Project Summary Hypothesis: Type 2 innate immunity protects the gut from C. difficile infection (CDI) via the coordinated actions of innate lymphoid cell type 2 (ILC2), eosinophils and alternatively-activated macrophages (AAM). Progress: The first 5 years of support from 5R01AI124214 demonstrated that microbiota-elicited type 2 immunity protects from CDI. It does so at two stages, protecting the epithelial barrier during acute CDI, and promoting healing. We discovered that key steps in this process are: (i) IL-25 and IL-33 production by the intestine in response to commensal microbes; (ii) IL-25 and IL-33 activation of ILC2; and (iii) protection by downstream type 2 cellular effectors eosinophils and AAM. The key knowledge gap is how ILC2, eosinophils and AAM act to protect. Significance: C. difficile is a CDC “Urgent Antibiotic Resistance Threat” as the number one hospital-acquired infection in North America. Current therapy with antibiotics is inadequate, as relapse occurs in up to 20% and death in 6%. The proposed studies, by identifying how innate immunity protects, offer the promise of immunotherapy as an addition to current approaches to treatment and as a replacement for fecal transplant. Investigators: The PI William Petri discovered the role of type 2 immunity in CDI in the prior 5 year period of support. Key personnel include Maureen Carey (systems biology), Stacey Burgess (spectral flow cytometry), Katia Sol-Church (scRNAseq) and Jennie Ma and Pankag Kumar (bioinformatics). Innovative aspects are foremost the hypothesis that type 2 innate immunity protects from C. difficile and that the immune system can be harnessed to treat CDI. Approach: We will describe during the course of CDI the individual and collective mechanisms by which ILC2 (Aim 1), eosinophils (Aim 2) and AAM (Aim 3) respond to CDI in the murine model. We will identify common underlying mechanisms of protection by the coordinated action of these 3 cell effectors of type 2 innate immunity. The studies will include single cell transcriptomics (scRNAseq) in the presence or absence of protective upstream activation by the intestinal epithelial cytokines IL-25 and IL-33, and interventional studies to directly test the contributions of innate immune effector cells on protection. The environment for the work is highly interactive with all of the key personnel located within a few minutes walk of Dr. Petri’s C. difficile research lab. Successful completion of these studies will identify the mechanisms by which cellular type 2 responses protect from CDI and lay the foundation for immunotherapy for CDI as an adjunct to antibiotics and as a replacement for fecal microbiota transplant (FMT).