutic potential.

T57

9434-9441

Sentence

denotes

Results

T4724

9443-9495

Sentence

denotes

A20 inhibits RIG-I-induced NF-κB and IRF3 activation

T58

9443-9495

Sentence

denotes

A20 inhibits RIG-I-induced NF-κB and IRF3 activation

T4725

9496-9696

Sentence

denotes

RIG-I signaling induces the activation of NF-κB, IRF3 and IRF7 transcription factors, which promote the expression of proinflammatory cytokines and type I IFNs that restrict further viral propagation.

T59

9496-9696

Sentence

denotes

RIG-I signaling induces the activation of NF-κB, IRF3 and IRF7 transcription factors, which promote the expression of proinflammatory cytokines and type I IFNs that restrict further viral propagation.

T4726

9697-9836

Sentence

denotes

Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52].

T60

9697-9836

Sentence

denotes

Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52].

T4727

9837-10161

Sentence

denotes

Similarly, we show that A20 overexpression in HEK293T cells prevents NF-κB- and IRF3-dependent luciferase reporter gene activation induced by transfection of a truncated constitutive active form of RIG-I [53], corresponding to only the two N-terminal CARD domains of RIG-I [RIG-I (2CARD)] (figure 1A, left and middle graph).

T61

9837-10161

Sentence

denotes

Similarly, we show that A20 overexpression in HEK293T cells prevents NF-κB- and IRF3-dependent luciferase reporter gene activation induced by transfection of a truncated constitutive active form of RIG-I [53], corresponding to only the two N-terminal CARD domains of RIG-I [RIG-I (2CARD)] (figure 1A, left and middle graph).

T4728

10162-10225

Sentence

denotes

We next investigated whether A20 also inhibits IRF7 activation.

T62

10162-10225

Sentence

denotes

We next investigated whether A20 also inhibits IRF7 activation.

T4729

10226-10388

Sentence

denotes

Unlike IRF3, IRF7 is not or weakly expressed under naïve conditions and IRF7 protein levels are rapidly upregulated upon virus-induced IRF3 activation [54], [55].

T63

10226-10388

Sentence

denotes

Unlike IRF3, IRF7 is not or weakly expressed under naïve conditions and IRF7 protein levels are rapidly upregulated upon virus-induced IRF3 activation [54], [55].

T4730

10389-10617

Sentence

denotes

To determine the effect of A20 on IRF7 activation, we therefore transfected minor amounts of an IRF7 expression plasmid together with plasmids encoding RIG-I (2CARD), A20 and an IRF7-specific IFNα4 luciferase reporter construct.

T64

10389-10617

Sentence

denotes

To determine the effect of A20 on IRF7 activation, we therefore transfected minor amounts of an IRF7 expression plasmid together with plasmids encoding RIG-I (2CARD), A20 and an IRF7-specific IFNα4 luciferase reporter construct.

T4731

10618-10842

Sentence

denotes

RIG-I (2CARD) expression in the absence of IRF7 co-expression showed negligible IFNα4 promoter activation (grey bar, figure 1A, right graph), whereas significant reporter gene expression was observed in the presence of IRF7.

T65

10618-10842

Sentence

denotes

RIG-I (2CARD) expression in the absence of IRF7 co-expression showed negligible IFNα4 promoter activation (grey bar, figure 1A, right graph), whereas significant reporter gene expression was observed in the presence of IRF7.

T4732

10843-10980

Sentence

denotes

Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph).

T66

10843-10980

Sentence

denotes

Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph).

T4733

10981-11081

Sentence

denotes

These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.

T67

10981-11081

Sentence

denotes

These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.

T68

11082-11198

Sentence

denotes

10.1371/journal.ppat.1002570.g001 Figure 1 A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.

T19347

11126-11198

Sentence

denotes

A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.

T19348

11199-11415

Sentence

denotes

(A) HEK293T cells were transfected with NF-κB (left), IRF3 (middle) or IRF7 dependent luciferase reporter (right) plasmids, together with plasmids expressing RIG-I (2CARD), IRF7 (right) and increasing amounts of A20.

T69

11199-11415

Sentence

denotes

(A) HEK293T cells were transfected with NF-κB (left), IRF3 (middle) or IRF7 dependent luciferase reporter (right) plasmids, together with plasmids expressing RIG-I (2CARD), IRF7 (right) and increasing amounts of A20.

T19349

11416-11483

Sentence

denotes

Luciferase activity in cell extracts was analyzed in a luminometer.

T70

11416-11483

Sentence

denotes

Luciferase activity in cell extracts was analyzed in a luminometer.

T19350

11484-11814

Sentence

denotes

Numbers are averages +/− SD of 3 samples per set-up. (B–D) A20myel-KO and wild type (A20myel-WT) BMDM were transfected with LMW poly(I:C) and analyzed at different time points (minutes) after the start of transfection for IκBα phosphorylation and degradation, and IRF3 phosphorylation by immunoblotting of total cell extracts (B).

T71

11484-11814

Sentence

denotes

Numbers are averages +/− SD of 3 samples per set-up. (B–D) A20myel-KO and wild type (A20myel-WT) BMDM were transfected with LMW poly(I:C) and analyzed at different time points (minutes) after the start of transfection for IκBα phosphorylation and degradation, and IRF3 phosphorylation by immunoblotting of total cell extracts (B).

T19351

11815-11946

Sentence

denotes

The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control.

T72

11815-11946

Sentence

denotes

The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control.

T19352

11947-12064

Sentence

denotes

Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C).

T73

11947-12064

Sentence

denotes

Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C).

T19353

12065-12214

Sentence

denotes

Quantification of band density for nuclear p65 and IRF3 is shown in bar graphs (expressed as relative density compared to the first lane; = time 0).

T74

12065-12214

Sentence

denotes

Quantification of band density for nuclear p65 and IRF3 is shown in bar graphs (expressed as relative density compared to the first lane; = time 0).

T19354

12215-12397

Sentence

denotes

IL-6, TNF and IFNβ levels in the supernatant of cells used in (B) were determined by bioassay (IL-6) and ELISA (TNF and IFNβ) as described in the ‘materials and methods’ section (D).

T75

12215-12397

Sentence

denotes

IL-6, TNF and IFNβ levels in the supernatant of cells used in (B) were determined by bioassay (IL-6) and ELISA (TNF and IFNβ) as described in the ‘materials and methods’ section (D).

T19355

12398-12451

Sentence

denotes

Data are representative of 2 independent experiments.

T76

12398-12451

Sentence

denotes

Data are representative of 2 independent experiments.

T4734

12452-12641

Sentence

denotes

To study the effect of endogenously expressed A20 on RIG-I-induced signaling in a more immunological relevant context, we performed further experiments in A20 deficient primary macrophages.

T77

12452-12641

Sentence

denotes

To study the effect of endogenously expressed A20 on RIG-I-induced signaling in a more immunological relevant context, we performed further experiments in A20 deficient primary macrophages.

T4735

12642-12856

Sentence

denotes

Since A20 full knockout mice die prematurely as a result of severe multi-organ inflammation [37], we generated mice carrying a conditional A20 allele in which exon IV and exon V were flanked by two loxP sites [56].

T78

12642-12856

Sentence

denotes

Since A20 full knockout mice die prematurely as a result of severe multi-organ inflammation [37], we generated mice carrying a conditional A20 allele in which exon IV and exon V were flanked by two loxP sites [56].

T4736

12857-13082

Sentence

denotes

Crossing these mice with transgenic mice expressing Cre recombinase under control of the lysozyme M promoter leads to specific deletion in myeloid cells and allowed us to generate myeloid cell specific A20 knockout mice [48].

T79

12857-13082

Sentence

denotes

Crossing these mice with transgenic mice expressing Cre recombinase under control of the lysozyme M promoter leads to specific deletion in myeloid cells and allowed us to generate myeloid cell specific A20 knockout mice [48].

T4737

13083-13316

Sentence

denotes

To stimulate the RIG-I receptor, we transfected A20myel-KO BMDM and wild type control cells with minimal amounts of low molecular weight (LMW) poly(I:C), which is known to preferentially bind and activate RIG-I rather than MDA5 [57].

T80

13083-13316

Sentence

denotes

To stimulate the RIG-I receptor, we transfected A20myel-KO BMDM and wild type control cells with minimal amounts of low molecular weight (LMW) poly(I:C), which is known to preferentially bind and activate RIG-I rather than MDA5 [57].

T4738

13317-13472

Sentence

denotes

Of note, this concentration of poly(I:C) was unable to induce significant TLR3 dependent NF-κB and IRF3 activation or cytokine production (data not shown).

T81

13317-13472

Sentence

denotes

Of note, this concentration of poly(I:C) was unable to induce significant TLR3 dependent NF-κB and IRF3 activation or cytokine production (data not shown).

T4739

13473-13611

Sentence

denotes

As expected, poly(I:C) transfection induced the rapid expression of A20 in wild type, but not in A20myel-KO BMDM (figure 1B, upper panel).

