Epistemic_Statements  

CORD-19:11372d2b62ec98cab9cf3f9be6b7cf967627a14a JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 636-810 Epistemic_statement denotes Here, we have found that CARMA3, a scaffold protein previously shown to mediate NF-kB activation induced by GPCR and EGFR, positively regulates MAVS-induced NF-kB activation.
T2 811-953 Epistemic_statement denotes However, our data suggest that CARMA3 sequesters MAVS from forming high-molecular-weight aggregates, thereby suppressing TBK1/IRF3 activation.
T3 954-1112 Epistemic_statement denotes Interestingly, following NF-kB activation upon virus infection, CARMA3 is targeted for proteasome-dependent degradation, which releases MAVS to activate IRF3.
T4 1850-2092 Epistemic_statement denotes The innate immune system is the first line of host defense against infection, which is essential for initial detection and recognition of pathogens, activation of acute anti-microbial responses, and subsequent activation of adaptive immunity.
T5 2375-2448 Epistemic_statement denotes The RLR family of proteins is crucial for detecting viral RNA in cytosol.
T6 4549-4770 Epistemic_statement denotes Elevated levels of proinflammatory cytokines are closely correlated with severity of clinical diseases, including airway inflammation and acute lung injury during influenza infection in children (Chiaretti et al., 2013) .
T7 5245-5547 Epistemic_statement denotes Recent mechanistic studies have found that, upon activation, MAVS forms a prion-like fibril at mitochondria and acts as an active platform for the recruitment of E3 ligases, including TRAF6, TRAF2, and TRAF5, through distinct TRAF-binding domains (Hou et al., 2011; Liu et al., 2013; Xu et al., 2014) .
T8 5752-5931 Epistemic_statement denotes It has been an important question why different individuals display highly variable systemic symptoms across the infected populations during outbreaks of seasonal virus infection.
T9 5932-6010 Epistemic_statement denotes To study how genetic polymorphisms contribute to this variation, Ferris et al.
T10 6011-6243 Epistemic_statement denotes (2013) crossed different incipient lines of mice, which exhibit a broad range of susceptibility to IAV infection, and identified three novel quantitative trait loci (QTLs) that may contribute to the susceptibility for IAV infection.
T11 6938-7262 Epistemic_statement denotes Upon activation, CARMA proteins form a complex with B cell lymphoma 10 (BCL10) and caspase-like protein mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and the CARMA-BCL10-MALT1 (CBM) complex functions to activate the downstream IKK complex, leading to activation of NF-kB (Jiang and Lin, 2012) .
T12 7263-7551 Epistemic_statement denotes Previous studies have shown that CARMA3 is crucial in mediating G protein-coupled receptor (GPCR)-and epidermal growth factor receptor (EGFR)-, but not TLR-or TNFR-, induced NF-kB activation (Grabiner et al., 2007; Jiang et al., 2011b; Klemm et al., 2007; McAllister-Lucas et al., 2007) .
T13 7552-7659 Epistemic_statement denotes However, it is unknown whether CARMA3 also is involved in regulating the host responses to viral infection.
T14 8051-8198 Epistemic_statement denotes Our data suggest that CARMA3 contributes to inflammatory and antiviral responses via regulating RIG-I/MAVS-induced TBK1/ IRF3 and NF-kB activation.
T15 8199-8498 Epistemic_statement denotes We have found that CARMA3 deficiency resulted in the defect in VSV-and RNA-induced NF-kB activation and production of pro-inflammatory cytokines, but, surprisingly, enhanced TBK1/IRF3 activation and production of type I IFN, thereby displaying a reduced viral load in VSV-infected cells and tissues.
T16 8499-8615 Epistemic_statement denotes Mechanistic studies showed that CARMA3 inhibited IRF3 activation through blocking the formation of MAVS aggregation.
T17 8791-8965 Epistemic_statement denotes Recent genetic studies indicate that the CARD10 gene is located in the genomic locus that may contribute to the host's susceptibility to IAV infection (Ferris et al., 2013) .
T18 9461-9654 Epistemic_statement denotes Similarly, lung injury caused by IAV infection was greatly attenuated in CARMA3 KO mice ( Figure S1A ), suggesting that CARMA3 plays a negative role in antiviral response against IAV infection.
T19 9655-9998 Epistemic_statement denotes Consistently, we found that CARMA3 KO mice produced more type I IFN IFNb in lungs compared to WT mice ( Figure 1C ), but expressed less pro-inflammatory cytokines IL-6, IL-1a, and IL-1b following IAV infection (Figures 1D and S1B-S1D), suggesting that CARMA3 also plays a positive role in inflammation in response to influenza virus infection.
T20 10103-10244 Epistemic_statement denotes To examine whether CARMA3 also contributes to the host responses to VSV infection, we challenged WT and CARMA3 KO mice intranasally with VSV.
