GROUP 3 INNATE LYMPHOID CELLS RELY ON THE UNFOLDED PROTEIN RESPONSE FOR CYTOKINE PRODUCTION IN INFLAMMATORY BOWEL DISEASE

Backgrounds: Group 3 innate lymphoid cells (ILC3s) have recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 serves as the regulatory hub of the unfolded protein response (UPR) that plays a vital role in intestinal inflammation.
Methods: We generated conditional knockout Ire1a^flo/flox,Rorc-Cre (Ire1a^ΔRorc) and Rag^-/-Ire1a^ΔRorc mice with Ire1a deleted in ILC3s. Intestinal infections were induced by Citrobacter rodentium and Clostridium difficile. ILC3s-derived cytokines including IL-22 is crucial for host defense in the acute phase of C. rodentium and C. difficile infection. Dextran sodium sulfate (DSS) and T-cell transfer were given to induce colitis. Ileal biopsies were collected from patients with active Crohn’s disease (CD) before starting ustekinumab (anti-IL-12/IL-23) for flow cytometry.
Results: In murine intestinal ILC3s, the activity of IRE1a-XBP1 pathway followed a robust 24h circadian rhythm which parallels that of clock genes and cytokines including IL-22. XBP1s was activated in ILC3s ex vivo by vasoactive intestinal peptide, which was previous shown to orchestrate the circadian expression of IL-22 by ILC3s. IRE1a-XBP1 in intestinal ILC3s was further activated in response to IL-23 and colitis in mice, as well as in inflamed IBD tissues compared to normal tissues from healthy individuals. We further showed that IRE1a-XBP1 activation in stimulated ILC3s required mitochondrial reactive oxygen species (mtROS) (Figure 1). Using the Ire1a^ΔRorc mice, we demonstrated that IRE1a-XBP1 is critical for cytokine expression by ILC3s. Ire1a^ΔRorc and Rag^-/-Ire1a^ΔRorc mice were highly susceptible to acute C. rodentium and C. difficile infection and acute DSS colitis; while Rag^-/-Ire1a^ΔRorc mice developed more severe chronic colitis and fibrosis upon T-cell transfer. Ire1a^ΔRorc and Rag^-/-Ire1a^ΔRorc mice exhibit reduced IL-22 and IL-17A in ILC3s and impaired epithelial barrier function with diminished expression of mucins and antimicrobial peptides. The susceptibilities of Ire1a^ΔRorc and Rag^-/-Ire1a^ΔRorc mice to colitis were rescued by administration of recombinant IL-22 (Figure 2). Moreover, the frequency of XBP1s+ ILC3s in ileal mucosa from CD patients before initiation of ustekinumab positively correlates with response to therapy.
Conclusions: We demonstrate that a non-canonical mtROS-IRE1a-XBP1 pathway augments cytokine production by intestinal ILC3s. Loss of IRE1a-XBP1 in ILC3s impairs the optimal and rhythmic expression of protective cytokines and renders the mice more susceptible to intestinal infections and colitis. We also identify XBP1+ ILC3s as a potential biomarker for predicting responsiveness to anti-IL-23 therapies in IBD.