T82

13473-13611

Sentence

denotes

As expected, poly(I:C) transfection induced the rapid expression of A20 in wild type, but not in A20myel-KO BMDM (figure 1B, upper panel).

T4740

13612-13773

Sentence

denotes

At early time points, slightly slower migrating forms of A20 were observed, indicating that A20 undergoes a yet unknown modification upon poly(I:C) transfection.

T83

13612-13773

Sentence

denotes

At early time points, slightly slower migrating forms of A20 were observed, indicating that A20 undergoes a yet unknown modification upon poly(I:C) transfection.

T4741

13774-13943

Sentence

denotes

Compared to wild type BMBM, A20 deficient cells showed enhanced NF-κB activation as indicated by increased phosphorylation and sustained degradation of IκBα (figure 1B).

T84

13774-13943

Sentence

denotes

Compared to wild type BMBM, A20 deficient cells showed enhanced NF-κB activation as indicated by increased phosphorylation and sustained degradation of IκBα (figure 1B).

T4742

13944-14141

Sentence

denotes

Furthermore, nuclear translocation of the p65 NF-κB subunit was enhanced in poly(I:C) transfected A20myel-KO BMDM, reaching a maximum at earlier time points compared to wild type cells (figure 1C).

T85

13944-14141

Sentence

denotes

Furthermore, nuclear translocation of the p65 NF-κB subunit was enhanced in poly(I:C) transfected A20myel-KO BMDM, reaching a maximum at earlier time points compared to wild type cells (figure 1C).

T4743

14142-14363

Sentence

denotes

IRF3 is known to be activated upon phosphorylation of a series of carboxyl terminal serine residues by the IKK-like kinases TBK1 and IKKε [58], leading to its dimerization and subsequent translocation to the nucleus [59].

T86

14142-14363

Sentence

denotes

IRF3 is known to be activated upon phosphorylation of a series of carboxyl terminal serine residues by the IKK-like kinases TBK1 and IKKε [58], leading to its dimerization and subsequent translocation to the nucleus [59].

T4744

14364-14565

Sentence

denotes

Using immunoblotting with an antibody directed against phosphorylated Ser396, maximum IRF3 phosphorylation was detected at earlier time points in A20myel-KO BMDM compared to wild type BMDM (figure 1B).

T87

14364-14565

Sentence

denotes

Using immunoblotting with an antibody directed against phosphorylated Ser396, maximum IRF3 phosphorylation was detected at earlier time points in A20myel-KO BMDM compared to wild type BMDM (figure 1B).

T4745

14566-14716

Sentence

denotes

Similar to p65, IRF3 nuclear translocation reached its maximum at an earlier time point in A20 deficient BMDM compared to wild type cells (figure 1C).

T88

14566-14716

Sentence

denotes

Similar to p65, IRF3 nuclear translocation reached its maximum at an earlier time point in A20 deficient BMDM compared to wild type cells (figure 1C).

T4746

14717-14814

Sentence

denotes

NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ.

T89

14717-14814

Sentence

denotes

NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ.

T4747

14815-14967

Sentence

denotes

In line with the enhanced activation of NF-κB and IRF3 as described above, A20myel-KO BMDM secreted increased amounts of IL-6, TNF and IFNβ (figure 1D).

T90

14815-14967

Sentence

denotes

In line with the enhanced activation of NF-κB and IRF3 as described above, A20myel-KO BMDM secreted increased amounts of IL-6, TNF and IFNβ (figure 1D).

T4748

14968-15044

Sentence

denotes

Similar results were obtained using peritoneal macrophages (data not shown).

T91

14968-15044

Sentence

denotes

Similar results were obtained using peritoneal macrophages (data not shown).

T4749

15045-15207

Sentence

denotes

Together, these results demonstrate that A20 plays an important role in the negative regulation of RIG-I-induced NF-κB and IRF3 activation in primary macrophages.

T92

15045-15207

Sentence

denotes

Together, these results demonstrate that A20 plays an important role in the negative regulation of RIG-I-induced NF-κB and IRF3 activation in primary macrophages.

T7698

15209-15269

Sentence

denotes

A20 negatively regulates IAV-induced gene expression in BMDM

T93

15209-15269

Sentence

denotes

A20 negatively regulates IAV-induced gene expression in BMDM

T7699

15270-15439

Sentence

denotes

To investigate the role of A20 in the IAV-induced proinflammatory and antiviral innate immune responses, we infected A20 deficient and control BMDM with IAV X-47 (H3N2).

T94

15270-15439

Sentence

denotes

To investigate the role of A20 in the IAV-induced proinflammatory and antiviral innate immune responses, we infected A20 deficient and control BMDM with IAV X-47 (H3N2).

T7700

15440-15560

Sentence

denotes

A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A).

T95

15440-15560

Sentence

denotes

A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A).

T7701

15561-15695

Sentence

denotes

Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A).

T96

15561-15695

Sentence

denotes

Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A).

T7702

15696-15831

Sentence

denotes

In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).

T97

15696-15831

Sentence

denotes

In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).

T98

15832-15962

Sentence

denotes

10.1371/journal.ppat.1002570.g002 Figure 2 A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.

T20279

15876-15962

Sentence

denotes

A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.

T20280

15963-16053

Sentence

denotes

(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1).

T99

15963-16053

Sentence

denotes

(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1).

T20281

16054-16182

Sentence

denotes

At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A).

T100

16054-16182

Sentence

denotes

At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A).

T20282

16183-16408

Sentence

denotes

18 hours post infection cell culture supernatant was analyzed for TNF and IFNβ protein levels by ELISA and multiplexing technologies (B). (C–D) Alveolar macrophages were mock treated or infected with IAV (moi 1) for 18 hours.

T101

16183-16408

Sentence

denotes

18 hours post infection cell culture supernatant was analyzed for TNF and IFNβ protein levels by ELISA and multiplexing technologies (B). (C–D) Alveolar macrophages were mock treated or infected with IAV (moi 1) for 18 hours.

T20283

16409-16466

Sentence

denotes

IL-6 and IFNβ mRNA expression was determined by qPCR (C).

T102

16409-16466

Sentence

denotes

IL-6 and IFNβ mRNA expression was determined by qPCR (C).

T20284

16467-16581

Sentence

denotes

Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D).

T103

16467-16581

Sentence

denotes

Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D).

T20285

16582-16640

Sentence

denotes

Error bars represent mean values (+/− SEM) of 2–3 samples.

T104

16582-16640

Sentence

denotes

Error bars represent mean values (+/− SEM) of 2–3 samples.

T20286

16641-16710

Sentence

denotes

Results are representative for 2 independent experiments. ***p<0,001.

T105

16641-16710

Sentence

denotes

Results are representative for 2 independent experiments. ***p<0,001.

T7703

16711-16896

Sentence

denotes

Upon host infection with IAV, alveolar macrophages are an important source of cytokines and chemokines, attracting innate immune cells to the lung during the primary phase of infection.

T106

16711-16896

Sentence

denotes

Upon host infection with IAV, alveolar macrophages are an important source of cytokines and chemokines, attracting innate immune cells to the lung during the primary phase of infection.

T7704

16897-17102

Sentence

denotes

To test whether A20 directly controls IAV-induced gene expression in alveolar macrophages, we isolated these cells from lungs of A20myel-KO and control littermates and infected them in vitro with IAV X-47.

T107

16897-17102

Sentence

denotes

To test whether A20 directly controls IAV-induced gene expression in alveolar macrophages, we isolated these cells from lungs of A20myel-KO and control littermates and infected them in vitro with IAV X-47.

T7705

17103-17374

Sentence

denotes

Expression and secretion of the proinflammatory cytokines IL-6 and TNF, the type-I IFN IFNβ and IFNα4 and the chemokines MCP-1 (ccl2) and KC (cxcl1) was significantly higher in IAV infected cells lacking A20 compared to infected wild type cells (figure 2C and figure S1).

T108

17103-17374

Sentence

denotes

Expression and secretion of the proinflammatory cytokines IL-6 and TNF, the type-I IFN IFNβ and IFNα4 and the chemokines MCP-1 (ccl2) and KC (cxcl1) was significantly higher in IAV infected cells lacking A20 compared to infected wild type cells (figure 2C and figure S1).

T7706

17375-17622

Sentence

denotes

Taken together these results demonstrate that A20 negatively regulates IAV-induced proinflammatory and antiviral gene expression in alveolar macrophages, consistent with the inhibitory effect of A20 seen on RIG-I-induced NF-κB and IRF3 activation.

T109

17375-17622

Sentence

denotes

Taken together these results demonstrate that A20 negatively regulates IAV-induced proinflammatory and antiviral gene expression in alveolar macrophages, consistent with the inhibitory effect of A20 seen on RIG-I-induced NF-κB and IRF3 activation.

