NLRC5 Negatively Regulates LTA-induced Inflammation Via TLR2/NF-?B And Participates In TLR2-mediated Allergic Airway Inflammation

Toll-like receptors(TLRs)are type I membrane-bound proteins,which participate in innate immune responses,can recognize and defend against invading pathogens and microorganisms.So far,11 members of the TLRs family have been found in mammals.Of which,TLR2 is a key innate immune receptor that is primarily expressed in immune cells and airway epithelial cells.TLR2 has been reported to participate in the pathogenesis of multiple inflammatory and immune diseases.NLRC5 is a newly found protein of the NLRs family,which is capable of regulating inflammatory responses and cell death.Both human and mouse NLRC5 are highly expressed in lungs,spleen,and thymus.Numerous studies have demonstrated that NLRC5 acts as a regulator of homeostasis in innate immunity.In this study,We investigated the role of NLRC5 in TLR2-mediated inflammatory response and allergic airway inflammation.1.Molecular mechanisms of NLRC5 to regulate the LTA-induced inflammatory response in macrophages via TLR2/NF-?B signaling pathwayBackground NOD-like receptors(NLRs)represent a family of intracellular PRRs that play a role in host defense against relevant pathogen-associated molecular patterns(PAMPs).NLRC5 is a largest member of the NLRs family,which was first discovered in 2010 and hasbeen shown to play a key role in innate immunity and multiple inflammatory responses.In addition,it can also participate and play an immune surveillance role in the development of tumors.Cui et al.found that in vitro,NLRC5 can negatively modulate the TLR4-mediated NF-?B activation and type I interferon production.Both TLR2 and TLR4 are transmembrane proteins,which can participate and play a regulatory role in the development of inflammatory responses.However,in vitro,whether NLRC5 can also participate in tlr2-mediated inflammatory responses and how NLRC5 regulates inflammation through the TLR2/NF-?B signaling pathway are still unclear,and it remains to be investigated.Objective1)Investigate the changes of NLRC5 in TLR2-mediated macrophage inflammatory response induced by LTA.2)Investigate the regulatory effect of NLRC5 in TLR2-mediated cellular inflammatory response and its potential targets for inflammatory regulation in downstream signaling pathways.Methods1)Observe the expression levels of TLR2,NLRC5 and inflammation-related signaling molecules in LTA-stimulated macrophages at different points in time Mouse macrophage line RAW264.7 were stimulated with LTA for 0h,4h,8h,12 h,24h,respectively.The expression levels of TLR2,NLRC5 and multiple inflammation-related signaling molecules were analyzed by Western Blot.2)Evaluate the effect of TLR2 siRNA on the LTA-induced macrophage inflammatory response was interfered with by siRNA in macrophages,as well as detect the changes in NLRC5 expression levels.RAW264.5 cells were transfected with TLR2 siRNA,and then were stimulated with LTA for 12 h.Knockdown efficiency as well as the levels of NLRC5 protein expression and inflammatory response were both determined by Western Blot analysis.Immunofluorescence staining was used to observe the distribution of NF-?B and its expression level by CLSM.3)Target intervention of NLRC5 in cells,and observed changes in TLR2-mediated macrophage inflammatory response and the changes of TLR2 expression level under LTA induction.RAW264.5 cells were transfected with NLRC5 siRNA,and then were stimulated with LTA for 12 h.Knockdown efficiency and the levels of TLR2 protein expression and inflammatory response were both determined by Western Blot analysis.The distribution of NF-?B and its expression level were observed by CLSM after NLRC5 siRNA intervention and LTA induction.Results1)LTA induces an inflammatory response and NLRC5 activation in RAW264.7cells RAW264.7 cells were stimulated by LTA,and Western blot analysis showed the expressions of inflammation-related proteins P-NF-?B p65,NLRP3 and ASC were significantly increased.Meanwhile,the expression level of NLRC5 also was increased in a time-dependent manner.At the same time,we found that protein expression level of NLRC5 peaked at 12 hours.2)LTA-induced activation of NLRC5 is TLR2-dependent RAW264.7 cells were transfected with TLR2 siRNA or Negative siRNA.After LTA stimulated,the level of inflammatory response was significantly reduced in TLR2 siRNA groups,meanwhile,the expressions of inflammatory proteins P-NF-?B p65,NLRP3 and ASC were notably decreased,More importantly,the expression level of NLRC5 also decreased.To further test this finding,the expression levels of NF-?B p65 and P-NF-?B p65 were determined by immunocytochemistry,Fluorescence imaging results showed that after TLR2 knockdown,the phosphorylation level of NF-?B p65 was significantly decreased.3)NLRC5 negatively regulates inflammatory responses and activation of NF-?B p65 in TLR2-mediated macrophages RAW264.7 cells were knocked down with NLRC5 siRNA and stimulated with LTA for12 h,Western blot analysis showed no significant change in TLR2 expression,but the phosphorylation level of NF-?B p65 was significantly increased in cells transfecting with NLRC5.In addition,the expressions of inflammasome-related proteins,such as NLRP3 was increased significantly,while ASC expression was decreased in NLRC5 