Role of Retinoic Acid signaling in cDC1 and cDC2 on intestinal immune homeostasis and disease

Informations projet

Retinoic AC-DC

N° de convention de subvention: 844950

DOI

10.3030/844950

Projet clôturé

Date de signature de la CE 10 Avril 2019

Date de début 1 Janvier 2020

Date de fin 25 Avril 2022

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 191 852,16

Contribution de l’UE

€ 191 852,16

Coordonné par

LUNDS UNIVERSITET
Sweden

Periodic Reporting for period 1 - Retinoic AC-DC (Role of Retinoic Acid signaling in cDC1 and cDC2 on intestinal immune homeostasis and disease)

Période du rapport: 2020-01-01 au 2021-12-31

Résumé du contexte et des objectifs généraux du projet

The intestinal mucosa is the largest surface of the body in contact with the external environment and is constantly exposed to foreign antigens and metabolites derived from the diet and resident microbiota. The intestinal microbiota is essential to our health, and continual and mutualistic dialogue between the microbiota and the intestinal immune system is required to maintain tissue homeostasis. Breakdown in such crosstalk, for example from nutritional deficiencies or microbiota alterations (dysbiosis), can rewire immune cell development and function, contributing to Inflammatory Bowel Disease (IBD).
Conventional dendritic cells (cDC) are the key antigen presenting cells of the immune system, essential for the initiation and regulation of adaptive immune responses. cDC comprise of two major subsets, cDC1 and cDC2 and we have previously shown that intestinal these subsets play distinct non-redundant roles in intestinal adaptive immune responses. It is also becoming increasingly clear that the phenotype and function of cDC is regulated by signals these cells receive within their local tissue environment. Identifying such environmental factors and the underlying mechanisms by which they regulate cDC function may provide opportunities to regulate cDC dependent immune responses for the benefit of human health.
The dietary derived Vitamin A metabolite, retinoic acid (RA) is a major immune modulator, in particular in the small intestine, where its concentrations are highest. We have shown that cDC receive RA signals within the small intestine however the impact of such signaling on cDC transcription and function is unclear. The overall objective of the project was to understand the impact of RA signaling on intestinal cDC1 and cDC2 transcription, phenotype and function in vivo and its importance in small intestinal immune homeostasis.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

In the initial stages of the project we generated mice that lacked RA signaling either in cDC1 or in cDC2 (Fig1). Mice whose cDC1 could not respond to RA signaling had normal numbers of intestinal cDC and normal adaptive immune cell composition throughout the small intestine and intestinal draining mesenteric lymph nodes (MLN). For this reason we focused the remaining part of the project on assessing mice that lacked RA signaling in small intestinal cDC2.
Mice that lacked RA signaling selectively in small intestinal cDC2 had increased numbers of activated CD4+ T cells in MLN in steady state. Consistent with this finding, we found that cDC2 isolated from the MLN of these mice were more efficient at priming CD4+ T cells in vitro compared with wildtype MLN cDC2. Thus, tonic RA signaling in intestinal cDC2 appears to regulate their capacity to prime CD4+ T cells.

To identify potential mechanisms underlying the enhanced CD4+ T cell priming capacity of intestinal cDC2 that could not respond to RA, and the broad impact of RA signaling on small intestinal cDC2, we performed bulk RNA-seq on intestinal cDC2 isolated from mice whose cDC2 could not respond to RA and compared these with intestinal cDC2 isolated from wildtype mice. Using this approach we identified numerous transcriptional differences between small intestinal cDC2 that had and had not received an RA signal in vivo. This approach also lead to the identification of candidate genes (some of which we confirmed at the protein level) that were upregulated in cDC2 in the absence of RA signaling, that might underlie their increased capacity to prime CD4+ T cells.

Interestingly, one candidate gene was expressed by a small proportion of intestinal cDC2 in wildtype mice but by a much larger proportion of cDC2 in mice whose cDC2 were unable to receive an RA signal (Fig2). Sorting of wildtype cDC2 based on surface expression of this protein in combination with in vitro T cell priming assays demonstrated that cDC2 expressing this protein were far more efficient at priming CD4+ T cells in vitro (Fig3). Blocking the activity of this surface protein in vitro had no impact on CD4+ T cell priming indicating that expression of this protein did not underlie their increased capacity to drive CD4+ T cell activation but rather was a marker for cDC2 that had an increased capacity to drive CD4+ T cell activation.

Collectively, the results of the current project demonstrate that tonic RA signaling in small intestinal cDC2 has a marked impact on the transcriptional profile of these cells that is associated with a reduced capacity to drive CD4+ T cell proliferation. Mechanistically, we have identified a marker that distinguishes a subset of small intestinal cDC2 present in wildtype mice that have an increased capacity to drive CD4+ T cell proliferation and found this population to be markedly increased in mice whose cDC2 are not capable of responding to RA (Fig3). Our results suggest that RA signaling in cDC2 may promote a tolerogenic phenotype in these cells and suggest that RA may be a potential target for regulating cDC2 function, for example in cDC centric therapeutic approaches. Future studies aim to identify the underlying pathways driving such RA dependent responses in cDC2.

The results of this project are currently being compiled for publication in an open access journal to ensure efficient dissemination to the scientific community. Results have been presented at regular intervals during the project to the group at laboratory meetings and at Immunology section meetings within the Department. Dissemination at conferences was not possible due to the COVID pandemic.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

Not applicable

Transgenic mouse models

Figure 3: Subpopulation of cDC2 in absence of RA is more potent to induce CD4+ T cell proliferation

Figure 2: Impact of RA deprivation on cDC2 at steady state