T8720

17624-17691

Sentence

denotes

A20 deficiency in myeloid cells protects against IAV lung infection

T110

17624-17691

Sentence

denotes

A20 deficiency in myeloid cells protects against IAV lung infection

T8721

17692-17968

Sentence

denotes

To determine the role of A20 expression in myeloid cells during an IAV infection in vivo, we intranasally inoculated both A20myel-KO mice and control littermates with a sublethal dose of the mouse adapted IAV X-47 (H3N2) strain and monitored morbidity in terms of weight loss.

T111

17692-17968

Sentence

denotes

To determine the role of A20 expression in myeloid cells during an IAV infection in vivo, we intranasally inoculated both A20myel-KO mice and control littermates with a sublethal dose of the mouse adapted IAV X-47 (H3N2) strain and monitored morbidity in terms of weight loss.

T8722

17969-18112

Sentence

denotes

A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A).

T112

17969-18112

Sentence

denotes

A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A).

T8723

18113-18368

Sentence

denotes

Also, total protein concentration in BAL fluid, which reflects lung damage and vascular leakage, was increased significantly at 7 and 10 days post infection in both wild type and A20myel-KO mice, and was slightly lower in A20myel-KO mice (data not shown).

T113

18113-18368

Sentence

denotes

Also, total protein concentration in BAL fluid, which reflects lung damage and vascular leakage, was increased significantly at 7 and 10 days post infection in both wild type and A20myel-KO mice, and was slightly lower in A20myel-KO mice (data not shown).

T8724

18369-18445

Sentence

denotes

Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection.

T114

18369-18445

Sentence

denotes

Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection.

T8725

18446-18562

Sentence

denotes

No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection.

T115

18446-18562

Sentence

denotes

No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection.

T8726

18563-18764

Sentence

denotes

However, after 10 days, almost no virus could be detected in the lungs of A20myel-KO mice while abundant infectious viral particles could still be isolated from lungs of all wild type mice (figure 3B).

T116

18563-18764

Sentence

denotes

However, after 10 days, almost no virus could be detected in the lungs of A20myel-KO mice while abundant infectious viral particles could still be isolated from lungs of all wild type mice (figure 3B).

T8727

18765-18922

Sentence

denotes

This indicates that loss of A20 in myeloid cells does not affect early viral replication but contributes to viral clearance at later stages during infection.

T117

18765-18922

Sentence

denotes

This indicates that loss of A20 in myeloid cells does not affect early viral replication but contributes to viral clearance at later stages during infection.

T118

18923-19035

Sentence

denotes

10.1371/journal.ppat.1002570.g003 Figure 3 A20 deficiency in myeloid cells protects against IAV lung infection.

T20779

18967-19035

Sentence

denotes

A20 deficiency in myeloid cells protects against IAV lung infection.

T20780

19036-19157

Sentence

denotes

A20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A).

T119

19036-19157

Sentence

denotes

A20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A).

T20781

19158-19271

Sentence

denotes

At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B).

T120

19158-19271

Sentence

denotes

At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B).

T20782

19272-19319

Sentence

denotes

The dashed line represents the detection limit.

T121

19272-19319

Sentence

denotes

The dashed line represents the detection limit.

T20783

19320-19339

Sentence

denotes

ND, not detectable.

T122

19320-19339

Sentence

denotes

ND, not detectable.

T20784

19340-19513

Sentence

denotes

BAL was isolated from IAV infected mice at 4, 7 and 10 days p.i. and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were analyzed by ELISA and multiplexing technologies (C).

T123

19340-19513

Sentence

denotes

BAL was isolated from IAV infected mice at 4, 7 and 10 days p.i. and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were analyzed by ELISA and multiplexing technologies (C).

T20785

19514-19659

Sentence

denotes

Absolute numbers of resident alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 4, 7 and 10 days p.i. (D).

T124

19514-19659

Sentence

denotes

Absolute numbers of resident alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 4, 7 and 10 days p.i. (D).

T20786

19660-19789

Sentence

denotes

Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. *p<0.05; **p<0.01.

T125

19660-19789

Sentence

denotes

Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. *p<0.05; **p<0.01.

T8728

19790-20022

Sentence

denotes

To verify if A20 deficiency in myeloid cells affects IAV-induced gene expression in the lung, we analyzed the levels of several chemokines and cytokines in the bronchoalveolar lavage (BAL) at day 4, 7 and day 10 following infection.

T126

19790-20022

Sentence

denotes

To verify if A20 deficiency in myeloid cells affects IAV-induced gene expression in the lung, we analyzed the levels of several chemokines and cytokines in the bronchoalveolar lavage (BAL) at day 4, 7 and day 10 following infection.

T8729

20023-20207

Sentence

denotes

Levels of KC and MIP-2 chemokines, as well as IL-6 were significantly higher at day 7 p.i. in BAL from IAV infected A20myel-KO mice compared to IAV infected wild type mice (figure 3C).

T127

20023-20207

Sentence

denotes

Levels of KC and MIP-2 chemokines, as well as IL-6 were significantly higher at day 7 p.i. in BAL from IAV infected A20myel-KO mice compared to IAV infected wild type mice (figure 3C).

T8730

20208-20395

Sentence

denotes

Unlike our observations with in vitro stimulated primary macrophages we could not detect a significant increase in MCP-1 or IFNα production in the lungs of A20myel-KO animals (figure 3C).

T128

20208-20395

Sentence

denotes

Unlike our observations with in vitro stimulated primary macrophages we could not detect a significant increase in MCP-1 or IFNα production in the lungs of A20myel-KO animals (figure 3C).

T8731

20396-20645

Sentence

denotes

KC is the murine orthologue of IL-8 and serves together with MIP-2 as a chemoattractant for neutrophils, while MCP-1 is mainly known as a chemoattractant for monocytes, which eventually develop into macrophages upon entering the alveolar lumen [60].

T129

20396-20645

Sentence

denotes

KC is the murine orthologue of IL-8 and serves together with MIP-2 as a chemoattractant for neutrophils, while MCP-1 is mainly known as a chemoattractant for monocytes, which eventually develop into macrophages upon entering the alveolar lumen [60].

T8732

20646-20935

Sentence

denotes

Consistent with the higher KC and MIP-2 levels in A20myel-KO mice, the number of neutrophils (CD11b+ Ly6C+ Ly6G+ F4/80−) that were recruited in the bronchoalveolar spaces upon IAV infection was clearly higher throughout infection in A20myel-KO mice compared to control animals (figure 3D).

T130

20646-20935

Sentence

denotes

Consistent with the higher KC and MIP-2 levels in A20myel-KO mice, the number of neutrophils (CD11b+ Ly6C+ Ly6G+ F4/80−) that were recruited in the bronchoalveolar spaces upon IAV infection was clearly higher throughout infection in A20myel-KO mice compared to control animals (figure 3D).

T8733

20936-21197

Sentence

denotes

Although we could detect a significant increase in monocyte (CD11b+ Ly6C+) recruitment at day 4 post infection, this was not evident at later time points after infection (figure 3D), which is consistent with the equal expression of MCP-1 in both groups of mice.

T131

20936-21197

Sentence

denotes

Although we could detect a significant increase in monocyte (CD11b+ Ly6C+) recruitment at day 4 post infection, this was not evident at later time points after infection (figure 3D), which is consistent with the equal expression of MCP-1 in both groups of mice.

T8734

21198-21407

Sentence

denotes

The number of resident alveolar macrophages (autofluorescent+ CD11c+ F4/80+ CD11b−) was also elevated in A20myel-KO mice but did not differ significantly between IAV infected or mock infected mice (figure 3D).

T132

21198-21407

Sentence

denotes

The number of resident alveolar macrophages (autofluorescent+ CD11c+ F4/80+ CD11b−) was also elevated in A20myel-KO mice but did not differ significantly between IAV infected or mock infected mice (figure 3D).

T8735

21408-21538

Sentence

denotes

Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63].

T133

21408-21538

Sentence

denotes

Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63].

T8736

21539-21787

Sentence

denotes

To test whether A20 expression in myeloid cells regulates the antiviral CTL response, total CD8+ T cells and virus specific Granzyme B (GrB) and IFNγ expressing CD8+ T cells were measured in BAL and lung parenchyma of wild type and A20myel-KO mice.

T134

21539-21787

Sentence

denotes

To test whether A20 expression in myeloid cells regulates the antiviral CTL response, total CD8+ T cells and virus specific Granzyme B (GrB) and IFNγ expressing CD8+ T cells were measured in BAL and lung parenchyma of wild type and A20myel-KO mice.

T8737

21788-21970

Sentence

denotes

A clear increase in CD8+ T cells could be detected at day 7 and 10 post infection, but no differences were observed between A20myel-KO and wild type mice (figure S2A and figure S2C).

T135

21788-21970

Sentence

denotes

A clear increase in CD8+ T cells could be detected at day 7 and 10 post infection, but no differences were observed between A20myel-KO and wild type mice (figure S2A and figure S2C).

T8738

21971-22135

Sentence

denotes

Also, the number of GrB and IFNγ CD8+ T cells as well as IFNγ expression levels in the lungs were not altered by the absence of A20 in myeloid cells (figure S2A–C).