T21 10383-10650 Epistemic_statement denotes Interestingly, we found that viral loads were significantly higher in the olfactory bulbs (OBs) of the brain in WT mice than those in CARMA3 KO mice ( Figures 1E and 1F ), although smaller differences were found in spleen and lung of these mice (Figures S1E and S1F).
T22 11186-11449 Epistemic_statement denotes Together, these results further support the above observations that CARMA3 plays a negative role in regulating antiviral responses in the host, but it plays a positive role in regulating the expression of pro-inflammatory cytokines in response to viral infection.
T23 11450-11641 Epistemic_statement denotes Since CARMA3 is only expressed in non-hematopoietic cells, the observed effect of CARMA3 deficiency on viral infection in vivo might be compromised by the contribution of hematopoietic cells.
T24 13159-13273 Epistemic_statement denotes It has been shown that dsRNA virus infection can induce NF-kB through both TLR signaling and RIG-I/MAVS signaling.
T25 13274-13531 Epistemic_statement denotes Given the fact that CARMA3 is not required for TLR-induced NF-kB activation (Grabiner et al., 2007) , we hypothesized that CARMA3 only mediates RIG-I/ MAVS-induced NF-kB, which can explain why VSV-induced NF-kB was only partial defective in CARMA3 KO cells.
T26 13532-13678 Epistemic_statement denotes To test this hypothesis, we stimulated WT and CARMA3 KO MEF cells with RIG-I-specific ligands poly(I:C) or 5 0 triphosphate dsRNA (5 0 ppp-dsRNA).
T27 14829-15003 Epistemic_statement denotes To determine if this observation is specific for viral infection, we stimulated these cells with RIG-I-specific ligands, 5 0 triphosphate dsRNA (5 0 ppp-dsRNA), or poly(I:C).
T28 15710-16061 Epistemic_statement denotes Furthermore, we isolated primary lung cells from CARMA3 WT and KO mice, and higher IFNb was detected in the CARMA3 KO lung cells compared to that in WT cells ( Figure S3G ), although we were not able to detect any significant difference in IL-6 production, which likely was due to the high basal level of IL-6 production in these cells ( Figure S3H ).
T29 16062-16345 Epistemic_statement denotes Previous studies have shown that BCL10 and MALT1 bind to CARMA3 to form the CBM complex upon EGFR-and GPCRinduced NF-kB activation (Grabiner et al., 2007; RNA was isolated and cytokine production in mRNA level was measured by real-time qPCR, normalized to the internal control GAPDH.
T30 17504-17616 Epistemic_statement denotes Next we wanted to determine if BCL10 and MALT1 play a similar function as CARMA3 in response to virus infection.
T31 17958-18054 Epistemic_statement denotes These results indicate that BCL10 plays a similar role as CARMA3 in response to virus infection.
T32 18055-18140 Epistemic_statement denotes However, MALT1 deficiency did not result in defective IL-6 production ( Figure S4A ).
T33 18427-18598 Epistemic_statement denotes Although IRF3 phosphorylation was slightly enhanced in MALT1 KO MEF cells ( Figure S4F ), it was not as significant as what we observed in CARMA3 KO or BCL10 KO MEF cells.
T34 18599-18733 Epistemic_statement denotes Together, these data suggest that MALT1 does not play a dominant role in regulating viral infection-induced NF-kB and IRF3 activation.
T35 18734-19067 Epistemic_statement denotes CARMA3 Regulates RIG-I/MAVS-Mediated IKK/NF-kB Activation and TBK1/IRF3 Activation in an Independent Manner Interestingly, we found that VSV infection-induced NF-kB activation could be detected as early as the first hour post-infection, whereas IRF3 phosphorylation was not detectable until 4 hr post-infection ( Figures 2C and 3C ).
T36 19068-19320 Epistemic_statement denotes Since VSV infectioninduced NF-kB activation was partially defective but IRF3 activation was enhanced in CARMA3 KO MEF cells, we decided to examine whether NF-kB-targeted genes expressed at early time points post-infection might inhibit IRF3 activation.
T37 19321-19459 Epistemic_statement denotes To test this possibility, WT MEF cells were pre-treated with NF-kB nuclear translocation inhibitor (NF-kBi) for 1 hr before VSV infection.
T38 19709-19987 Epistemic_statement denotes To further support this conclusion, we transfected IkBa super-repressor (SR) mutant into immortalized MEF cells, and we found that, although expression of IkBa SR mutant could suppress VSV-induced NF-kB activation, it could not enhance IRF3 phosphorylation (Figures 5C and 5D) .
T39 19988-20188 Epistemic_statement denotes Finally, to determine whether newly synthesized proteins regulate IRF3 activation, primary MEF cells were pretreated with protein synthesis inhibitor cycloheximide (CHX) before poly(I:C) transfection.
