Patient suffering from inflammatory bowel diseases (IBD) manifest an increased risk of developing colitis-associated colon cancer (CAC). Interleukin 22 (IL22) palys an important function in restoring IBD status. However, given its ability to induce epithelial cell proliferation, IL22 correlates with the development of tumoral lesions. IL22 is secreted by different immune cell types, including iNKT cells, but their function in in this context is still highly debated.
Here, we studied the role of IL22-secreting iNKT cells during gut chronic inflammation and CAC development and the stimuli responsible for iNKT cells’ IL22 secretion. Lamina propria mononuclear cells from a discovery cohort of 27 CAC patients and controls (IBD and sporadic CRC) were isolated from surgical specimens obtained from the IRCCS Policlinico Hospital, Milan (Italy). Multiparametric flow cytometry analyses, RNA and protein levels were evaluated through qPCR and ELISA and mucus-associated microbiota composition was established through 16S rRNA gene sequencing. Stable human intestinal- and peripheral blood-derived iNKT cell lines and C57bl/6 mice were used to evaluate the molecular mechanisms involved in dictating iNKT cells to produce IL22. Metabolomic analyses were performed to identify the molecules involved in this differentiation. To spatially define iNKT cells phenotype, gene expression and function within or in proximity of cancer cells spatial proteomics and transcriptomics were performed by Cosmix (Nanostring, Seattle) on FFPE tissues of 20 CAC patients and controls.
Collectively, we observed increased expression of IL22 and a higher NKT22 infiltration in CAC tissues, as well as an enrichment of Odoribacter, a gram-negative bacteria implicated in tryptophan metabolism and in Aryl hydrocarbon receptor (AhR) ligands production. We demonstrated that stimulation of iNKT cell lines with Odoribacter-derived metabolites induced IL22 production by iNKT cells and that this secretion is dependent on AhR function.
Metabolomic analyses revealed increased presence of few tryptophan metabolites in the Odoribacter supernatant. These metabolites were capable of increasing NKT22 differentiation both in vitro and in vivo models. The administration of Odoribacter Supernatant or the single purified metabolites were sufficient to increase tumorigenesis in C57bl/6 colorectal cancer mouse models but not in iNKT-deficient TRAJ18KO mice. Spatial Trascriptomic analyses confirmed the pro-tumorigenic function of NKT22 cells in close proximity of cancer cells.ì, but not of untrasformed epithelail cells or in IBD samples.
These data suggest that iNKT cells are induced by the recognition of microbiota-derived metabolites implicated in tryptophan metabolism and Aryl hydrocarbon receptor (AhR) stimulation. NKT22-microbiota interaction in CAC patients plays a potential crucial role in tumor development.

Alberto Baeri

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