T136

21971-22135

Sentence

denotes

Also, the number of GrB and IFNγ CD8+ T cells as well as IFNγ expression levels in the lungs were not altered by the absence of A20 in myeloid cells (figure S2A–C).

T8739

22136-22217

Sentence

denotes

Protection against IAV infection is also provided by the humoral immune response.

T137

22136-22217

Sentence

denotes

Protection against IAV infection is also provided by the humoral immune response.

T8740

22218-22399

Sentence

denotes

To test whether loss of A20 in myeloid cells affects B cell mediated immunity, we determined hemagglutinin (HA) antibody titers in the serum of A20myel-KO and wild type littermates.

T138

22218-22399

Sentence

denotes

To test whether loss of A20 in myeloid cells affects B cell mediated immunity, we determined hemagglutinin (HA) antibody titers in the serum of A20myel-KO and wild type littermates.

T8741

22400-22581

Sentence

denotes

However, no differences could be detected between wild type and A20myel-KO animals (figure S2D), indicating that humoral immunity is not affected by A20 expression in myeloid cells.

T139

22400-22581

Sentence

denotes

However, no differences could be detected between wild type and A20myel-KO animals (figure S2D), indicating that humoral immunity is not affected by A20 expression in myeloid cells.

T8742

22582-22861

Sentence

denotes

Together, these data suggest that mechanisms other than adaptive immunity, such as an increased innate immune response, characterized by an increased influx of neutrophils and increased numbers of alveolar macrophages, contribute to the better viral clearance in A20myel-KO mice.

T140

22582-22861

Sentence

denotes

Together, these data suggest that mechanisms other than adaptive immunity, such as an increased innate immune response, characterized by an increased influx of neutrophils and increased numbers of alveolar macrophages, contribute to the better viral clearance in A20myel-KO mice.

T8743

22862-22974

Sentence

denotes

It is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung.

T141

22862-22974

Sentence

denotes

It is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung.

T8744

22975-23197

Sentence

denotes

We therefore analyzed whether the increased proinflammatory cytokine production and infiltration of proinflammatory cells in A20myel-KO mice affects mortality induced by intranasal infection with a lethal dose of IAV X-47.

T142

22975-23197

Sentence

denotes

We therefore analyzed whether the increased proinflammatory cytokine production and infiltration of proinflammatory cells in A20myel-KO mice affects mortality induced by intranasal infection with a lethal dose of IAV X-47.

T8745

23198-23340

Sentence

denotes

Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A).

T143

23198-23340

Sentence

denotes

Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A).

T8746

23341-23607

Sentence

denotes

A20myel-KO mice still showed significant weight loss and lung damage (as reflected by increased total protein concentration in the BAL; data not shown) during the course of infection but were able to recover, in contrast to wild type mice that succumbed (figure 4B).

T144

23341-23607

Sentence

denotes

A20myel-KO mice still showed significant weight loss and lung damage (as reflected by increased total protein concentration in the BAL; data not shown) during the course of infection but were able to recover, in contrast to wild type mice that succumbed (figure 4B).

T8747

23608-23904

Sentence

denotes

Similar to our observations with sublethal IAV infection, pulmonary KC and MIP-2 production was stronger in A20myel-KO animals compared to wild type mice following lethal IAV infection (figure 4C), which correlates with the increased numbers of neutrophils in the lungs of these mice (figure 4C).

T145

23608-23904

Sentence

denotes

Similar to our observations with sublethal IAV infection, pulmonary KC and MIP-2 production was stronger in A20myel-KO animals compared to wild type mice following lethal IAV infection (figure 4C), which correlates with the increased numbers of neutrophils in the lungs of these mice (figure 4C).

T8748

23905-24140

Sentence

denotes

Also levels of the proinflammatory cytokines IL-6, TNF and IL-1β, which are often associated with immunopathogenesis in humans [64], were increased in the lungs of A20myel-KO mice compared to control animals (figure 4C and figure S3A).

T146

23905-24140

Sentence

denotes

Also levels of the proinflammatory cytokines IL-6, TNF and IL-1β, which are often associated with immunopathogenesis in humans [64], were increased in the lungs of A20myel-KO mice compared to control animals (figure 4C and figure S3A).

T8749

24141-24306

Sentence

denotes

Again, MCP-1 production was not increased and even lower in A20myel-KO mice (figure 4C), and also monocyte recruitment was not different between both groups of mice.

T147

24141-24306

Sentence

denotes

Again, MCP-1 production was not increased and even lower in A20myel-KO mice (figure 4C), and also monocyte recruitment was not different between both groups of mice.

T8750

24307-24435

Sentence

denotes

We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F).

T148

24307-24435

Sentence

denotes

We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F).

T8751

24436-24607

Sentence

denotes

Collectively these data indicate that A20 deficiency in myeloid cells is associated with an increased innate immune response and protection against a lethal IAV infection.

T149

24436-24607

Sentence

denotes

Collectively these data indicate that A20 deficiency in myeloid cells is associated with an increased innate immune response and protection against a lethal IAV infection.

T150

24608-24722

Sentence

denotes

10.1371/journal.ppat.1002570.g004 Figure 4 A20 deficiency in myeloid cells protects against lethal IAV infection.

T21219

24652-24722

Sentence

denotes

A20 deficiency in myeloid cells protects against lethal IAV infection.

T21220

24723-24930

Sentence

denotes

A20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p<0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i.

T151

24723-24930

Sentence

denotes

A20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p<0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i.

T21221

24931-25124

Sentence

denotes

BAL fluid was collected from wild type (A20myel-WT) and A20myel-KO mice 6 days following infection with a lethal dose IAV and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were determined (C).

T152

24931-25124

Sentence

denotes

BAL fluid was collected from wild type (A20myel-WT) and A20myel-KO mice 6 days following infection with a lethal dose IAV and KC, MIP-2, MCP-1, IL-6 and IFNα protein levels were determined (C).

T21222

25125-25251

Sentence

denotes

Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D).

T153

25125-25251

Sentence

denotes

Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D).

T21223

25252-25310

Sentence

denotes

Numbers are averages +/− SEM of at least 5 mice per group.

T154

25252-25310

Sentence

denotes

Numbers are averages +/− SEM of at least 5 mice per group.

T21224

25311-25395

Sentence

denotes

Data are representative of 2 independent experiments. *p<0.05; **p<0.01, ***p<0.001.

T155

25311-25395

Sentence

denotes

Data are representative of 2 independent experiments. *p<0.05; **p<0.01, ***p<0.001.

T156

25397-25407

Sentence

denotes

Discussion

T11703

25408-25554

Sentence

denotes

In the present study we have investigated the contribution of A20 expression in myeloid cells in the innate immune response to IAV lung infection.

T157

25408-25554

Sentence

denotes

In the present study we have investigated the contribution of A20 expression in myeloid cells in the innate immune response to IAV lung infection.

T11704

25555-25701

Sentence

denotes

In the pulmonary environment, macrophages populate both lung parenchyma and the alveolar lumen where they are referred to as alveolar macrophages.

T158

25555-25701

Sentence

denotes

In the pulmonary environment, macrophages populate both lung parenchyma and the alveolar lumen where they are referred to as alveolar macrophages.

T11705

25702-25801

Sentence

denotes

Under naïve conditions, alveolar macrophages exert important immunomodulatory functions [65], [66].

T159

25702-25801

Sentence

denotes

Under naïve conditions, alveolar macrophages exert important immunomodulatory functions [65], [66].

T11706

25802-25977

Sentence

denotes

However, alveolar macrophages are also crucial in the innate immune response against IAV as they are one of the first cells that encounter infectious IAV particles [67], [68].

T160

25802-25977

Sentence

denotes

However, alveolar macrophages are also crucial in the innate immune response against IAV as they are one of the first cells that encounter infectious IAV particles [67], [68].

T11707

25978-26195

Sentence

denotes

They are an important source of proinflammatory cytokines and chemokines that attract innate immune cells to the lung during the primary phase of infection [69], and they are the primary producers of type I IFNs [70].

T161

25978-26195

Sentence

denotes

They are an important source of proinflammatory cytokines and chemokines that attract innate immune cells to the lung during the primary phase of infection [69], and they are the primary producers of type I IFNs [70].

T11708

26196-26355

Sentence

denotes

Alveolar macrophages are also known to phagocytose virus infected apoptotic cells and antibody coated viral particles, further contributing to viral clearance.

T162

26196-26355

Sentence

denotes

Alveolar macrophages are also known to phagocytose virus infected apoptotic cells and antibody coated viral particles, further contributing to viral clearance.

T11709

26356-26558

Sentence

denotes

We could show that BMDM or alveolar macrophages derived from A20myel-KO mice express higher amounts of chemokines, cytokines and type I IFNs compared to wild type mice in response to in vitro infection.

T163

26356-26558

Sentence

denotes

We could show that BMDM or alveolar macrophages derived from A20myel-KO mice express higher amounts of chemokines, cytokines and type I IFNs compared to wild type mice in response to in vitro infection.

