ILC3s-derived RANKL contributes to tuft cell hyperplasia and anti-helminth immunity

Intestinal tuft cells are reported to orchestrate parasite type 2 immunity through producing IL-25 and LTC4. Meanwhile, type 2 cytokines IL-13 and IL-4 can induce tuft cell hyperplasia from intestinal stem cells during helminth infection. Here, using intestinal organoids culture, we show that RANKL synergizes with both IL-13 and IL-4 to enhance tuft cell expansion. Genetic mouse models revealed that RANK-RelB axis in intestinal epithelial cells (IECs) is required for optimal tuft cell-ILC2 circuit activation during helminth infection. Mechanistically, RANKL enhances IL-13-activated IL-4RA/STAT6 signaling in IECs, promoting tuft cell differentiation. Using genetic mouse models, we further demonstrated that ILC3s-derived RANKL is indispensable for optimal tuft cell hyperplasia during helminth infection. The scRNA-seq data revealed that N. brasiliensis infection will modulate ILC3 subsets distribution and RANKL expression. We cultured the intestinal organoids from Vil-creERT2; Rankfl/fl mice with or without 4-OHT (termed as WT and KO respectively). To explore the signaling pathways modulated by RANKL, we further treated the WT and KO organoids with IL-13 and with or without RANKL for 4 days. Then the intestinal organoids were applied for bulk RNA sequencing. To explore the adaptations of ILC3s during helminth infection, intestinal lamina propria leukocytes(LPL) from naïve mice or N. brasiliensis-infected mice were isolated and applied for single-cell RNA sequencing. Three subsets of ILC3s were identified and the transcriptional transitions of ILC3s from naïve state to N. brasiliensis infection were analyzed.