br In sporadic colorectal cancer CRC
In sporadic colorectal cancer (CRC), the breakdown of the epithelial barrier subsequent to oncogenic transformation leads to the translocation of bacterial products and triggers the interleukin (IL)-23–IL-17A signaling pathway (Grivennikov et al., 2012). Increased amounts of these cytokines within tumors por-tends poor prognosis in CRC patients (Tosolini et al., 2011) and promotes CRC in various animal models (Blatner et al., 2012; Gri-vennikov et al., 2012; Huber et al., 2012; Kirchberger et al., 2013). In humans and mice, however, not all tumors expressing high amounts of the pro-tumorigenic cytokine IL-23 have commensu-rately high IL-17A expression, implying that other regulators of TEI exist. Overall, a complete mechanistic understanding of TEI induction, its maintenance, and its contribution to tumorigen-esis is lacking.
IL-1 plays an essential role in inducing IL-17 production by T helper (Th17) ST 2825 in both humans and mice in the context of inflammation and autoimmunity (Coccia et al., 2012; Zhou and Littman, 2009). Approaches to block the IL-1 pathway are being used to treat inflammatory conditions such as rheumatoid arthritis, gout, and familial Mediterranean fever (Dinarello et al., 2012). Despite its well-known role in maintaining immune-medi-ated inflammatory responses, the role of IL-1 signaling as a regu-lator of TEI and CRC in autochthonous models is obscure. IL-1 signaling is implicated in the generation of both pro- or anti-tu-mor immune responses, as well as in the development of intes-tinal inflammation linked to tumorigenesis (Bersudsky et al., 2014; Malik et al., 2016; Voronov et al., 2003). A recent CANTOS trial testing the ability of the anti-IL-1b antibody canakinumab to prevent adverse cardiovascular events revealed a protective effect of systemic IL-1b inhibition in lung cancer, but not colon cancer (Ridker et al., 2017).
Figure 1. Global Inactivation of IL-1R1 Reduced IL-17A Responses but Only Minimally Affects CRC Tumorigenesis
(A) Il1a, Il1b, and Il17a mRNA expression was normalized to housekeeping RpL32 gene in normal colon and CRC tumors from CDX2 (CPC)-APC mice. N R 10.
(B) ELISA for IL-1a, IL-1b, and IL-17A protein concentration in ex vivo normal colon and tumor culture supernatants (24 hr incubation), N R 5.
(C–E) CRC tumors in CPC-APC-Il17aGFP mice treated with Anakinra or PBS control. (C) qRT-PCR analysis of Il17a, Rorc, and Il22 mRNA expression in tumors, N R 7. (D) Representative FACS plot (N R 5) of GFP expression. (E) Quantification of GFP+ cells in LPL and IEL fractions from CRC-bearing mice ± Anakinra. Gating strategy is indicated on the panels.
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Here, we used cell-type-specific inactivation of IL-1R1 to examine the impact of IL-1 on distinct cells within the CRC tumor microenvironment. IL-1 signaling controlled CRC development and progression by three distinct mechanisms. In epithelial cells, IL-1 signaling directly drove tumorigenesis without affecting in-flammatory responses. Deletion of Il1r1 in T cells blocked IL-17 and IL-22 production, reducing TEI and blunting CRC tumorigen-esis. These pro-tumorigenic effects of IL-1 were countered by IL-1 signaling in myeloid cells, specifically neutrophils. Deletion of Il1r1 in myeloid cells resulted in increased infiltration of bacte-ria into the tumor tissue, and the triggering of a pro-tumorigenic inflammatory response resembling tumor elicited inflammation. The complex effects of IL-1 in distinct cell type within the tumor microenvironment may underlie the distinct results in bacteria-rich and other cancers upon IL-1 blockade.
Increased IL-1a and IL-1b Expression in CRC Tumors Promotes IL-17A Responses
We operated under the premise that potential regulators of TEI should be increased in tumor tissue and should be able to control
processes associated with inflammatory responses in a variety of cell types. We used the CDX2Cre-Apcf/wt (CPC-APC) mouse
model of conditional monoallelic APC loss in the colon to induce CRC. We found elevated Il1a, Il1b, and Il17a gene expression in tumors compared to normal colon tissue (Figure 1A). Multiplex assay of tumor culture supernatants showed increased protein concentration of all three cytokines, compared to matched normal tissues (Figure 1B). Other members of the IL-1 cytokine family were detected in CRC (Figure S1A and S1B). FACS sorting of specific cell populations from normal and tumor tissue with subsequent qRT-PCR analysis revealed that monocytes are the main source of IL-1a and IL-1b (Figure S1C), although tumor epithelial and stromal cells also were producing IL-1 (Figure S1D).
Data from in vitro models of mouse and human Th17 cell differ-entiation, and from in vivo models of chronic inflammation, posi-tions IL-1 as an important regulator of Th17 differentiation and IL-17A production (Chung et al., 2009; Coccia et al., 2012). There-fore, we sought to examine whether IL-1 signaling is required for generating and maintaining the IL-17A TEI responses in CRC. To check whether pharmacological blockade of IL-1 signaling re-sulted in decrease of TEI cytokines in vivo, we injected reporter CPC-APC-Il17aGFP+/ mice i.p. with the recombinant IL-1R antagonist Anakinra (IL-1Ra) for 3 consecutive days. Two hours after the last injection, tumors and normal tissues were isolated and subjected to qRT-PCR and flow cytometry analyses. In tu-mor tissue, qRT-PCR analysis revealed a significant drop in mRNA expression for Il17a, its transcription factor Rorc and another TEI-related cytokine Il22 compared to normal colon (Fig-