T11710

26559-26815

Sentence

denotes

Similarly, in vivo infection with IAV resulted in higher levels of primarily neutrophil attracting chemokines such as KC and MIP-2 and several proinflammatory cytokines such as IL-6, TNF and IL-1β in the lungs of A20myel-KO mice compared to wild type mice.

T164

26559-26815

Sentence

denotes

Similarly, in vivo infection with IAV resulted in higher levels of primarily neutrophil attracting chemokines such as KC and MIP-2 and several proinflammatory cytokines such as IL-6, TNF and IL-1β in the lungs of A20myel-KO mice compared to wild type mice.

T11711

26816-26994

Sentence

denotes

This was associated with a selective enhancement of neutrophil recruitment to the bronchoalveolar compartment, and resulted in improved viral clearance later on during infection.

T165

26816-26994

Sentence

denotes

This was associated with a selective enhancement of neutrophil recruitment to the bronchoalveolar compartment, and resulted in improved viral clearance later on during infection.

T11712

26995-27168

Sentence

denotes

Although the role of neutrophils during viral infection is still under debate, recent evidence supports a protective function of these cells during IAV infection [71], [72].

T166

26995-27168

Sentence

denotes

Although the role of neutrophils during viral infection is still under debate, recent evidence supports a protective function of these cells during IAV infection [71], [72].

T11713

27169-27374

Sentence

denotes

MCP-1 levels were not affected by the absence of A20 in myeloid cells, which is consistent with the notion that airway epithelial cells are the primary source of MCP-1 production during IAV infection [73].

T167

27169-27374

Sentence

denotes

MCP-1 levels were not affected by the absence of A20 in myeloid cells, which is consistent with the notion that airway epithelial cells are the primary source of MCP-1 production during IAV infection [73].

T11714

27375-27567

Sentence

denotes

Mice deficient for the MCP-1 receptor CCR2, which is expressed on a subset of circulating monocytes, are protected against IAV infection and display reduced signs of immunopathology [74]–[76].

T168

27375-27567

Sentence

denotes

Mice deficient for the MCP-1 receptor CCR2, which is expressed on a subset of circulating monocytes, are protected against IAV infection and display reduced signs of immunopathology [74]–[76].

T11715

27568-27757

Sentence

denotes

During IAV infection these monocytes develop into inflammatory dendritic cells or proinflammatory macrophages [77] and are considered major contributors to IAV-induced immunopathology [78].

T169

27568-27757

Sentence

denotes

During IAV infection these monocytes develop into inflammatory dendritic cells or proinflammatory macrophages [77] and are considered major contributors to IAV-induced immunopathology [78].

T11716

27758-28022

Sentence

denotes

A20myel-KO mice were protected against a lethal IAV infection, which is rather surprising since an excessive proinflammatory cytokine response and an excessive influx of inflammatory cells is generally believed to be associated with increased lethality [64], [79].

T170

27758-28022

Sentence

denotes

A20myel-KO mice were protected against a lethal IAV infection, which is rather surprising since an excessive proinflammatory cytokine response and an excessive influx of inflammatory cells is generally believed to be associated with increased lethality [64], [79].

T11717

28023-28301

Sentence

denotes

However, the selective effect of A20 deficiency on neutrophil recruitment, without altering inflammatory monocyte (Ly6C+ CD11b+) recruitment, further support the idea that monocytes and not neutrophils are major contributors to IAV-associated immunopathology and lethality [78].

T171

28023-28301

Sentence

denotes

However, the selective effect of A20 deficiency on neutrophil recruitment, without altering inflammatory monocyte (Ly6C+ CD11b+) recruitment, further support the idea that monocytes and not neutrophils are major contributors to IAV-associated immunopathology and lethality [78].

T11718

28302-28447

Sentence

denotes

We show that A20 deficient BMDM display enhanced NF-κB and IRF3 activation in response to RIG-I stimulation by synthetic LMW double stranded RNA.

T172

28302-28447

Sentence

denotes

We show that A20 deficient BMDM display enhanced NF-κB and IRF3 activation in response to RIG-I stimulation by synthetic LMW double stranded RNA.

T11719

28448-28663

Sentence

denotes

RIG-I has previously been shown to play a key role in the innate immune response to IAV [13], suggesting that the increased immune response of A20myel-KO mice to IAV lung infection reflects enhanced RIG-I signaling.

T173

28448-28663

Sentence

denotes

RIG-I has previously been shown to play a key role in the innate immune response to IAV [13], suggesting that the increased immune response of A20myel-KO mice to IAV lung infection reflects enhanced RIG-I signaling.

T11720

28664-28906

Sentence

denotes

We propose that A20 inhibits IAV-induced proinflammatory gene expression (as shown in our manuscript for TNF, IL-6, KC, MIP-2, and IFNβ) by negatively regulating NF-κB and IRF3 activation, which are the major pathways controlling these genes.

T174

28664-28906

Sentence

denotes

We propose that A20 inhibits IAV-induced proinflammatory gene expression (as shown in our manuscript for TNF, IL-6, KC, MIP-2, and IFNβ) by negatively regulating NF-κB and IRF3 activation, which are the major pathways controlling these genes.

T11721

28907-29049

Sentence

denotes

However, this does not exclude an additional effect of A20 on other signaling pathways that may contribute to proinflammatory gene expression.

T175

28907-29049

Sentence

denotes

However, this does not exclude an additional effect of A20 on other signaling pathways that may contribute to proinflammatory gene expression.

T11722

29050-29192

Sentence

denotes

A20 is believed to exert its NF-κB and IRF3 inhibitory functions by modulating the ubiquitination status of different signaling proteins [80].

T176

29050-29192

Sentence

denotes

A20 is believed to exert its NF-κB and IRF3 inhibitory functions by modulating the ubiquitination status of different signaling proteins [80].

T11723

29193-29465

Sentence

denotes

In this context, it was recently shown that A20 cooperates with the ubiquitin-binding proteins TAX1BP1 and ABIN1 to to disrupt the TRAF3-TBK1-IKKε complex, thereby negatively affecting K63-polyubiquitination of TBK1 and IKKε, and their ability to activity IRF3 [81], [82].

T177

29193-29465

Sentence

denotes

In this context, it was recently shown that A20 cooperates with the ubiquitin-binding proteins TAX1BP1 and ABIN1 to to disrupt the TRAF3-TBK1-IKKε complex, thereby negatively affecting K63-polyubiquitination of TBK1 and IKKε, and their ability to activity IRF3 [81], [82].

T11724

29466-29575

Sentence

denotes

Whether similar mechanisms are involved in the regulation of RIG-I induced NF-κB activation is still unclear.

T178

29466-29575

Sentence

denotes

Whether similar mechanisms are involved in the regulation of RIG-I induced NF-κB activation is still unclear.

T11725

29576-29760

Sentence

denotes

So far we were unable to clearly detect ubiquitination of TBK1 and IKKε in primary macrophages, preventing us from studying the effect of A20 deficiency on their ubiquitination status.

T179

29576-29760

Sentence

denotes

So far we were unable to clearly detect ubiquitination of TBK1 and IKKε in primary macrophages, preventing us from studying the effect of A20 deficiency on their ubiquitination status.

T11726

29761-29886

Sentence

denotes

It cannot be excluded however that A20 also targets other substrates that mediate NF-κB and IRF3 activation in myeloid cells.

T180

29761-29886

Sentence

denotes

It cannot be excluded however that A20 also targets other substrates that mediate NF-κB and IRF3 activation in myeloid cells.

T11727

29887-29987

Sentence

denotes

The identification of these substrates will be the topic of future investigations in our laboratory.

T181

29887-29987

Sentence

denotes

The identification of these substrates will be the topic of future investigations in our laboratory.

T11728

29988-30211

Sentence

denotes

Multiple other deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30], OTUB1/2 [31], and A20 [49]–[52] have been shown to negatively regulate RIG-I signaling to NF-κB and IRF-3, implicating possible redundancy.

T182

29988-30211

Sentence

denotes

Multiple other deubiquitinating enzymes (DUBs), such as DUBA [28], CYLD [29], [30], OTUB1/2 [31], and A20 [49]–[52] have been shown to negatively regulate RIG-I signaling to NF-κB and IRF-3, implicating possible redundancy.

T11729

30212-30318

Sentence

denotes

However, evidence so far was limited to in vitro data and was obtained under non-physiological conditions.

T183

30212-30318

Sentence

denotes

However, evidence so far was limited to in vitro data and was obtained under non-physiological conditions.

T11730

30319-30606

Sentence

denotes

The clear protective phenotype of A20myel-KO mice that we here describe indicates that A20 expression in myeloid cells is a central gatekeeper of RIG-I induced signaling in response to IAV infection and that other DUBs cannot substitute for A20 deficiency under physiological conditions.