T40 20256-20488 Epistemic_statement denotes Therefore, the impaired NF-kB activation at early hours postinfection is not the cause for the enhanced IRF3 activation in CARMA3 KO MEF cells, indicating that CARMA3 regulates NF-kB and IRF3 activation through an independent event.
T41 21005-21270 Epistemic_statement denotes However, when TBK1 or IKKε was overexpressed in these cells, knockdown of CARMA3 or BCL10 did not alter activation of NF-kB or IRF3 ( Figures 5G, 5H , 5J, 5K, S5B, and S5C), indicating that CARMA3 and BCL10 function downstream of MAVS but upstream of TBK1 and IKKε.
T42 21271-21419 Epistemic_statement denotes Since CARMA3 is a scaffold protein with multiple protein-protein interaction domains, we examined whether CARMA3 is physically associated with MAVS.
T43 21584-21677 Epistemic_statement denotes In contrast, MALT1 could not bind to MAVS when overexpressed in HEK293T cells ( Figure S6B ).
T44 21800-22023 Epistemic_statement denotes More interestingly, inducible interactions, but not constitutive interactions, were observed between endogenous MAVS and BCL10 or between endogenous MAVS and CARMA3 in CARMA3-reconstituted KO MEF cells (Figures 6C and 6D) .
T45 22024-22298 Epistemic_statement denotes The dynamic interaction between MAVS and CARMA3 could be detected in both primary BCL10 Het and KO MEF cells ( Figure 6E ), whereas the MAVS-BCL10 interaction could only be detected in CARMA3 KO cells reconstituted with CARMA3, but not with its vector control ( Figure 6F ).
T46 22299-22412 Epistemic_statement denotes These data suggested that BCL10 might be recruited to the MAVS-containing complex via CARMA3, but not vice versa.
T47 22413-22584 Epistemic_statement denotes To address whether CARMA3 binds to MAVS through CARD-CARD interaction, we co-transfected HEK293T cells with MAVS and CARMA3 WT or truncate mutants ( Figures S6C and S6D ).
T48 22710-22979 Epistemic_statement denotes It has been shown that infection with SeV, another negative, single-stranded virus, induces the formation of large MAVS aggregates, which is functionally important in mediating IRF3 activation (He et al., 2015; Hou et al., 2011; Moresco et al., 2011; Xu et al., 2014) .
T49 22980-23067 Epistemic_statement denotes Since CARMA3 binds to MAVS, we hypothesized that CARMA3 may block MAVS oligomerization.
T50 23331-23407 Epistemic_statement denotes This suggests that CARMA3 can be recruited to mitochondria and bind to MAVS.
T51 23680-23819 Epistemic_statement denotes To further confirm that CARMA3 blocks MAVS from forming aggregates, we infected primary MEF cells with VSV and isolated crude mitochondria.
T52 24125-24248 Epistemic_statement denotes Together, these results indicate that CARMA3 plays a negative role in the assembly of MAVS aggregates upon virus infection.
T53 24437-24549 Epistemic_statement denotes Interestingly, we found that HA-tagged CARMA3 protein was gradually reduced upon VSV infection (Figures 7D-7G) .
T54 24550-24684 Epistemic_statement denotes However, this reduction in CARMA3 protein was rescued by pretreatment of cells with proteasome inhibitor MG132 ( Figures 7E and 7F ).
T55 24685-24777 Epistemic_statement denotes This indicated that, following VSV infection, CARMA3 is targeted for proteasome degradation.
T56 24983-25110 Epistemic_statement denotes However, in contrast to CARMA3, the protein level of BCL10 was not significantly altered following VSV infection ( Figure 7G ).
T57 25111-25232 Epistemic_statement denotes Together, these results suggest that CARMA3 is targeted to proteasome-mediated degradation following RNA virus infection.
T58 25439-25602 Epistemic_statement denotes Upon RIG-I activation, MAVS is first activated at early time points following viral infection, which can activate IKKa/IKKb/NEMO in a CARMA3/BCL10dependent manner.
T59 25603-25852 Epistemic_statement denotes However, in this early phase of post-infection, MAVS is sequestered by the CARMA3/BCL10 complex via interaction, and, therefore, it cannot form the high-molecular weight aggregates, which is required for downstream activation of TBK1/IRF3 signaling.
T60 26497-26636 Epistemic_statement denotes However, the precise mechanism as to how CARMA3 is targeted for ubiquitination-based proteasome degradation requires further investigation.
T61 26637-26823 Epistemic_statement denotes In addition, it will be interesting to see if CARMA3 plays a general effect in response to other RNA viruses as well as DNA viruses that are typically not recognized by cytoplasmic RLRs.
