Research On The Mechanism And Effect Of Increased NKp46~+ILC3s In The Heart Of Viral Myocarditis

Objective:Viral myocarditis(VMC)is the main cause of dilated cardiomyopathy(DCM),among which up to 25%of VMC is caused by enterovirus,and Coxsackie virus B3(CVB3)is the most common virus.Studies have confirmed that NK cells,macrophages and T cells are recruited to the site of infection in the acute phase of myocarditis,secreting tumor necrosis factor(TNF),IL-1α,IFN-γand other cytokines,to aggravate heart damage and impair its contractile function.Preliminary experiments have shown that Type 3 innate lymphoid cells(ILC3s)increase in the stages of cardiac inflammation in CVB3,but the mechanism is still unclear.Therefore,this study explored the phenotype and function of ILC3s in the heart of the acute phase of VMC,and tried to find the reasons for the infiltration and increase of ILC3s in the heart.Methods:(1)Detection of the expression of ILC3s and related genes in the heart and small intestine of the VMC model at different time points:Construct VMC mouse model,weigh the mice every day before being sacrificed,and take the eyeball blood on the 3,5,7,and 10 days respectively.Mice were killed by cervical dislocation after eyeball blood was taken.Take the mouse heart,save the middle section of the mouse heart,and fix it with paraformaldehyde for histopathological examination.The whole mouse fresh heart tissue was used to prepare a cardiac cell suspension,and the changes in the number of cardiac ILCs,ILC3s and ILC3s subgroups were detected by flow cytometry(FCM).The small intestine tissues of mice were taken to extract the lymphocytes in the lamina propria of the small intestine,the changes in the number of ILC3s in the lamina propria of the small intestine were detected by FCM,and the expression levels of S1PR1,CXCR6 and KLF2 m RNA were detected by q RT-PCR.(2)The effect of Anti-CD90.2 on VMC:Starting four days before the induction of the VMC model,anti-CD90.2 was injected into the tail vein every two days.And after the induction of the model,anti-CD90.2 was injected into the tail vein every other day.On the 7th day of the model,the mice were sacrificed by the cervical dislocation method,and the mouse heart was taken,and the middle section of the mouse heart was retained and fixed with paraformaldehyde for histopathological examination.The collected mouse eye blood was placed in a 37°C incubator for 30 minutes to be coagulated and then centrifuged to collect serum.Save fresh heart tissue for m RNA extraction,and q RT-PCR to detect the expression levels of IFN-α,IFN-β,and IFN-γm RNA in the heart tissue.The heart cell suspension was prepared from fresh heart tissue,and the changes in the number of cardiac ILCs,ILC1s,ILC2s and ILC3s were detected by FCM.The spleen cell suspension was prepared from the mouse,and the changes in the number of cardiac ILCs were detected by FCM.(3)Adoptive transfer of small intestine ILCs to detect heart tissue infiltration:Magnetic beads were used to sort the small intestinal ILCs of VMC model CD45.1donor mice,and adoptively transfer the small intestinal ILCs to VMC model CD45.2recipient mice through tail vein injection.After 48 hours,the mice were sacrificed by cervical dislocation.The mouse heart tissue was taken to prepare cardiac cell suspension,and the change of CD45.1~+cell ratio was detected by FCM.(4)The effect of cardiac macrophages on the differentiation of cardiac ILCs to ILC3s:The VMC mouse model was constructed.On the 5th day of the model,the mice were killed by cervical dislocation.The heart tissue was taken to prepare a cardiac cell suspension,and F4/80~+cardiac macrophages were sorted by magnetic beads.Take normal mouse heart tissue to prepare cardiac cell suspension,and magnetic beads to sort cardiac ILCs.The sorted cardiac macrophages and cardiac ILCs were co-cultured for 48 hours,and the changes in the proportion of ILC3s were detected by FCM.Result:(1)FCM results showed that compared with the control group,the number of total ILCs increased significantly in the heart on the 5th and 7th day of the model,and the number of ILC3s in the heart tissue on the 5th day of VMC significantly higher which are mainly NKp46~+CCR6~-ILC3s in the heart.The number of ILC3s cells in the small intestine tissue was significantly increased in VMC.The q RT-PCR results showed that compared with control group,the expression levels of S1PR1,KLF2,and CXCR6m RNA in the lamina propria lymphocytes of the small intestine of the VMC model mice increased.(2)H&E results showed that the inflammatory cell infiltration of the heart tissue in the CVB3+anti-CD90.2 group was relieved compared with the CVB3 group;compared with the control group,the bodyweight of mice in the CVB3 group and CVB3+anti-CD90.2 group was significantly lower than that in the Control group.The FCM results showed that compared with the CVB3 group,the total number of ILCs in the heart and spleen tissues of the CVB3+anti-CD90.2 group mice was significantly reduced;ILC3s in the heart tissue of VMC model mice treated with anti-CD90.2neutralizing antibody were effectively depleted;while the cell reduction of ILC1s and ILC2s was not statistically significant.The q RT-PCR results showed that the expression of interferon in the heart tissue of mice in the CVB3 model group and CVB3+anti-CD90.2 treatment group was significantly increased,and the expression level of interferon I in the heart of the CVB3+anti-CD90.2 treatment group was significantly higher than that of the other groups.(3)The results of flow cytometry showed that there were infiltrating ILCs in the heart tissue of the VMC mouse model by adoptive transfer of small intestinal ILCs for48 hours,but they were not detected in the spleen.(4)The results of flow cytometry showed that the proportion of ILC3s increased after 48 hours of co-culture of cardiac macrophages and cardiac ILCs compared with the control group.Conclusion:(1)ILC3s,mainly NKp46~+cells,increase in the acute phase of VMC,and promote the development of myocarditis inflammation.(2)There are two possible sources of cardiac ILC3s in viral myocarditis:(1)ILCs in the small intestine tissue migrate into the heart tissue and cause the increase of ILC3s;(2)Cardiac macrophages promote the differentiation of cardiac progenitor-like ILCs into ILC3s.