Exploring The Mechanisms Of Intestinal Epithelial Cells Affecting Intestinal Immune System

The intestinal epithelial cells(IECs)is continually exposed to diverse foreign antigens from commensals,pathogens,and even food,so animals must have a way to induce immune responses against pathogens while retaining immune tolerance towards commensals and food.Beyond acting as a physical barrier,the function of IECs in sensing and responding to microbial signals is increasingly appreciated.After sensing different stimuli,IECs in different segments of the small intestine has regional implications for the vast network of immune cells within and below the intestinal epithelium.The mechanisms involved in IEC-environmental stimuli-immune interactions,however,are not fully characterized.In this paper,we subregionally explore the ability of IECs to direct intestinal homeostasis during physiological and inflammatory conditions.In the first part,we explored how the protein Gasdermin D(GSDMD)affects intestinal immune tolerance to food in the upper small intestine IECs.Previous studies have investigated the role of GSDMD as the executioner protein of pyroptosis in myeloid cells such as macrophages and dendritic cells(DCs).It’s a lytic form of cell death that initiates suicide of the cell.When encountered with pathoges or threatened by self-derived danger signal,cells will activate downstream inflammatory CASPs(Cysteinyl Aspartate Specific Proteinases)through inflammasome pathways to cleave GSDMD.During this process,self-inhibited full-length GSDMD will be cleaved into a 30 kD fragment that punches holes in the membrane and carries out its function.Current studies on GSDMD mostly focus on myeloid cells.At first,the high expression of GSDMD in small intestinal epithelial cells drawed our attention.However,as an immune-cell rich organ,strong inflammatory death may leds to an unpredictable consequence,which made us curios about the exact function of GSDMD in the small intestine.Here,we found that GSDMD can function in non-pyroptotic role in the small intestine intestinal epithelial cells(siIECs)by generating a different 13/42 cleavage.Genetic knockout this 13/42 GSDMD cleavage or conditional knock out GSDMD in intestinal epithelial cells disrupts immune tolerance to food in the small intestine.We first discovered that siIECs accumulate a less recognized 13 kD N-terminal fragment of GSDMD(GSDMD-N13)and a corresponding C-terminus fragment(GSDMD-C42)in the upper small intestines.And among several environmental stimuli in intestine we tested,only proteinaceous dietary antigens but not commensal bacteria,intestine viruses nor bile acid can influence the generation of this non-pyroptotic 13 kD GSDMD fragment in epithelial cells of the upper small intestine.Importantly,we experimentally confirmed in the siIECs that the distribution of GSDMD-N13 and Full-length GSDMD was unaffected by deficiency of inflammasome components or upon inflammasome activation,confirming that the induction of this cleavage is an inflammasome-independent process.We next performed immunoprecipitation followed by mass spectrometry(IP-MS)to identify potential protease(s)responsible for the 13/42 cleavage of GSDMD.The result confirmed that CASP3 and CASP7 in response to dietary antigens are proteases capable of catalyzing 13/42 GSDMD cleavage both in vitro and in vivo.Moreover,dietary antigens can lead to the activaiton of CASP3 and CASP7 in vivo.Unlike the 30 kD GSDMD cleavage fragment that executes pyroptosis by translocating from the cytosol and rupturing cell membranes,we demonstrate that the 13 kD N-terminal GSDMD cleavage fragment translocates to the nucleus.To explore which pathway(s)may be affected by nuclear GSDMD-N13 in siIECs,we used bulk RNA sequencing and single-cell RNA sequencing technology.The data suggest GSDMD-N13 induces the transcription of CIITA and MHCII molecules in siIECs,which in turn induces the Type 1 regulatory T cells(Trl)in upper small intestine.In light of our observations and the fact that dietary antigens is known to induce tolerance,we reasoned that 13/42 GSDMD cleavage in siIECs might be involved in proteinaceous dietary-antigen-induced tolerance in the small intestine.Indeed,mice given amino acid diet devoid of proteins,mice treated with a CASP3/7 inhibitor Ac-DEVD-CHO,and a small intestinal cleavage resistant variant(SICR)mutant mice(GsdmdSICR)and mice with MHCII deficiency in IECs(H2-Ab1fl/fl VilCre)both displayed a dysregulated food tolerance phenotype.Viewed alongside GSDMD’s pyroptosis-executioner function,our study supports that the differential cleavage of GSDMD can be understood as a regulatory hub controlling immunity versus tolerance in the small intestine.These insights concerning GSDMD’s divergent roles in immunity-versus-tolerance deepen our understanding of innate immunity generally and may facilitate the development of new therapies to treat food allergies.In the second part,we explored the role of RNA helicase DHX15 in the defense against gut pathogens in the terminal small intestine.We generated Dhx15fl/fl VilCre mice to specifically deplete Dhx 15 in IECs.We infected these mice with the enteric bacterium Citrobacter rodentium(C.rod)and elucidated the antibacterial immune response by RNA sequencing.We found that Dhx15 plays a critical role in the Wnt signaling pathway to induce α-defensin production in Paneth cells.Defensins play a crucial role in the development of inflammatory bowel diseases(IBD).Consistent with this,significantly reduced DHX15 expression was observed in intestinal specimens from patients with ulcerative colitis(UC).Here,we found that DHX15 has an unreported function in Wnt signaling in IECs,which plays a critical role in the antibacterial response in the intestine.IECs form the boundary between the internal body and the outside environment.In addition to epithelial cell-mediated defence mechanisms,IECs also coordinate the development of immune cells residing in the lamina propria and underlying lymphoid tissues and downstream immune responses.Our work explored two specific mechanisms by which IECs affect the intestinal immune system,which are of great significance for understanding intestinal immunity and treating related diseases.