Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - GutILC3 (Cell-cell interactions critical to ILC3 function in the human gut)

Teaser

The overall goal of the GutILC3 project was to advance our understanding of group 3 innate lymphoid cells (ILC3) function in inflammatory bowel disease (IBD). The exact etiology of IBD is unknown, however it is considered to result from an inappropriate inflammatory response...

Summary

The overall goal of the GutILC3 project was to advance our understanding of group 3 innate lymphoid cells (ILC3) function in inflammatory bowel disease (IBD). The exact etiology of IBD is unknown, however it is considered to result from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Currently, only subgroups of patients benefit from existing therapies. The two major forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC), both conferring a dramatically increased risk for development of colorectal cancer (CRC). These intestinal diseases constitute a significant economic and health burden in Europe.
In recent years, it has become clear that the inflammatory axis of IL-23–IL-17/IL-22 cytokines plays a crucial role in the intestinal mucosal inflammation. The recently described ILCs have been reported to be key players in mucosal homeostasis and inflammation. The IL-23- and IL-1β-responsive ILC3 produce IL-17 and IL-22 cytokines. Strikingly, NKp44+ ILC3 are crucial in maintaining gut homeostasis by secreting IL-22, while the IL-17-producing NKp44- ILC3 are present in human IBD and cause inflammation in a mouse model of colitis.
There is an urgent need for novel therapeutic strategies in order to improve outcomes of IBD. The GutILC3 project made conceptual advancements in targeting intestinal ILC3 activity under inflammatory condition and investigated the functional heterogeneity of the various ILC3 subpopulations. Outcomes of the project will impact the European society by proposing measures for new therapeutic targets in intestinal immune-pathologies like IBD in humans.


Specific objectives of GutILC3 are to:

investigate cellular function of human ILC3 derived from tonsils and gut
pinpoint crucial molecules or processes for targeting ILC3 function
interrogate the phenotypic and functional heterogeneity of human ILC3 subsets
set a base for novel therapeutic approaches in the treatment of IBD by directly targeting ILC3 activity

Work performed

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 (

Final results

In the GutILC3 project we identified vitamin D as a novel modulator of human intestinal ILC3 function by downregulation of the IL-23R pathway on ILC3. The IL-23R pathway, including IL22, IL17A and IL17F genes, is one of the most extensively studied risk pathways for IBD and were found upregulated in colonic mucosa both in CD and ulcerative colitis. Indeed, targeting IL-23/IL-12 by ustekinumab is beneficial for patients with CD who failed to respond to anti-TNF therapy. In the frame of the GutILC3 project we show evidence for the first time that vitamin D downregulates the IL-23-responsiveness of intestinal ILC3. Hence, vitamin D is expected to exert similar therapeutic effects as ustekinumab. This finding will potentially impact the European society as it proposes therapeutic targeting of ILC3 in IL-23-driven inflammatory diseases such as IBD, CRC and psoriasis. Thus, it projects wider societal implications of vitamin D supplementation that could be beneficial in treating these inflammatory diseases by directly interfering with the ILC3 function.
Additionally, we described functional heterogeneity of the newly identified human ILC3 subpopulations. This progress projects beyond state of the art as it provides new incitements about the complexity of human ILCs. This could be exploited by selective targeting of the various human ILC3 subsets in intestinal inflammatory diseases such as IBD.