Interleukin-23 receptor signaling in regulatory T cells in a mouse model of inflammation-associated cancer.

The pro-inflammatory cytokine interleukin-23 (IL-23) has been implicated in colorectal carcinogenesis (CRC). Yet, the cell-specific contributions of IL-23 receptor (IL-23R) signaling in CRC remain unknown. One of the cell types that highly expresses IL-23R are colonic regulatory T cells (Treg cells). The aim of this study was to define the contribution of Treg cell-specific IL-23R signaling in sporadic and inflammation-associated CRC. In mice, the role of IL-23R in Treg cells in colitis-associated cancer (CAC) was investigated using azoxymethane/dextran sodium sulphate in wild-type Treg cell reporter mice (WT, Foxp3YFP-Cre), and mice harboring a Treg cell-specific deletion of IL-23 (Il23rΔTreg). Il23rΔTreg mice had increased dysplasia compared to WT mice associated with decreased tumor-infiltrating macrophages. The role of Treg cell IL-23R in sporadic CRC was examined via orthotopic injection of the syngeneic colon cancer cell line MC-38 submucosally into the colon/rectum of mice. In the sporadic cancer model, Il23rΔTreg mice had increased survival and decreased tumor size compared to WT mice. Additionally, MC-38 tumors of Il23rΔTreg mice had a higher frequency of pro-inflammatory macrophages and IL-17 producing CD4+ T cells. This data suggests that loss of IL-23R signaling in Treg cells permits IL-17 production by CD4+ T cells that in turn promotes pro-inflammatory macrophages to clear tumors. These findings further support and highlight the importance of selecting a physiologically relevant model based on the type of cancer in which to evaluate the role of specific genes in late tumorigenesis. Finally, single-cell RNA-seq analysis of a previously published dataset in human sporadic cancer, revealed that IL23R was highly expressed in CRC compared to other cancers and specifically in tumor-associated Treg cells. C57BL/6 Foxp3YFP-Cre (The Jackson Laboratory, Bar Harbor, ME, stock #016959, designated WT) and ll23rΔTreg (designated KO), lacking IL-23R in FOXP3+ Treg cells, were used. Inflammation-associated carcinogenesis: 7-8-week-old mice were co-housed, bedding was mixed 14 days prior to the start of the protocol, and 3 days prior to start of the protocol mice were placed on deprivation caps to acclimate mice to drinking from water bottles exclusively. Mice were intraperitoneally injected with 10 mgkg-1 azoxymethane (Sigma-Aldrich) and exposed to three 5-day cycles of 3.5% dextran sodium sulphate (TdB consultancy, Uppsala, Sweden). Each DSS cycle was followed by a 16-day recovery period of regular autoclaved water. At the experimental endpoint, tumor and adjacent normal (full thickness colon) was collected for RNA-sequencing.