T184

30319-30606

Sentence

denotes

The clear protective phenotype of A20myel-KO mice that we here describe indicates that A20 expression in myeloid cells is a central gatekeeper of RIG-I induced signaling in response to IAV infection and that other DUBs cannot substitute for A20 deficiency under physiological conditions.

T11731

30607-30769

Sentence

denotes

If A20 has a similar non-redundant role in other cell types that are implicated in the response to IAV, such as lung epithelial cells, remains to be investigated.

T185

30607-30769

Sentence

denotes

If A20 has a similar non-redundant role in other cell types that are implicated in the response to IAV, such as lung epithelial cells, remains to be investigated.

T11732

30770-30896

Sentence

denotes

Understanding and controlling the activation of innate antiviral immune responses is an important step toward novel therapies.

T186

30770-30896

Sentence

denotes

Understanding and controlling the activation of innate antiviral immune responses is an important step toward novel therapies.

T11733

30897-31066

Sentence

denotes

About a fifth of world's population is infected by IAV annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised.

T187

30897-31066

Sentence

denotes

About a fifth of world's population is infected by IAV annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised.

T11734

31067-31175

Sentence

denotes

The high mutation rate of IAV turns all attempts of vaccine and antiviral design into a never ending battle.

T188

31067-31175

Sentence

denotes

The high mutation rate of IAV turns all attempts of vaccine and antiviral design into a never ending battle.

T11735

31176-31315

Sentence

denotes

In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen.

T189

31176-31315

Sentence

denotes

In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen.

T11736

31316-31421

Sentence

denotes

Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication.

T190

31316-31421

Sentence

denotes

Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication.

T11737

31422-31698

Sentence

denotes

Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism [83].

T191

31422-31698

Sentence

denotes

Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism [83].

T11738

31699-31896

Sentence

denotes

Our results suggest that not only siRNA targeting IAV components, but boosting the antiviral immune response by intranasal administration of siRNA against A20 might be a valid therapeutic approach.

T192

31699-31896

Sentence

denotes

Our results suggest that not only siRNA targeting IAV components, but boosting the antiviral immune response by intranasal administration of siRNA against A20 might be a valid therapeutic approach.

T11739

31897-31971

Sentence

denotes

Also small compound inhibitors of A20 might be an interesting alternative.

T193

31897-31971

Sentence

denotes

Also small compound inhibitors of A20 might be an interesting alternative.

T11740

31972-32107

Sentence

denotes

Finally, similar targeting of A20 might be of interest in the battle against other viral infections including RSV and SARS coronavirus.

T194

31972-32107

Sentence

denotes

Finally, similar targeting of A20 might be of interest in the battle against other viral infections including RSV and SARS coronavirus.

T195

32109-32130

Sentence

denotes

Materials and Methods

T14796

32132-32148

Sentence

denotes

Ethics statement

T196

32132-32148

Sentence

denotes

Ethics statement

T14797

32149-32358

Sentence

denotes

All experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations.

T197

32149-32358

Sentence

denotes

All experiments on mice were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations.

T14798

32359-32482

Sentence

denotes

Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001).

T198

32359-32482

Sentence

denotes

Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091, approval ID 2010/001).

T14799

32483-32540

Sentence

denotes

All efforts were made to ameliorate suffering of animals.

T199

32483-32540

Sentence

denotes

All efforts were made to ameliorate suffering of animals.

T14800

32541-32660

Sentence

denotes

Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).

T200

32541-32660

Sentence

denotes

Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine (12 mg/kg) and xylazine (60 mg/kg).

T14992

32662-32666

Sentence

denotes

Mice

T201

32662-32666

Sentence

denotes

Mice

T14993

32667-32725

Sentence

denotes

A20fl/fl mice were generated as previously described [56].

T202

32667-32725

Sentence

denotes

A20fl/fl mice were generated as previously described [56].

T203

32726-32792

Sentence

denotes

A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I.

T14994

32726-32940

Sentence

denotes

A20fl/fl mice were crossed with LysM-Cre mice [84] (provided by I. Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48].

T204

32793-32940

Sentence

denotes

Förster, Institute of Genetics, University of Cologne, Germany) to generate A20fl/fl LysMCre transgenes and are described in detail elsewhere [48].

T14995

32941-33090

Sentence

denotes

Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities.

T205

32941-33090

Sentence

denotes

Mice were housed in individually ventilated cages at the VIB Department of Molecular Biomedical Research in specific pathogen-free animal facilities.

T14996

33091-33191

Sentence

denotes

Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates.

T206

33091-33191

Sentence

denotes

Influenza infections were performed on age- (between 7 and 9 weeks old) and sex-matched littermates.

T14997

33192-33273

Sentence

denotes

A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background.

T207

33192-33273

Sentence

denotes

A20fl/fl LysM-Cre animals were backcrossed three times to the C57Bl/6 background.

T14998

33274-33396

Sentence

denotes

A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.

T208

33274-33396

Sentence

denotes

A20fl/fl mice expressing or lacking the LysM-Cre transgene were termed A20myel-KO and wild type (A20myel-WT) respectively.

T15282

33398-33447

Sentence

denotes

Viral infection and determination of viral titers

T209

33398-33447

Sentence

denotes

Viral infection and determination of viral titers

T15283

33448-33528

Sentence

denotes

Mouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells.

T210

33448-33528

Sentence

denotes

Mouse adapted IAV X-47 (H3N2; PR8×A/Victoria/3/75) was propagated in MDCK cells.

T15284

33529-33702

Sentence

denotes

For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally.

T211

33529-33702

Sentence

denotes

For viral inoculation, mice were anesthetized by i.p. injection with ketamine (12 mg/kg) and xylazine (60 mg/kg) and 50 µl X-47 diluted in PBS was administered intranasally.

T15285

33703-33791

Sentence

denotes

For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47.

T212

33703-33791

Sentence

denotes

For lethal and sublethal infection, mice received respectively 2-LD50 or 0.05-LD50 X-47.

T15286

33792-33972

Sentence

denotes

To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS.

T213

33792-33972

Sentence

denotes

To determine pulmonary viral titers, median tissue culture infectious dose (TCID50) was measured as follows: lungs were homogenized with a Polytron homogenizer (Kinematica) in PBS.

T15287

33973-34163

Sentence

denotes

Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.

T214

33973-34163

Sentence

denotes

Eight-fold serial dilutions of lung homogenates were incubated on MDCK cells for 5 days in DMEM supplemented with trypsin (1 µg/ml), 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.

T15288

34164-34281

Sentence

denotes

For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored.

T215

34164-34281

Sentence

denotes

For read-out, 0.5% chicken red blood cells (RBC) were added and end-point dilution of hemagglutination was monitored.

T15289

34282-34365

Sentence

denotes

TCID50 titers were then calculated according to the method of Reed and Muench [85].

T216

34282-34365

Sentence

denotes

TCID50 titers were then calculated according to the method of Reed and Muench [85].

T15660

34367-34424

Sentence

denotes

Determination of HAI (hemagglutination inhibition) titers

T217

34367-34424

Sentence

denotes

Determination of HAI (hemagglutination inhibition) titers

T15661

34425-34652

Sentence

denotes

To determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination.

T218

34425-34652

Sentence

denotes

To determine the HAI titers in infected mice, sera of these were treated with receptor-destroying enzyme (RDE/Cholera filtrate; Sigma) to remove sialic acids from serum proteins capable of aspecific inhibition of agglutination.

T15662

34653-34787

Sentence

denotes

After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min.

T219

34653-34787

Sentence

denotes

After incubation overnight at 37°C, the RDE was inactivated by addition of 0.75% sodium citrate in PBS and heating to 56°C for 30 min.

T15663

34788-34879

Sentence

denotes

To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC.

T220

34788-34879

Sentence

denotes

To remove sialic acid binding proteins, sera were cleared with 1/10 volume 50% chicken RBC.

T15664

34880-35036

Sentence

denotes

Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates.

T221

34880-35036

Sentence

denotes

Titration was done by incubating a two-fold dilution series of sera with 4 HA units of X-47 virus for 1 hour at room temperature in 96-well U-bottom plates.

T15665

35037-35126

Sentence

denotes

Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later.

T222

35037-35126

Sentence

denotes

Finally, an equal volume of 0.5% chicken RBC was added and titers were read 30 min later.

T15666

35127-35373

Sentence

denotes

Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.

T223

35127-35373

Sentence

denotes

Negative controls included PBS instead of immune serum (agglutination control) or PR8 instead of X-47 virus (control for agglutination effect of sera); as positive control, serum from a mouse infected twice with a sublethal dose of X-47 was used.

T16109

35375-35444

Sentence

denotes

In vivo intracellular GrB and IFNγ staining of activated CD8+ T cells

T224

35375-35444

Sentence

denotes

In vivo intracellular GrB and IFNγ staining of activated CD8+ T cells

T16110

35445-35605

Sentence

denotes

Granzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86].

