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test3

2_test

{"project":"2_test","denotations":[{"id":"22396652-16306043-98461847","span":{"begin":393,"end":395},"obj":"16306043"},{"id":"22396652-15661910-98461848","span":{"begin":398,"end":400},"obj":"15661910"},{"id":"22396652-15208624-98461849","span":{"begin":608,"end":610},"obj":"15208624"},{"id":"22396652-10893229-98461850","span":{"begin":944,"end":946},"obj":"10893229"},{"id":"22396652-11070172-98461851","span":{"begin":950,"end":952},"obj":"11070172"},{"id":"22396652-11009421-98461852","span":{"begin":3301,"end":3303},"obj":"11009421"},{"id":"22396652-20530205-98461853","span":{"begin":3418,"end":3420},"obj":"20530205"},{"id":"22396652-21841782-98461854","span":{"begin":3644,"end":3646},"obj":"21841782"},{"id":"22396652-18591409-98461855","span":{"begin":3878,"end":3880},"obj":"18591409"},{"id":"22396652-15210742-98461856","span":{"begin":4847,"end":4849},"obj":"15210742"},{"id":"22396652-9566918-98461857","span":{"begin":4925,"end":4927},"obj":"9566918"},{"id":"22396652-17277103-98461858","span":{"begin":11207,"end":11209},"obj":"17277103"},{"id":"22396652-6206190-98461859","span":{"begin":12088,"end":12090},"obj":"6206190"},{"id":"22396652-1552285-98461860","span":{"begin":12094,"end":12096},"obj":"1552285"},{"id":"22396652-17579060-98461861","span":{"begin":12100,"end":12102},"obj":"17579060"},{"id":"22396652-17213831-98461862","span":{"begin":14599,"end":14601},"obj":"17213831"}],"text":"Results\n\nA20 inhibits RIG-I-induced NF-κB and IRF3 activation\nRIG-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. Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52]. 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). We next investigated whether A20 also inhibits IRF7 activation. 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]. 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. 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. Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph). These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.\n10.1371/journal.ppat.1002570.g001 Figure 1 A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.\n(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. Luciferase activity in cell extracts was analyzed in a luminometer. 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). The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control. Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C). 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). 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). Data are representative of 2 independent experiments. 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. 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]. 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]. 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]. 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). 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). 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. 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). 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). 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]. 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). 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). NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ. 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). Similar results were obtained using peritoneal macrophages (data not shown). 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.\n\nA20 negatively regulates IAV-induced gene expression in BMDM\nTo 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). A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A). Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A). In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).\n10.1371/journal.ppat.1002570.g002 Figure 2 A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.\n(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1). At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A). 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. IL-6 and IFNβ mRNA expression was determined by qPCR (C). Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D). Error bars represent mean values (+/− SEM) of 2–3 samples. Results are representative for 2 independent experiments. ***p\u003c0,001. 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. 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. 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). 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.\n\nA20 deficiency in myeloid cells protects against IAV lung infection\nTo 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. A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A). 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). Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection. No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection. 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). 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.\n10.1371/journal.ppat.1002570.g003 Figure 3 A20 deficiency in myeloid cells protects against IAV lung infection.\nA20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A). At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B). The dashed line represents the detection limit. ND, not detectable. 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). 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). Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01. 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. 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). 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). 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]. 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). 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. 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). Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63]. 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. 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). 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).\nProtection against IAV infection is also provided by the humoral immune response. 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. 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. 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.\nIt is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung. 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. Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A). 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). 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). 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). 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. We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F). 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.\n10.1371/journal.ppat.1002570.g004 Figure 4 A20 deficiency in myeloid cells protects against lethal IAV infection.\nA20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p\u003c0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i. 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). Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D). Numbers are averages +/− SEM of at least 5 mice per group. Data are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01, ***p\u003c0.001."}

