Investigating the impact of antibiotic-induced dysbiosis on protection from C. difficile colitis by mouse colonic Innate Lymphoid Cells
Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During C. difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILC. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective duringC. difficileinfection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function. Overall design: Here, samples were mouse colon ILCs. We flow-sorted colon ILCs at three different conditions. Conditions were -No Abx (Baseline), Abx (mice were administered an antibiotic cocktail -45 mg/L Vancomycin, 35 mg/L Colistin, 35 mg/L Gentamycin, and 215 mg/L Metronidazole, in drinking water for three days and followed by ip injection of Clindamycin, 0.016mg/g of body weight), Abx+IL-33. We pooled samples from 10 mice in each condition. The whole colon from mice was dissected and opened longitudinally, followed by washing with PBS. Tissues were then left in buffer 1 (HBSS with 25 mM HEPES and 5% FBS) until harvesting all the mice. Colon tissues were incubated at 37°C temperature with gentle shaking for 40 min to remove the epithelial layer into 40 ml Buffer 2 (HBSS with 15 mM HEPES, 5 mM EDTA, 10% FBS, and 1 mM DTT). Tissue sections were cut into small pieces with a fine scissor and incubated into 5 ml digestion buffer (RPMI 1640 containing 0.17 mg/ml liberase, 30 mg/ml DNase) for 30-35 minutes at 37°C temperature with gentle shaking. Solutions were then passed through 100-micron and 40-micron cell strainers to collect single-cell suspensions. Suspensions were centrifuged at 500g for 5 minutes to pellet cells, followed by resuspension into 300 ml FACS buffer (2% FBS in PBS). From the cell suspensions, ILCs were enriched using a mouse Lineage cell depletion kit (Milteny Biotec) following the manufacturer's protocol