T225

35445-35605

Sentence

denotes

Granzyme B (GrB) and IFNγ expressing CD8+ T cells were determined by treating the mice intranasally with 50 µg Brefeldin A (Sigma) as previously described [86].

T226

35606-35740

Sentence

denotes

6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A.

T16111

35606-35865

Sentence

denotes

6 h later, BAL and lungs were isolated and single cell suspensions were prepared from the lung in the presence of 3 µg/ml Brefeldin A. Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions.

T227

35741-35865

Sentence

denotes

Cells were stained, fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences) according to the manufacturer's instructions.

T16112

35866-35962

Sentence

denotes

Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression.

T228

35866-35962

Sentence

denotes

Activated CD8+ T cells were analyzed by flow cytometry based on CD62lo CD3+ and CD8+ expression.

T16113

35963-36071

Sentence

denotes

Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.

T229

35963-36071

Sentence

denotes

Live/Dead fixable aqua dead cell stain kit (Molecular Probes) was used to discriminate live from dead cells.

T16543

36073-36095

Sentence

denotes

Cells and transfection

T230

36073-36095

Sentence

denotes

Cells and transfection

T16544

36096-36230

Sentence

denotes

HEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.

T231

36096-36230

Sentence

denotes

HEK293T and MDCK cells were grown in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.

T232

36231-36451

Sentence

denotes

HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T.

T16545

36231-36793

Sentence

denotes

HEK293T cells were transfected using the calcium phosphate precipitate transfection method with specific expression vectors (pCAGGS-E-hA20 (LMBP 3778), pCAGGS-E-RIG-I-CARD (LMBP 6517), pEF-HA-IRF-7 (kindly provided by T. Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J. Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization.

T233

36452-36668

Sentence

denotes

Taniguchi, Graduate School of Medicine and Faculty of Medicine, University of Tokyo)), NF-κB, IRF3, IRF7 reporter plasmids (respectively pConLuc (LMBP3248), pISRE-luc (LMBP4011), pGL3-IFNα4-luc (kindly provided by J.

T234

36669-36793

Sentence

denotes

Hiscott, McGill University, Montreal, Quebec, Canada), and pACTbetagal (LMBP4341) for transfection efficiency normalization.

T16546

36794-36928

Sentence

denotes

Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.

T235

36794-36928

Sentence

denotes

Details of plasmids are presented along with detailed sequence maps at the BCCM-LMBP plasmid databank http://bccm.belspo.be/index.php.

T16547

36929-37129

Sentence

denotes

For the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF.

T236

36929-37129

Sentence

denotes

For the generation of BMDM, bone marrow cells were cultured 7 days in RPMI 1640 (Gibco) supplemented with 10% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate, antibiotics and 40 ng/ml recombinant M-CSF.

T16548

37130-37227

Sentence

denotes

BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies.

T237

37130-37227

Sentence

denotes

BMDM were of ≥95% purity as measured by flow cytometry using F4/80 and CD11b specific antibodies.

T16549

37228-37361

Sentence

denotes

For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA.

T238

37228-37361

Sentence

denotes

For the isolation of alveolar macrophages, the trachea was canulated and the lung was flushed 4 times with HBSS containing 1 mM EDTA.

T16550

37362-37501

Sentence

denotes

Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.

T239

37362-37501

Sentence

denotes

Alveolar macrophages were cultured in RPMI 1640 (Gibco) supplemented with 1% FCS, 2 mM L-glutamine, 0.4 mM sodium pyruvate and antibiotics.

T17160

37503-37519

Sentence

denotes

Western blotting

T240

37503-37519

Sentence

denotes

Western blotting

T17161

37520-37712

Sentence

denotes

For total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors.

T241

37520-37712

Sentence

denotes

For total lysates, cells were lysed at 4°C for 15 min in lysis buffer (200 mM NaCl, 1% NP-40, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 2 mM DTT) supplemented with protease and phosphatase inhibitors.

T242

37713-37945

Sentence

denotes

Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C.

T17162

37713-38058

Sentence

denotes

Nuclear and cytoplasmic lysates were prepared by resuspending cells in B1 (10 mM Hepes pH 7.5, 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min.

T243

37946-38058

Sentence

denotes

Next, NP-40 detergent was added to a final concentration of 0.65% and cells were centrifuged at 500 g for 5 min.

T244

38059-38296

Sentence

denotes

The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C.

T17163

38059-38429

Sentence

denotes

The nuclear fraction containing pellet was lyzed in B2 (20 mM Hepes pH 7.5, 1% NP-40, 400 mM NaCl, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.5 mM EDTA and 0.1 mM EGTA supplemented with protease and phosphatase inhibitors) for 15 min at 4°C. The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences).

T245

38297-38429

Sentence

denotes

The lysates were subsequently separated by SDS-PAGE and analyzed by western blotting and ECL detection (Perkin Elmer Life Sciences).

T17164

38430-38638

Sentence

denotes

Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals).

T246

38430-38638

Sentence

denotes

Immunoblots were revealed with anti-A20, anti-IκBα, anti-p65, and anti-histon H1 (Santa Cruz), anti-IRF3 (Invitrogen), anti-phospho-IRF3 and anti-phospho-IκBα (Cell Signaling) and anti-actin (MP Biomedicals).

T17165

38639-38757

Sentence

denotes

The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool.

T247

38639-38757

Sentence

denotes

The density of the bands was quantified (fold induction) with the ImageJ (http://rsbweb.nih.gov/ij) Gel analyzer tool.

T17166

38758-38829

Sentence

denotes

All intensities were calculated relative to the first lane ( = time 0).

T248

38758-38829

Sentence

denotes

All intensities were calculated relative to the first lane ( = time 0).

T17826

38831-38845

Sentence

denotes

Flow cytometry

T249

38831-38845

Sentence

denotes

Flow cytometry

T17827

38846-38970

Sentence

denotes

Lungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min.

T250

38846-38970

Sentence

denotes

Lungs were dissected and incubated with collagenase type IV (1 mg/ml; Sigma) and DNAse (100 U/ml; Roche) at 37°C for 30 min.

T17828

38971-39044

Sentence

denotes

Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh.

T251

38971-39044

Sentence

denotes

Subsequently, samples were filtered through a 70 µm and 40 µm nylon mesh.

T17829

39045-39158

Sentence

denotes

For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA.

T252

39045-39158

Sentence

denotes

For the preparation of BAL, trachea were canulated and airway lumen was flushed 4 times with HBSS with 1 mM EDTA.

T253

39159-39558

Sentence

denotes

Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen.

T17830

39159-39656

Sentence

denotes

Cells were stained with monoclonal antibodies directed against MHC-II (I-A/I-E) FITC (M5/114.15.2), CD11c PerCP-Cy5.5 (N418), F4/80 APC (BM8), CD62L PE (MEL-14), Granzyme B FITC (NGZB) from eBiosciences and CD3 Molecular Complex Horizon v450 (17A2), Ly6C Horizon v450 (AL-21), Ly6G PE (1A8), CD11b APC-Cy7 (M1/70), CD8α PerCP (53-6.7), IFNγ Alexa 647 (XMG1.2) and CD16/32 (2.4G2) from BD Pharmingen. Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences).

T254

39559-39656

Sentence

denotes

Samples were acquired on a LSRII Cytometer and analyzed using FACSDiva software (BD Biosciences).

T17831

39657-39727

Sentence

denotes

Propidium iodide was used to discriminate between live and dead cells.

T255

39657-39727

Sentence

denotes

Propidium iodide was used to discriminate between live and dead cells.

T18266

39729-39752

Sentence

denotes

Cytokine quantification

T256

39729-39752

Sentence

denotes

Cytokine quantification

T18267

39753-39881

Sentence

denotes

For TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies.

T257

39753-39881

Sentence

denotes

For TNF ELISA, 96-well plates were coated with TNF coating (TN3-19, eBioscience) and detection (R4-6A2, eBioscience) antibodies.

T18268

39882-39975

Sentence

denotes

IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories).

T258

39882-39975

Sentence

denotes

IFNα and IFNβ protein levels were determined with an ELISA kit (PBL Biomedical Laboratories).

T18269

39976-40095

Sentence

denotes

For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences).

T259

39976-40095

Sentence

denotes

For IFNγ ELISA, 96-well plates were coated with IFNγ coating (XMG1.2) and detection (R4-6A2) antibodies (eBiosciences).

T18270

40096-40246

Sentence

denotes

Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions.

T260

40096-40246

Sentence

denotes

Detection of MCP-1, KC, TNF, IL-1β and IL-6 in BAL fluid was performed using Bioplex (BioRad) technology according to the manufacturer's instructions.

T18271

40247-40329

Sentence

denotes

Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.

T261

40247-40329

Sentence

denotes

Milliplex technology (Millipore) was used for the detection of MIP-2 in BAL fluid.

