The Role Of IL-33 Induced-group 2 Innate Lymphoid Cells In Sepsis And Its Molecular Mechanism

Part 1 Expression of IL-33 in sepsis and its relationship to ILC2Objective:IL-33,a member of IL-1-related cytokines,is derived from impaired endothelial,epithelial or myeloid cells.As an alarmin cytokine,IL-33 regulates T cell,mast cell and innate lymphoid cells(ILCs)through its specific receptor ST2.ILC2 is an important member of ILCs,and are the main ILCs subtype in lung.It has been identified that IL-33 is a potent cytokine in activating ILC2.And activated ILC2 play significant role in immune response,inflammation regulation and tissue repair.However,the role and relationship of IL-33 and ILC2 in sepsis remains unclear.This study aims to elucidate the expression of IL-33 and its relationship to ILC2 in the lungs following sepsis.Methods:Sepsis was induced by CLP.Sham surgery(SS)operated mice were included as a control group.Lung and peripheral blood were collected at 0h,3h,6h,12h,24h and 36h after the surgery.ILC2 in lungs were detected by flow cytometry.Il-25,Il-33 and x Tslp expression in the lung was assayed by RT-qPCR.The IL-33 level in blood was measured by ELISA.Mice were treated intratracheally(i.t.)with recombinant murine IL-25 or IL-33,TSLP in a volume of 50 μl PBS per mouse for 24 h and lung ILC2 were detected.Results:ILC2 in the lungs at baseline and time points up to 36 h after CLP or SS were detected by flow cytometry.The percentage and absolute number of ILC2 in the lungs at 12 h after CLP was significantly increased as compared with that in SS mice,and the ILC2 number remained elevated for at least additional 24 h.The percentage and absolute number of ILC2 in the lungs of SS mice did not show significant changes.We measured Il-25,Tslp and Il-33 mRNA expression,using RT-qPCR,in the lungs of CLP mice for up to 24 h;and found that the expression of all these cytokines were significant increased and peaked at 6 h after CLP.Then,we measured IL-33 protein concentrations in the blood and found that CLP significantly increased plasma IL-33 level,which peaked at 6 h,and decreased by 24 h.Treatment with rmIL-33 induced significant increase of ILC2 in the lungs as compared with PBS control group.IL-25 also increased ILC2 in the lungs,but the levels were significantly lower than that in the rmIL-33-treated mice and CLP mice.Despite the increase in Tslp mRNA expression in the lungs after CLP,treatment with rmTSLP did not increase the percentage and number of ILC2 in the lungs.Conclusions:In septic mice,ILC2 recruited in lungs following the increasing expression of IL-33 in lungs and circulation.These data indicate that IL-33 may paly a significant role in ILC2 expansion in the lungs in response to sepsis.Part 2 The impact of IL-33 and its receptor ST2 in sepsis induced ILC2 expansion and functionObjective:In view of the expression pattern of IL-33 and ILC2 expansion in lungs in the first part,moreover,IL-33 is a potent activator of ILC2 and can induce the cytokines secretion.ILC2 is the main source of IL-4,IL-9 and IL-13 in lung.Thus,this part of the study was designed to investigate the impact of IL-33 and its receptor ST2 on sepsis induced ILC2 expansion and cytokine secretion.Methods:WT,IL-33 deficiency(Il-33-/-)mice and ST2 deficiency(Il1rl1-/-)mice were subjected to CLP and the percentage and number of ILC2 were measured by flow cytometry after CLP.Il-33-/-mice were also administrated with rmIL-33,and then ILC2 in lung of septic mice was detected.The expression of cytokine(IL-4,IL-9 and IL-13)in lung ILC2 were measured by intracellular staining and flow cytometry.The IL-9 and IL-13 concentration in BALF and plasma of mice after the surgery were detected by ELISA.Results:Deficiency of either IL-33 or ST2 prevented ILC2 expansion in the lungs following sepsis.Moreover,intratracheally administration of rmIL-33 to Il-33-/-mice restored the increase of ILC2 in the lungs at 24 h after CLP and even SS mice.We further found that IL-9 positive(IL-9+)ILC2 significantly increased by 24 h after CLP,and IL-13+ ILC2 transiently increased after CLP and reached a peak at 6 h.IL-4+ILC2,however,did not significantly increase following sepsis.Also,both of the cytokines were markedly increased at 24 h and 12 h after CLP.However,both Il33-/-and Illrl1-/-mice exhibited reduced levels of IL-9 and IL-13 in plasma and BALF after sepsis as compared with those in WT mice.Conclusions:ILC2 expalsion in the lungs and the specific pattern of cytokine expression and secretion from lung ILC2 following sepsis were in an IL-33/ST2-dependent manner.Part 3 The protective role of recruited ILC2 in sepsisObjective:Endothelium dysfunction has been reported to play an important role in activation of a number of inflammatory cells,release of inflammatory mediators,and induction of inflammation in sepsis.Previous report showed that endothelial cell death occurs in sepsis.ILC2,an important immune cells,play a central role in host response to inflammation,infection and epithelial cell protection.Our data showed that CLP induced ILC2 expansion in lungs and genetic deficiency of IL-33 or ST2 prevented ILC2 expansion.Therefore,this part of the study investigated the role of recruited ILC2 in sepsis induced endothelium dysfunction and its mechanism.Methods:WT and Il-33-/-mice were subjected to CLP and evans blue dye(EBD)was applied to assess blood vessel permeability in vivo.The cell death rate of lung endothelial cell was measured by Annexin V/7-AAD stain at 24 h after CLP.Using ILC2 and endothelial cell co-culture model,we explored the endothelial cell death after LPS/TNFa stimulation.The staining of Caspase-1 and TUNEL were used to measure and define endothelial cell pyroptosis.The Caspase-1 activation was also measured by flow cytometry and Western blot.After adminstration of anti-IL-9 antibody in lung,the endothelial cell death was accessed after CLP for 24h.Results:We demonstrated that at 24 h after CLP,the lungs of Il-33-/-mice had increased permeability shown as more blue coloration in the lungs than that in WT lungs.EBD concentration was 2-fold higher in the lungs of Il-33-/-mice than that in WT mice.At 24 h after CLP,MLEC death in Il-33-/-mice increased as compared with that in WT mice.After co-cultured with ILC2 in vitro in the presence and absence of LPS and TNFa,lung endothelial cell death induced by LPS and TNFa was markedly reduced.However,addition of rmIL-33 in the culture medium failed to protect MLEC death.We farther defined the MLEC death type by staining the cells with TUNEL and Caspase-1 following LPS and TNFa challenge.We found that TUNEL and activated Caspase-1 double positive lung endothelial cells,which represent pyroptosis,occupied-70%of the MLEC death.And endothelial cell death was markedly reduced in Caspase-1-/-endothelial cells.IL-9 reduced MLEC pyroptosis by reducing Caspase-1 activation.After administration of anti-IL-9 antibody in lung,the endothelial cell death increased after CLP for 24 h compared to SS mice.Conclusions:IL-33 protects sepsis induced endothelial cell death by recruiting ILC2 in lung tissue.ILC2-derived IL-9 mediates the protection of MLEC from pyroptosis by suppressing Caspase-1 activation.