pmc-enju-pas

bionlp-st-ge-2016-coref

{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T4891","span":{"begin":4708,"end":4712},"obj":"Antecedent"},{"id":"T4890","span":{"begin":4863,"end":4866},"obj":"Anaphor"},{"id":"T4889","span":{"begin":1153,"end":1163},"obj":"Antecedent"},{"id":"T4888","span":{"begin":1346,"end":1359},"obj":"Anaphor"}],"relations":[{"id":"R3328","pred":"boundBy","subj":"T4888","obj":"T4889"},{"id":"R3329","pred":"boundBy","subj":"T4890","obj":"T4891"}],"namespaces":[{"prefix":"_base","uri":"https://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Results\n\nA20 inhibits RIG-I-induced NF-κB and IRF3 activation\nRIG-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. Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52]. 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). We next investigated whether A20 also inhibits IRF7 activation. 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]. 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. 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. Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph). These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.\n10.1371/journal.ppat.1002570.g001 Figure 1 A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.\n(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. Luciferase activity in cell extracts was analyzed in a luminometer. 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). The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control. Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C). 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). 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). Data are representative of 2 independent experiments. 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. 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]. 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]. 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]. 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). 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). 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. 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). 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). 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]. 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). 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). NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ. 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). Similar results were obtained using peritoneal macrophages (data not shown). 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.\n\nA20 negatively regulates IAV-induced gene expression in BMDM\nTo 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). A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A). Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A). In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).\n10.1371/journal.ppat.1002570.g002 Figure 2 A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.\n(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1). At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A). 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. IL-6 and IFNβ mRNA expression was determined by qPCR (C). Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D). Error bars represent mean values (+/− SEM) of 2–3 samples. Results are representative for 2 independent experiments. ***p\u003c0,001. 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. 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. 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). 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.\n\nA20 deficiency in myeloid cells protects against IAV lung infection\nTo 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. A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A). 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). Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection. No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection. 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). 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.\n10.1371/journal.ppat.1002570.g003 Figure 3 A20 deficiency in myeloid cells protects against IAV lung infection.\nA20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A). At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B). The dashed line represents the detection limit. ND, not detectable. 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). 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). Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01. 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. 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). 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). 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]. 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). 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. 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). Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63]. 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. 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). 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).\nProtection against IAV infection is also provided by the humoral immune response. 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. 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. 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.\nIt is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung. 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. Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A). 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). 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). 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). 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. We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F). 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.\n10.1371/journal.ppat.1002570.g004 Figure 4 A20 deficiency in myeloid cells protects against lethal IAV infection.\nA20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p\u003c0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i. 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). Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D). Numbers are averages +/− SEM of at least 5 mice per group. Data are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01, ***p\u003c0.001."}

bionlp-st-ge-2016-test-proteins

bionlp-st-ge-2016-uniprot

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T20778","span":{"begin":9785,"end":9789},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T20777","span":{"begin":9586,"end":9590},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8719","span":{"begin":12919,"end":12924},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T8718","span":{"begin":12309,"end":12319},"obj":"http://purl.obolibrary.org/obo/UBERON_0000353"},{"id":"T8717","span":{"begin":12304,"end":12319},"obj":"http://purl.obolibrary.org/obo/UBERON_0008946"},{"id":"T8716","span":{"begin":14626,"end":14631},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8715","span":{"begin":14438,"end":14443},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8714","span":{"begin":13964,"end":13968},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8713","span":{"begin":13535,"end":13539},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8712","span":{"begin":12304,"end":12308},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8711","span":{"begin":10444,"end":10448},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8710","span":{"begin":8742,"end":8746},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T8709","span":{"begin":8243,"end":8247},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T7697","span":{"begin":7583,"end":7588},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T7696","span":{"begin":7419,"end":7423},"obj":"http://purl.obolibrary.org/obo/UBERON_0002048"},{"id":"T4723","span":{"begin":3281,"end":3286},"obj":"http://purl.obolibrary.org/obo/UBERON_0000062"}],"text":"Results\n\nA20 inhibits RIG-I-induced NF-κB and IRF3 activation\nRIG-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. Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52]. 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). We next investigated whether A20 also inhibits IRF7 activation. 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]. 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. 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. Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph). These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.\n10.1371/journal.ppat.1002570.g001 Figure 1 A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.\n(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. Luciferase activity in cell extracts was analyzed in a luminometer. 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). The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control. Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C). 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). 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). Data are representative of 2 independent experiments. 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. 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]. 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]. 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]. 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). 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). 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. 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). 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). 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]. 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). 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). NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ. 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). Similar results were obtained using peritoneal macrophages (data not shown). 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.\n\nA20 negatively regulates IAV-induced gene expression in BMDM\nTo 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). A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A). Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A). In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).\n10.1371/journal.ppat.1002570.g002 Figure 2 A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.\n(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1). At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A). 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. IL-6 and IFNβ mRNA expression was determined by qPCR (C). Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D). Error bars represent mean values (+/− SEM) of 2–3 samples. Results are representative for 2 independent experiments. ***p\u003c0,001. 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. 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. 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). 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.\n\nA20 deficiency in myeloid cells protects against IAV lung infection\nTo 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. A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A). 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). Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection. No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection. 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). 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.\n10.1371/journal.ppat.1002570.g003 Figure 3 A20 deficiency in myeloid cells protects against IAV lung infection.\nA20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A). At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B). The dashed line represents the detection limit. ND, not detectable. 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). 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). Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01. 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. 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). 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). 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]. 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). 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. 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). Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63]. 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. 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). 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).\nProtection against IAV infection is also provided by the humoral immune response. 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. 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. 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.\nIt is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung. 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. Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A). 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). 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). 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). 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. We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F). 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.\n10.1371/journal.ppat.1002570.g004 Figure 4 A20 deficiency in myeloid cells protects against lethal IAV infection.\nA20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p\u003c0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i. 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). Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D). Numbers are averages +/− SEM of at least 5 mice per group. Data are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01, ***p\u003c0.001."}