T18657

40331-40369

Sentence

denotes

RNA isolation, cDNA synthesis and qPCR

T262

40331-40369

Sentence

denotes

RNA isolation, cDNA synthesis and qPCR

T18658

40370-40698

Sentence

denotes

Total RNA was extracted using Aurum Total RNA mini kit (BioRad) and reverse transcribed into cDNA with iScript cDNA synthesis kit (BioRad) according to the manufacturer's instructions. qPCR was performed by using SYBR Green I master mix I (Roche) in the Lightcycler 480 detection system (Roche) with the following primers: HPRT:

T263

40370-40698

Sentence

denotes

Total RNA was extracted using Aurum Total RNA mini kit (BioRad) and reverse transcribed into cDNA with iScript cDNA synthesis kit (BioRad) according to the manufacturer's instructions. qPCR was performed by using SYBR Green I master mix I (Roche) in the Lightcycler 480 detection system (Roche) with the following primers: HPRT:

T18659

40699-40764

Sentence

denotes

5′-AGTGTTGGATACAGGCCAGAC-3′ and 5′CGTGATTCAAATCCCTGAAGT-3′; IL-6:

T264

40699-40764

Sentence

denotes

5′-AGTGTTGGATACAGGCCAGAC-3′ and 5′CGTGATTCAAATCCCTGAAGT-3′; IL-6:

T18660

40765-40836

Sentence

denotes

5′-GAGGATACCACTCCCAACAGACC-3′ and 5′-AAGTGCATCATCGTTGTTCATACA-3′; IFNβ:

T265

40765-40836

Sentence

denotes

5′-GAGGATACCACTCCCAACAGACC-3′ and 5′-AAGTGCATCATCGTTGTTCATACA-3′; IFNβ:

T18661

40837-40904

Sentence

denotes

5′-TCAGAATGAGTGGTGGTTGC-3′ and 5′-GACCTTTCAAATGCAGTAGATTCA-3′; A20:

T266

40837-40904

Sentence

denotes

5′-TCAGAATGAGTGGTGGTTGC-3′ and 5′-GACCTTTCAAATGCAGTAGATTCA-3′; A20:

T18662

40905-40971

Sentence

denotes

5′-AAACCAATGGTGATGGAAACTG-3′ and 5′-GTTGTCCCATTCGTCATTCC-3′; CCL2:

T267

40905-40971

Sentence

denotes

5′-AAACCAATGGTGATGGAAACTG-3′ and 5′-GTTGTCCCATTCGTCATTCC-3′; CCL2:

T18663

40972-41043

Sentence

denotes

5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; CXCL1:

T268

40972-41043

Sentence

denotes

5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; CXCL1:

T18664

41044-41112

Sentence

denotes

5′-GAGCCTCTAACCAGTTCCAG-3′ and 5′-TGAGTGTGGCTATGACTTCG-3′ and IFNα4:

T269

41044-41112

Sentence

denotes

5′-GAGCCTCTAACCAGTTCCAG-3′ and 5′-TGAGTGTGGCTATGACTTCG-3′ and IFNα4:

T18665

41113-41175

Sentence

denotes

5′-TGATGAGCTACTACTGGTCAGC-3′ and 5′-GATCTCTTAGCACAAGGATGGC-3′.

T270

41113-41175

Sentence

denotes

5′-TGATGAGCTACTACTGGTCAGC-3′ and 5′-GATCTCTTAGCACAAGGATGGC-3′.

T18666

41176-41250

Sentence

denotes

Primers were designed with PerlPrimer (http://perlprimer.sourceforge.net).

T271

41176-41250

Sentence

denotes

Primers were designed with PerlPrimer (http://perlprimer.sourceforge.net).

T18667

41251-41319

Sentence

denotes

Quantification was performed using the comparative CT method (ΔΔCT).

T272

41251-41319

Sentence

denotes

Quantification was performed using the comparative CT method (ΔΔCT).

T18668

41320-41366

Sentence

denotes

Results are expressed relative to HPRT values.

T273

41320-41366

Sentence

denotes

Results are expressed relative to HPRT values.

T19100

41368-41378

Sentence

denotes

Statistics

T274

41368-41378

Sentence

denotes

Statistics

T19101

41379-41419

Sentence

denotes

Results are expressed as the mean ± SEM.

T275

41379-41419

Sentence

denotes

Results are expressed as the mean ± SEM.

T19102

41420-41493

Sentence

denotes

Statistical significance between groups was assessed using two-way ANOVA.

T276

41420-41493

Sentence

denotes

Statistical significance between groups was assessed using two-way ANOVA.

T19103

41494-41626

Sentence

denotes

The differences for in vivo experiments (at least 5 mice per group) were calculated using the Mann-Whitney U-test for unpaired data.

T277

41494-41626

Sentence

denotes

The differences for in vivo experiments (at least 5 mice per group) were calculated using the Mann-Whitney U-test for unpaired data.

T19104

41627-41812

Sentence

denotes

Statistical significance of differences between survival rates was analyzed by comparing Kaplan-Meier curves using the log-rank test (GraphPad Prism version 5, GraphPad, San Diego, CA).

T278

41627-41812

Sentence

denotes

Statistical significance of differences between survival rates was analyzed by comparing Kaplan-Meier curves using the log-rank test (GraphPad Prism version 5, GraphPad, San Diego, CA).

T279

41814-41836

Sentence

denotes

Supporting Information

T280

41837-41920

Sentence

denotes

Figure S1 A20 deficient alveolar macrophages are hyperresponsive to IAV infection.

T281

41921-42059

Sentence

denotes

Alveolar macrophages isolated from A20myel-KO and wild type (A20myel-WT) mice were mock treated or infected with IAV (moi 1) for 18 hours.

T282

42060-42143

Sentence

denotes

TNF, MCP-1 (ccl2), KC (cxcl1) and IFNα4 mRNA expression was determined by qPCR (A).

T283

42144-42262

Sentence

denotes

Cell culture supernatant was analyzed for TNF, MCP-1 and KC protein levels by ELISA and multiplexing technologies (B).

T284

42263-42321

Sentence

denotes

Error bars represent mean values (+/− SEM) of 2–3 samples.

T285

42322-42410

Sentence

denotes

Results are representative for 2 independent experiments. *p<0.05; **p<0.01; ***p<0.001.

T286

42411-42416

Sentence

denotes

(TIF)

T287

42417-42453

Sentence

denotes

Click here for additional data file.

T288

42454-42571

Sentence

denotes

Figure S2 Adaptive immunity is not altered by the absence of A20 in myeloid cells following sublethal IAV infection.

T289

42572-42671

Sentence

denotes

Wild type (A20myel-WT) and A20myel-KO mice were infected intranasally with a sublethal dose of IAV.

T290

42672-42856

Sentence

denotes

BAL was isolated from IAV infected mice at 4, 7 and 10 days p.i. and analyzed for total CD8+ T cells, Granzyme B (GrB) and IFNγ expressing CD8+ T cells (A) and IFNγ protein levels (B).

T291

42857-42980

Sentence

denotes

Total CD8+ T cells, GrB and IFNγ expressing CD8+ T cells in the lung parenchyma were also determined by flow cytometry (C).

T292

42981-43108

Sentence

denotes

Virus specific antibody titers in serum at 4, 7 and 10 days p.i. were determined via a hemagglutination inhibition (HAI) assay.

T293

43109-43170

Sentence

denotes

The dashed line depicts the detection limit of the assay (D).

T294

43171-43281

Sentence

denotes

Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments.

T295

43282-43287

Sentence

denotes

(TIF)

T296

43288-43324

Sentence

denotes

Click here for additional data file.

T297

43325-43439

Sentence

denotes

Figure S3 Adaptive immunity is not altered by the absence of A20 in myeloid cells following lethal IAV infection.

T298

43440-43536

Sentence

denotes

Wild type (A20myel-WT) and A20myel-KO mice were infected intranasally with a lethal dose of IAV.

T299

43537-43756

Sentence

denotes

6 days p.i. mice were sacrificed and analyzed. (A) TNF and IL-1β protein levels in BAL fluid were measured by ELISA and multiplexing technologies. (B) Viral titers in the lung were measured and expressed as mean TCID50.

T300

43757-44172

Sentence

denotes

The dashed line represents the detection limit. (C) Total CD8+, GrB and IFNγ expressing T cells in BAL fluid were measured using flow cytometry. (D) IFNγ protein levels in BAL fluid were measured by ELISA. (E) Total CD8+, GrB and IFNγ expressing T cells in lung parenchyma were measured using flow cytometry. (F) Virus specific antibody titers in serum were determined via a hemagglutination inhibition (HAI) assay.

T301

44173-44217

Sentence

denotes

The dashed line depicts the detection limit.

T302

44218-44295

Sentence

denotes

Numbers are averages +/− SEM of at least 5 mice per group. *p<0.05; **p<0.01.

T303

44296-44301

Sentence

denotes

(TIF)

T304

44302-44338

Sentence

denotes

Click here for additional data file.

PMC:3291650 JSON TXT 30 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function IAV-Glycan

PMC:3291650 JSONTXT 30 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function IAV-Glycan

PMC:3291650 JSON TXT 30 Projects