Genetic diversity of Collaborative Cross mice reveals FFAR3 as a target for ILC2 anti-inflammatory reprogramming

Pulmonary group 2 innate lymphoid cells (ILC2s) are key drivers of Type 2 inflammation in diseases like asthma, yet the molecular mechanisms regulating their function are incompletely understood. Using the genetically diverse Collaborative Cross (CC) mouse panel, we mapped a quantitative trait locus (QTL) that governs ILC2 prevalence in the lung after aeroallergen exposure. This QTL creates a large population of ILC2s in the lung that are resistant to activation and have diminished Type 2 effector function. We identified free-fatty acid receptor 3 (FFAR3) as a gene responsible for this effect and demonstrated that FFAR3 signaling reprograms ILC2s to an anti-inflammatory state by promoting their survival, reducing Type 2 cytokine production, and enhancing IL-10 expression. This reprogramming is mediated by epidermal growth factor receptor (EGFR) upregulation. We showed that FFAR3's anti-inflammatory effect is conserved in human ILC2s, making it a potential therapeutic target for Type 2 inflammation. Overall design: Naive group 2 innate lymphoid cells (ILC2s) were isolated from the unchallenged lungs of mice by flow cytometry. We obtained ILC2s from both female C57BL/6J mice and female CC030/GeniUnc (Collaborative Cross) mice. Experiment 1: ILC2s were sorted from unchallenged C57BL/6J and CC030/GeniUnc lungs directly into buffer for analysis, allowing for comparison of C57BL/6J and CC030/GeniUnc ILC2s in the naive state. Cells were sorted separately from 3 mice of each strain. Experiment 2: ILC2s were flow sorted from CC030/GeniUnc, and all ILC2s were cultured with IL-33, TSLP, and IL-2. ILC2s received either DMSO vehicle or AR420626 treatment (FFAR3 agonist, 10uM). ILC2s were isolated from 5 CC030/GeniUnc, and cells from each biological replicate were split between vehicle and treatment condition to allow for paired samples. Cells were harvested at Day 2 and Day 6 of culture for analysis.