GO-BP

GO-MF

GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T7785","span":{"begin":6098,"end":6113},"obj":"http://purl.bioontology.org/ontology/ICD10/B34.9"}],"text":"Results\n\nA20 inhibits RIG-I-induced NF-κB and IRF3 activation\nRIG-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. Previous studies have shown that ectopically expressed A20 negatively regulates NF-κB and IRF3 activation upon RIG-I stimulation [50]–[52]. 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). We next investigated whether A20 also inhibits IRF7 activation. 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]. 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. 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. Similar to its inhibitory effect on NF-κB and IRF3 activation, A20 also prevented RIG-I-induced IRF7 activation (figure 1A, right graph). These results demonstrate the potential of A20 to inhibit RIG-I-induced NF-κB and IRF3/7 activation.\n10.1371/journal.ppat.1002570.g001 Figure 1 A20 inhibits NF-κB and IRF3 activation in response to RIG-I stimulation.\n(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. Luciferase activity in cell extracts was analyzed in a luminometer. 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). The band that is non-specifically recognized by the anti-IκBα antibody (indicated by an asterisk) can be used as a loading control. Nuclear translocation of NF-κB (p65) and IRF3 was analyzed by immunoblotting of cytoplasmic or nuclear fractions (C). 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). 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). Data are representative of 2 independent experiments. 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. 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]. 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]. 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]. 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). 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). 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. 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). 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). 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]. 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). 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). NF-κB controls the expression of IL-6 and TNF, and NF-κB and IRF3 control the expression of IFNβ. 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). Similar results were obtained using peritoneal macrophages (data not shown). 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.\n\nA20 negatively regulates IAV-induced gene expression in BMDM\nTo 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). A20 mRNA levels were rapidly induced in wild type BMDM, but not in A20 deficient BMDM, upon viral infection (figure 2A). Furthermore, A20myel-KO BMDM show enhanced expression of IL-6 and IFNβ mRNA after IAV infection compared to control cells (figure 2A). In accordance with these data, cell culture supernatant collected from these cells contained higher levels of TNF and IFNβ (figure 2B).\n10.1371/journal.ppat.1002570.g002 Figure 2 A20 negatively regulates IAV-induced gene expression in BMDM and alveolar macrophages.\n(A–B) BMDM isolated from A20myel-WT and A20myel-KO animals were infected with IAV (moi 1). At different hours post infection (hours p.i.) cells were lysed and IL-6, IFNβ and A20 mRNA expression was analyzed by qPCR (A). 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. IL-6 and IFNβ mRNA expression was determined by qPCR (C). Cell culture supernatant was analyzed for IL-6 and IFNβ protein levels by ELISA and multiplexing technologies (D). Error bars represent mean values (+/− SEM) of 2–3 samples. Results are representative for 2 independent experiments. ***p\u003c0,001. 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. 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. 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). 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.\n\nA20 deficiency in myeloid cells protects against IAV lung infection\nTo 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. A20myel-KO mice showed reduced weight loss compared to wild type control littermates and recovered faster from the viral challenge (figure 3A). 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). Next, we measured pulmonary viral titers at 4, 7 and 10 days post infection. No differences in viral titers were observed in A20myel-KO mice versus wild type mice at day 4 and 7 post infection. 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). 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.\n10.1371/journal.ppat.1002570.g003 Figure 3 A20 deficiency in myeloid cells protects against IAV lung infection.\nA20myel-WT and A20myel-KO mice were infected intranasally with a sublethal dose of IAV and weight loss was monitored (A). At day 4, 7 and 10 post infection (p.i.) viral titers in the lung were measured and expressed as mean TCID50 (B). The dashed line represents the detection limit. ND, not detectable. 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). 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). Numbers are averages +/− SEM of at least 5 mice per group and are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01. 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. 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). 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). 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]. 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). 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. 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). Elimination of IAV infected cells depends on the clonal expansion of virus specific cytotoxic CD8+ T cells (CTL) [61], [62], [63]. 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. 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). 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).\nProtection against IAV infection is also provided by the humoral immune response. 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. 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. 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.\nIt is generally believed that IAV-induced mortality is due to an excessive proinflammatory response in the lung. 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. Surprisingly, almost all A20myel-KO mice survived (10/11), while all control mice succumbed (0/11) within 16 days after infection (figure 4A). 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). 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). 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). 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. We could also not detect any differences in viral clearance or antiviral adaptive immunity at 6 h post infection (figure S3B–F). 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.\n10.1371/journal.ppat.1002570.g004 Figure 4 A20 deficiency in myeloid cells protects against lethal IAV infection.\nA20myel-WT (n = 11) and A20myel-KO (n = 11) mice were intranasally infected with a lethal dose of IAV and survival (p\u003c0.001) (A) and weight loss +/− SEM (B) was monitored for respectively 21 and 15 days p.i. 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). Absolute numbers of alveolar macrophages, neutrophils and monocytes in BAL were analyzed by flow cytometry at 6 days p.i. (D). Numbers are averages +/− SEM of at least 5 mice per group. Data are representative of 2 independent experiments. *p\u003c0.05; **p\u003c0.01, ***p\u003c0.001."}

simple1

BioNLP16_DUT

BioNLP16_Messiy

DLUT931

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

testone

PMC:3291650 / 9434-25395 JSONTXT

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PMC:3291650 / 9434-25395 JSON TXT