In WP1 we examined localization of ILC3 in the human gut mucosa by using 4-color immunofluorescence and confocal microscopic analysis. Based on our previously obtained single cell RNA-sequencing transcriptional analysis of tonsil ILCs we detected vitamin D receptor (VDR) expression in ILC3, which was highly significantly upregulated in IL-23- and IL-1β-activated ILC3. Vitamin D is risk factor for IBD and CRC, while VDR-deficient mice develop more severe colitis.
In WP2.1 we evaluated the functional consequences of VDR stimulation on tonsil ILC3 and described the cellular mechanism of the vitamin D-induced reprograming of ILC3 activity. In particular, we observed downregulation of the IL-23 receptor (IL-23R) pathway, including the ILC3-specific transcription factors Ahr and RORγt, with dramatic loss of the IL-23-driven IL-22, IL-17F and GM-CSF cytokine production of ILC3. Besides downregulation of the IL-23R pathway, vitamin D simultaneously upregulated key components of the IL-1β-pathway, which skewed ILC3 cytokine secretion towards a more innate cell profile including production of IL-6, IL-8 and MIP-1α/β.
In WP2.a we observed that IBD patients with ongoing inflammation are vitamin D-deficient, while IBD patients in remission have normal vitamin D levels. This proposed that vitamin D might play important roles in the inflammatory status of IBD patients with a likely direct influence on ILC3 activity. Hence, we further investigated the impact of vitamin D on intestinal ILC3 function. After IL-23 and IL-1β stimulation, gut cell suspensions derived from biopsies of IBD patients secreted high amounts of IL-22, which was greatly inhibited by co-incubation with vitamin D. The NKp44+ ILC3 appeared as major cellular source of intestinal IL-22 in response to the cytokine stimulation. Furthermore, by sorting ILC3 from non-inflamed gut biopsies we could confirm our findings in WP2.b. Vitamin D reprogrammed the gut ILC3 cytokine production by suppressing the IL-23-driven IL-22 and GM-CSF production and at the same time it induced the IL-1β-triggered production of IL-6 cytokine.
In WP3 we analyzed the heterogeneity of human ILC3 that is highly relevant for ILC3 function in the gut. Based on the single cell RNA-sequencing analysis of tonsil ILC3, we examined the functional diversity of human ILC3 subpopulations. Importantly, we described three phenotypically distinct ILC3 subpopulations based on expression of NKp44, CD62L and HLA-DR markers. These newly described ILC3 subpopulations showed diverse pattern of inflammatory cytokine production, with the NKp44+ ILC3 subpopulation being the most multi-functional and the CD62L+ ILC3 subpopulation the least responsive subset. Importantly, we identified the HLA-DR+ ILC3 cells as major source of IL-17F production. These data point out the previously unappreciated transcriptional and functional heterogeneity of human ILC3. Notably, we were also able to show expression of these cytokines in gut ILC3 subsets reflecting the high functional diversity of tonsil ILC3.
Exploitation
In the GutILC3 project we set a base for a possible therapeutic role of vitamin D in IBD treatment. Beyond this project together with our clinical collaborators we are planning to set up an interventional clinical study for vitamin D supplementation of IBD patients. In this future study we will directly assess intestinal ILC3 function and will associate it to the benefits of vitamin D treatment in the pathogenesis of IBD.
Dissemination
All findings obtained in the frame of the GutILC3 project had been disseminated. Data had been presented at internal and joint meetings with collaborators as well as at international conferences and high quality immunology meetings. All findings have been communicated in peer-reviewed highly ranked journals in the field of immunology. There are 2 first-authored review articles, 1 top 10 first-authored original article (J Allergy Clin Immunol) and 1 top 10 co-authored original article (Nat Immunol) resulting from the GutILC3 project.
