IL10 Secretion Endows Intestinal Human iNKT Cells with Regulatory Functions Towards Pathogenic T Lymphocytes

Abstract

Background and aims Invariant Natural Killer T (iNKT) cells perform pleiotropic functions in different tissues by secreting a vast array of pro-inflammatory and cytotoxic molecules. However, the presence and function of human intestinal iNKT cells capable to secrete immunomodulatory molecules such as IL-10 has never been reported so far. Here we describe for the first time the presence of IL10-producing iNKT cells (NKT10 cells) in the intestinal lamina propria of healthy individuals and of Crohn’s Disease (CD) patients. Methods Frequency and phenotype of NKT10 cells were analyzed ex-vivo from intestinal specimens of Crohn’s disease (n=17) and controls (n=7). Stable CD-derived intestinal NKT10 cell lines were used to perform in vitro suppression assays and cocultures with patients'-derived mucosa-associated microbiota. Experimental colitis models were performed by adoptive cell transfer of splenic naïve CD4+ T cells in the presence or absence of IL10-sufficient or deficient iNKT cells. In vivo induction of NKT10 cells was performed by administration of SCFA by oral gavage. Results Patient’s derived intestinal NKT10 cells demonstrated suppressive capabilities towards pathogenic CD4+ T cells. The presence of increased proportions of mucosal NKT10 cells associated with better clinical outcomes in CD patients. Moreover, an intestinal microbial community enriched in SCFA-producing bacteria sustained the production of IL10 by iNKT cells. Finally, IL10- deficient iNKT cell failed to control the pathogenic activity of adoptively transferred CD4+ T cells in an experimental colitis model. Conclusions These results describe an unprecedent IL10-mediated immunoregulatory role of intestinal iNKT cells in controlling the pathogenic functions of mucosal T-helper subsets and in maintaining the intestinal immune homeostasis.

Harnessing invariant natural killer T cells to control pathological inflammation

“…Alternatively, if human iNKT cells generally retain functional plasticity, it may be important to identify methods to specifically promote their regulatory functions. To this end, a recent study found that the presence of IL-7 during in vitro expansion of human iNKT cells resulted in a CD4 + population with enhanced T H2 cytokine production ( 106 ), while exposure to short chain fatty acids, palmitate, or the mTOR inhibitor rapamycin may induce a regulatory phenotype ( 27 , 37 , 107 ). Another important consideration is that iNKT immunotherapy that engages T H2 pathways would likely be contraindicated in certain inflammatory diseases, including asthma, chronic obstructive pulmonary disease, and ulcerative colitis, where T H2 cytokine production by iNKT cells has been associated with disease-exacerbating effects (reviewed in ( 108 – 110 )).…”

“…Analyses of human iNKT cells have suggested that they may participate in similar regulatory processes. IL-10 producing iNKT cells recently identified in the intestinal lamina propria of Crohn’s Disease patients showed suppressive activity towards pathogenic CD4 + T cells, and the frequency of IL-10 producing iNKT cells in colon tissue of these patients correlated inversely with T H1 and T H17 cell frequency, and was associated with reduced disease severity, higher TGFB gene expression, and lower levels of inflammatory proteins ( 37 ). Moreover, co-culture of human T regs with iNKT cells led to increased T reg FOXP3 expression, enhanced IL-10 secretion, and more profound inhibition of conventional T cell proliferation ( 38 ).…”

Tumor infiltrating iNKT cells sustain neutrophil pro-tumorigenic functions influencing disease progression in human colorectal cancer

“…Single-cell suspensions were prepared from the colon of C57BL/6, Traj18 -/- and CD1d -/- mice as previously described 9 . Briefly, cells were isolated via incubation with 5 mM EDTA at 37°C for 30 min, followed by mechanical disruption with GentleMACS (Miltenyi Biotec).…”

“…Intestinal mucus scraped from the human and murine colons were stored at -80°C until DNA extraction. DNA extraction, 16S rRNA gene amplification, purification, library preparation and pair-end sequencing on the Illumina MiSeq platform were performed as previously described 9 . Reads were pre-processed using the MICCA pipeline (v.1.7.0) (http://www.micca.org) 48 as previously described 9 .…”

“…iNKT cells are present also in the intestinal lamina propria as tissue resident cells and variations in the gut microbiota composition can rapidly alter their phenotype 1,6 . Dysbiosis imprints iNKT cells toward a pro-inflammatory phenotype 7 whereas normobiosis restoration upon fecal microbiota transplantation 8,9 and exposure to Short Chain Fatty Acids (SCFA) 8,9 induce their production of regulatory cytokines, such as IL10. Along with the patrolling of tissue integrity, iNKT cells actively participate in the immune surveillance against malignant transformation and tumor progression, including human colorectal cancer (CRC) 10 .…”