Molecular Mechanism Of Auto-Antibodies Sustain A Cardiac Inflammatory Environment Through TLRs/NF-κB/NLRP3 Inflammasome Pathway In Late Myocardial Infarction

Objective:Myocardial infarction(MI)is a severe fatal disease,and it is one of the main reasons leading to heart failure(HF).Because of its high morbidity and mortality,HF secondary to MI threatens human health and increases the socioeconomic burden.The long-term activation of the auto-immune system could be the reason for persistent cardiac inflammation and maladaptive heart remodeling.It’s reported the humoral immune system can be activated consistently post-MI,secreting antibodies targeting diverse autoantigens of the cardiovascular system,aggravating the development of cardiovascular diseases.However,the influence of humoral immune system activation on persistent cardiac inflammation is still unclear.in this condition,we conduct a study in vivo and in vitro,trying to figure out the relationship between the persistent activation of the humoral immune system post-MI and long-term cardiac inflammatory reaction and the underlying mechanism,and investigating new targets to improve HF post-MI.Materials and methods:Our study consists of three parts to illuminate the relationship between the activation of the humoral immune system post-MI and the long-term cardiac inflammation and investigate the underlying molecular mechanism through in vivo and in vitro experiments.Part I: The MI model in mice(C57BL/6J,male,6-8 weeks,24-28g)were induced by permanent coronary artery occlusion.The mice were randomly divided into the Sham+PBS group,MI+PBS group,and MI+ CD20 monoclonal antibody(CD20 m Ab)group.Mice in the MI+CD20 m Ab group were intravenously treated with 10 mg/kg CD20 m Ab every other day from 7 to 28 days after surgery.Mice in the sham and MI+PBS groups received the same dose of PBS without CD20 m Ab.Cardiac tissue and plasma were harvested 4 weeks after surgery.The concentration of circulating auto-IgG was tested by enzyme-linked immunosorbent assay(ELISA).The IgG deposition and macrophage infiltration were evaluated by Western blot(WB),Hematoxylin-eosin staining,Immunofluorescence staining,and Immunohistochemistry staining.Moreover,the infarction area and fibrosis level were assessed by Masson staining and Sirius Red staining,and the cardiac morphology and function were detected using Transthoracic echocardiography.Part II: We investigated the interaction mechanism between auto-IgG and the cardiac tissue through the above animal model.The expression level of TLR2 and TLR4 were tested by WB and IHC.The interaction between auto-IgG and TLRs was tested using Co-Immunoprecipitation(Co-IP).Furthermore,the expression level of NLRP3 and its downstream molecules were assessed by WB and IF.Part III: We cultured cardiac cell line HL-1 in vitro for inducing cell model,and purified the auto-IgG from MI mice serum by Protein A/G magnetic beads.The purified IgG was used to treat the HL-1 cell.Besides,the small interfering RNA(si RNA)of TLR2,TLR4,and NF-κB were conducted to investigate the molecular mechanism.The cell was divided into three groups to assess the influence of auto-IgG on cardiomyocytes:Buffer group,auto-IgG group,and Control-IgG group.The expression level of TLR2/4,NF-ΚB,NLRP3,Caspase1,GSDMD,and IL-1β were evaluated by q PCR,WB,and IF.The cell was divided into four groups to investigate the influence of auto-IgG on the inflammatory signal pathway of cardiomyocytes: NC+Buffer group,NC+Control-IgG group,NC+auto-IgG group,and si RNA+auto-IgG group.The expression level of TLRs/NF-κB/NLRP3 inflammasome pathway was tested by WB.The differences in pyroptosis level of cardiomyocytes between groups were evaluated by Hoechst-PI bistaining.Results:Part I: Compared to mice in the Sham group,we found there were amounts of autoIgG in circulation in mice 4 weeks post-MI by ELISA,and the auto-IgG can deposit in the cardiac tissue in late MI by WB,IF,and IHC.The administration of CD20 m Ab can diminish the level of auto-IgG in circulation and cardiac tissue.Then we demonstrated there were more macrophages infiltrating the cardiac tissue in the MI+PBS group by WB,IF,and HE staining and the CD20 m Ab can remarkably decrease the infiltration of macrophages post-MI,which means there was a correlation between auto-IgG deposition and macrophages infiltration in late MI.Meanwhile,the Masson staining and Sirius Red staining showed that compared to the MI+PBS group,there were smaller infarction areas and fibrosis levels in the MI+CD20 m Ab group.And the transthoracic echocardiography showed the administration of CD20 m Ab can remarkably improve cardiac remodeling and heart function,which demonstrated that the auto-IgG deposition is correlated with myocardial fibrosis and heart function deteriorationPart II: based on the previous work,we found a high level of expression of TLR2 and TLR4 in the MI+PBS group,and the expression of TLR2/4 in MI+CD20 m Ab was similar to those in the Sham+PBS group.That means the auto-IgG could induce the upregulation of TLR2 and TLR4.The Co-IP experiment showed the interaction between auto-IgG and TLRs post-MI.Furthermore,compared with the Sham+PBS group,we found a distinct up-regulation of NLRP3 and its downstream molecules in the MI+PBS group by WB and IF,but there was little up-regulation in the MI+CD20 m Ab group.So we can conclude the deposition of IgG can activate the NLRP3 inflammasome pathway.Part III: In this part,we found that auto-IgG can induce the up-regulation of TLR2 and TLR4 in HL-1 by WB and IF,and activate and translocate NF-κB into nuclear.Meanwhile,we demonstrated that auto-IgG can induce the up-regulation of NLRP3 and its downstream molecules in HL-1,such as Caspase1,and IL-1β.We knock down TLR2,TLR4,and NF-κB by si RNA respectively,all of these can inhibit the auto-IgG upregulating the NLRP3 and its downstream molecules.Furthermore,we found that the auto-IgG can induce pyroptosis in HL-1 by Hoechst-PI bi-staining,and it can be salvaged by the knockdown of TLR2,TLR4,or NF-ΚB.Conclusion:1.There is abundant auto-IgG in circulation because of the activation of the humoral immune system in late MI of mice,and the IgG can deposit in cardiac tissue.There is a close relationship between long-term auto-IgG deposition and persistent macrophage infiltration.And the long-term IgG deposition is correlated with maladaptive cardiac remodeling and heart function deterioration.2.The long-term auto-IgG deposition is correlated with the high-level expression of TLR2/4 post-MI,and there is an interaction between IgG and TLR2/4.Meanwhile,the auto-IgG deposition has a correlation with the up-regulation of molecules of the NLRP3 pathway.3.The auto-IgG up-regulates the expression of NLRP3 and its downstream molecules through the TLRs/NF-κB pathway in cardiomyocytes and induces pyroptosis.