Therapeutic Effect And Mechanism Of Lactobacillus Reuteri On Immune Checkpoint Blockade-Associated Colitis

Objective: Immune checkpoint blockade(ICB)immunotherapy can increases antitumor immunity,but also triggers severe inflammatory side effects,known as immune-related adverse effects(ir AEs).One of the most common toxicities is immune checkpoint blockade-associated colitis(ICB-associated colitis).Studies have shown that ICB-associated colitis may be closely related to gut microbiome.Therefore,this study attempts to analyze the changes of microbiome in the mice with colitis and the effect of the microbiome to explain the mechanism,which will provide new ideas for alleviating ICB-associated colitis.Methods: 1.Withing DSS,ICB-associated colitis mouse model were established to simulate ICB treatment to monitor the changes in body weight,survival rate,intestinal pathology and serum inflammatory cytokines to determine the degree of intestinal inflammation in mice.2.Based on 16 S r RNA metagenomic sequencing,the intestinal flora diversity and dominant strains of colitis mice were analyzed to find the differences in gut microbiome.3.Vancomycin eliminating intestinal Lactobacillus and Lactobacillus reuteri by gavage were both used to determine the degree of intestinal inflammation in mice,which reflecting the effect of Lactobacillus reuteri on ICB-associated colitis.4.Flow cytometry was used to analyze the proportion and proliferation of immune cells in the colon of mice,especially the changes of subpopulations of innate lymphoid cells(ILCs)to investigate the mechanism of Lactobacillus reuteri alleviating ICB –associated colitis.Results: 1.Establishment of mouse model of ICB-associated colitis: ICB administration aggravated mouse DSS colitis.The body weight of the mice was significantly reduced,and the survival rate was reduced,inflammatory cells infiltrated the intestinal epithelium,and the serum inflammatory factor levels were significantly increased.2,ICB-related colitis is closely related to gut microbiome changes: the gut microbiome diversity of intestinal inflammation mice was decreased,the dominant species were changed,and the relative abundance of Parabacteroides,Lactobacillus and Turicibacter was changed in ICB-related colitis mice.3.Vancomycin eliminating Lactobacillus from the intestine of mice,significantly aggravated intestinal inflammation in mice.The body weight of the mice was significantly reduced,the survival rate was reduced,more inflammatory cells infiltrated intestinal epithelium and the serum inflammatory factor levels were significantly increased.L.reuteri therapeutically abrogates ICB-associated intestinal inflammation in DSS-treated mice.The weight is not significantly reduced.The intestinal epithelium was intact and the Lactobacillus reuteri treatment also decreased the serum levels of the inflammatory cytokines.Also,Lactobacillus reuteri does not affect the anti-tumor effect of ICB.4.The immune regulatory function of Lactobacillus reuteri is associated with intestinal ILC3 cells.Lactobacillus reuteri can reduce the proportion of ILC3 s in the intestine,and has no significant relationship with ILC1 s lymphocytes and ILC2 s cells.Conlution: The intestinal inflammation side effects of ICB-associated colitis was related to the changes of gut microbiome.Probiotics Lactobacillus reuteri can abrogate ICB-associated colitis by preserving group 3 innate lymphoid cells without affecting the anti-tumor effect of ICB.