siRNA groups.The expression levels of NF-?B p65 and P-NF-?B p65 were detected by immunocytochemistry.The results of fluorescence imaging in NF-?B p65 and P-NF-?B p65 were consistent with of western blot analysis.Conclusions1)NLRC5 is involved in TLR2-mediated macrophage inflammatory response2)NLRC5 negatively regulates the TLR2-mediated macrophage inflammatory response via NF-?B signaling pathway,which propose a new and effective mechanism of innate immune regulation.2.NLRC5 participates and may can regulate TLR2-mediated allergic airway inflammation in miceBackground As a serious health problem,the global incidence and mortality of bronchial asthma continues to increase.It is estimated in the word,that about 334 million people suffer from asthma,and the total number of patients is expected to increase by 100 million by2025.The pathogenesis of asthma is related to chronic allergic airway inflammation and immune abnormalities.At present,The role of TLR2 induced innate immune responses in the pathogenesis of asthma is still controversial.In addition,until now,whether NLRC5 participates in TLR2-mediated allergic airway inflammation and how it regulates inflammation through the TLR2/NF-?B signaling pathway were still unknown,the relevant mechanism remains to be explored.Objective1)Observe the expression levels of TLR2,NLRC5 and inflammasome related proteins in mice allergic airway inflammation.2)Investigate whether NLRC5 participates in TLR2-mediated allergic airway inflammation and how it regulates inflammation through the TLR2/NF-?B signaling pathway.Methods1)A model of allergic airway inflammation was established to detect the level of airway inflammation,TLR2 and NLRC5 expression in the lung tissues of C57 mice C57 mice were sensitized and challenged with OVA to establish a model of allergic airway inflammation.The protein expression levels of TLR2 and NLRC5 in mice lung tissues were analyzed by Western Blot.The airway wall thickness and the number of rings of inflammatory were observed by Hematoxylin-eosin(HE)staining.Periodic acid– Schiff(PAS)staining was used to assess for quantitative analysis of goblet cell hyperplasia and mucus production in lung tissues.The total number of cells and the number of different inflammatory cells in BALF were counted by inverted microscope with Wright ' s stain.2)Evaluate the effect of TLR2 gene knockout in the allergic airway inflammation in mice TLR2 gene knockout(TLR2-/-mice)was performed in C57 mice,then,TLR2-/-mice and C57 mice were sensitized and challenged with OVA,respectively.The histological analysis of Mice airways were performed by HE stain for airway inflammatory cell infiltration and by PAS stain for mucus production and goblet cell hyperplasia.The number of total cells and the inflammatory cells in BALF were detected by Wright's staining.The levels of IL 13,IL 6,IFN-? and TNF-? in BALF were detected by ELISA.3)Evaluate the effect of TLR2 knockout on the levels of NLRC5 protein expression and inflammation-related protein expression in allergic airway inflammation in mice OVA sensitized and challenged C57 and TLR2-/-mice respectively to establish a model of allergic airway inflammation.The total proteins of lung tissues were extracted and were detected by Western Blot.The changes of NLRC5 protein and inflammatory-related proteins expression were evaluated.Results1)OVA-induced mice allergic airway inflammation was accompanied by the increased expression of TLR2 and NLRC5 Compared with the saline control group,the number of total cells and inflammatory cells in BALF was significantly increased in OVA-induced mice.The bronchial lumens were narrow and inflammatory cell infiltration and mucus secretion were obviously found on airway wall in OVA groups.Meanwhile,western blot analysis showed that the protein expression levels of TLR2 and NLRC5 both were significantly increased in accord with the level of airway inflammation.2)In TLR2 knockout mice,OVA induced allergic airway inflammation was significantly reduced Under OVA sensitization and challenge,compared with WT mice,the bronchial wall thickening,inflammatory cell infiltration,mucus secretion and epithelial goblet cell proliferation were significantly reduced in TLR2-/-mice,as well as the number of total cells and inflammatory cells in BALF was notablely decreased.ELISA showed the levels of IL-6,IL-13 and TNF-? in BALF were significantly down-regulated,while the expression level of IFN-? was increased in TLR2-/-mice.3)TLR2 gene knockout can significantly reduce the expression levels of inflammation-related proteins and NLRC5 in allergic airway inflammation Under OVA sensitization and challenge,The phosphorylation level of NF-?B p65 was significantly increased in C57 mice.Meanwhile,the expression levels of inflammasome-related proteins NLRP3 and ASC were also increased.Compared with C57 mice,however,the expression levels of inflammation-related proteins such as P-NF-?B p65,NLRP3 and ASC were remarkablely decreased in TLR2-/-mice.More importantly,the level of NLRC5 protein expression was also notably decreased.Conclusions1)NLRC5,TLR2 and inflammasome-related proteins are involved in allergic airway inflammation2)NLRC5 is involved in TLR2-mediated allergic airway inflammation and may can regulate the inflammation through TLR2/NF-?B signaling pathway...