T62 26920-26995 Epistemic_statement denotes However, host factors contributing to this variability are largely unknown.
T63 26996-27198 Epistemic_statement denotes Recent studies suggest that the CARD10 gene, which encodes CARMA3, is located in a genomic locus that contributes to the host's susceptibility to RNA virus, such as IAV infection (Ferris et al., 2013) .
T64 27199-27407 Epistemic_statement denotes Our data, showing the distinct regulation of CARMA3 in the pro-inflammatory response and the antiviral response, suggest that CARMA3 may be a gene contributing to the host's susceptibility to viral infection.
T65 27408-27671 Epistemic_statement denotes Therefore, it will be important to find out whether CARMA3 protein expression levels or particular CARMA3 polymorphisms exist in different human populations, which may explain the variable susceptibility among different populations in response to virus infection.
T66 28298-28592 Epistemic_statement denotes In bone marrow-derived dendritic cells (BMDCs), CARD9 deficiency resulted in defects in NF-kB activation and production of pro-inflammatory cytokines, including IL-6 and IL-1b, in response to 5 0 ppp dsRNA treatment; however, it did not alter the production of type I IFN (Poeck et al., 2010) .
T67 28653-28811 Epistemic_statement denotes It will be interesting to find out how CARMA1, the counterpart of CARMA3 in hematopoietic cells, functions in mediating RIG-I/MAVS signaling in myeloid cells.
T68 29076-29166 Epistemic_statement denotes This suggests that BCL10 may regulate RIG-I/MAVS signaling in a cell-type-specific manner.
T69 29167-29288 Epistemic_statement denotes During RNA virus infection, NF-kB activation can be induced through TLR-dependent and TLR-independent signaling pathways.
T70 29289-29417 Epistemic_statement denotes It has been demonstrated that CARMA3 is not required for TLR-induced NF-kB activation (Jiang et al., 2011b; Pan and Lin, 2013) .
T71 29418-29638 Epistemic_statement denotes In this study, we found CARMA3 deficiency induced only partial defects in NF-kB activation, suggesting that CARMA3 may play an important role in TLR-independent NF-kB activation, which is induced by RIG-I/MAVS signaling.
T72 29639-29937 Epistemic_statement denotes TLR-dependent NF-kB activation may play dominant roles in the induction of local IL-6 production in non-hematopoietic cells following RNA viral infection, which may explain why we do not observe a significant effect of CARMA3 deficiency in local IL-6 mRNA production in OBs following VSV infection.
T73 29938-30108 Epistemic_statement denotes Virus infection-induced NF-kB activation promotes expression of pro-inflammatory cytokines and chemokine, which are crucial to trigger inflammatory responses in the host.
T74 30228-30314 Epistemic_statement denotes Overly robust NF-kB activation or inflammation can lead to severe health consequences.
T75 30315-30586 Epistemic_statement denotes For example, Ebola virus, a negative sense, singlestranded virus, is a highly virulent pathogen that causes a frequently lethal hemorrhagic fever syndrome, which is well correlated with the high inflammatory response that is induced in the host (Rasmussen et al., 2014) .
T76 30837-31176 Epistemic_statement denotes Challenging WT mice with IAV induced a strong production of pro-inflammatory cytokines, including IL-6, and severe inflammation, lung injury, and significant weight loss, which were attenuated in CARMA3 KO mice, indicating that CARMA3 is a key molecule to regulating inflammatory responses in the host and potentially a therapeutic target.
T77 31177-31371 Epistemic_statement denotes Previous studies indicated that NF-kB activation contributes to the production of type I IFN in response to viral infection (Basagoudanavar et al., 2011; Wang et al., 2010 Wang et al., , 2007b .
T78 31705-31962 Epistemic_statement denotes This indicates the importance of CARMA3 for inhibiting the expression of type I IFN and antiviral responses, suggesting that IRF3 activation plays a more dominant role in the expression of type I IFN than NF-kB activation in response to RNA virus infection.
T79 32211-32441 Epistemic_statement denotes It is interesting that CARMA3 is gradually turned over following VSV infection, which may serve as a mechanism to turn off NF-kB activation, and, meanwhile, it releases MAVS to form functional aggregates to induce IRF3 activation.
T80 32442-32686 Epistemic_statement denotes Further studies are necessary to determine the molecular mechanism as to how CARMA3 is targeted for K48 ubiquitination and degradation, and targeting CARMA3 for degradation may help to reduce inflammation while enhancing the antiviral response.
T81 32687-32843 Epistemic_statement denotes Therefore, understanding this mechanism may provide molecular insights for designing therapeutic agents for suppressing viral infectioninduced inflammation.
T82 34652-34748 Epistemic_statement denotes The real-time qPCR and ELISA assay were performed as recommended by the manufacturer's protocol.