Investigation Of Commensal-Dependent ILC3 Modulation Of Mucosal Healing In Ulceative Colitis

PART ONEAntibiotics targeting Gram+commensal can impair epithelium repairing after DSS induced colitis【Background】We first evaluated whether interfering of commensal by oral administering of antibiotics can moderate the susceptibility of DSS-induced colitis.【Method】Body weight recovery,vascular endothelial growth factor（VEGF）and epidermal growth factor（EGF）level were examined during the 7-14 days post DSS treatment.Microscopic analysis was performed to evaluate pathology changes,【Results】Elimination of Gram positive commensals using vancomycin resulted in mice losing less body weight and colon length during the acute colitis phase,however,there was notably delayed body weight recovery.Colonic growth cytokine VEGF and EGF were also find to be expressed in a relatively low level.IL-22/STAT3 signaling were less activated in vancomycin treated mice as evidenced by decreased IL-22 and phosphorylation of STAT3 in recovery stage【Conclusion】vancomycin-sensitive gram-positive bacteria,but not gram-negative nor anaerobic bacteria,were required during the recovery from DSS-induced experimental colitisPART TWOEliminating Gram+leads to decreased tissue IL-22 level accompanied by reduced RORγt+ILC3【Background】To validate the role of ILCs in the regulation of IL-22/STAT3 axis.【Method】Mononuclear cells were extracted from colonic lamina propria and markers of interest were analyzed using flow cytometry.The absolute number and proportion of ILC3 as well as its expression level of IL-22 were compared among antibiotics groups and SPF mice 5 days after cease of DSS treatment.【Results】The absolute number of ILC3s significantly decreased in vancomycin group.The IL-22 expression by ILC3 ex vivo was also impaired in vancomycin treated mice compared with that in SPF mice and neomycin treated mice.【Conclusion】The decreased IL-22 production is a more specific response of RORγt⁺ILC3 to G+commensal elimination rather than muffled epithelial immunity caused by decreased luminal microbiome richness.PART THREEDecreased colonic IL-22 level was related to undersupply of IL-23expressing dendritic cell.【Background】IL-23 is the most potent inducer of colonic ILC3 production of IL-22 along with other less effective pro-inflammatory cytokines such as IL-1β.To make sure if decreased colonic IL-23 expression could be related to the shrink of CX₃CR1⁺dendritic cell（DC）population,we analyzed the lamina propria DC composition after DSS termination【Method】The lamina propria DC composition after DSS termination was analyzed using flowcytometry.Cytokines required for recruitment and stimulation of lamina propria DC were also examined.【Results】We found that the absolute number of CX₃CR1⁺DC in vancomycin group were significantly less than neomycin treated and SPF mice.In comparison,there was no obvious difference of CD103⁺DC population among those groups.The CCR2receptors on the surface of monocytes migrated to the colon remained intact after antibiotics treatment,while qPCR analysis revealed that the mucosal CCL2 and CCL7were lower in vancomycin group【Conclusion】These results suggest Gram+commensal may contribute to the homeostasis of IL-22 producing ILC3 in a CX₃CR1⁺DC dependent way.PART FOURSupplement of DSS only partially restore IL-22 secretion of mucosal healing【Background】To investigate whether substitute of G+bacteria derivatives could rescue the recovery defect of vancomycin treated mice.【Method】One time administration of MDP was given intraperitoneally after completing DSS treatment to mice in the vancomycin group.Judging from body weight recovery and histology【Results】However,Mice received MDP expressed significantly higher mucosal CCL2 and CCL7,the absolute number of CX₃CR1⁺DC also increased by day 12,Though the RORγt⁺ILC3 number didn’t expand as expected.【Conclusion】Those results indicated that stimulation of NOD2 by MDP alone during the acute inflammation was enough to recruit CX₃CR1⁺DC,however,there were other driven force（s）missing for the complete recovery of the population of IL-22 expressing RORγt⁺ILC3.