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  • br Analysis of epithelial specific IL R deletion revealed a


    Analysis of epithelial-specific IL-1R1 deletion revealed a decrease in CRC tumor multiplicity, slower proliferation of early tumor seeds, and decreased activation of NF-kB. NF-kB is an essential regulator of cell survival and proliferation, and its inac-tivation in intestinal epithelial VX765 by deletion of IKKb kinase during the development of colitis-associated cancer decreases tumor multiplicity (Greten et al., 2004). These observations are in line with reports that IL-1a and IL-1b stimulate growth in colon cancer cell lines in vitro and in xenograft transplants in vivo (Voronov et al., 2003; Holen et al., 2016). While NF-kB can be activated by a plethora of stimuli, IL-1R signaling may be essen-tial for activating NF-kB in CRC because IL-1a and IL-1b are rapidly released by transformed and stressed cells, as well as by cells stimulated with microbes infiltrating early tumors. IL-1 signaling in epithelium regulated early CRC, while being dispens-able for the expression of inflammatory cytokines, indicating that IL-1 signaling controlled CRC in an inflammation-independent manner.
    In T cells, IL-1 signaling regulated CRC in an inflammation-dependent manner. Lymphoid cells, including T cells and ILC3, are important producers of IL-17A and other TEI cytokines dur-ing host defense and chronic inflammation. These lymphoid cells infiltrate CRC tumors and their increased presence correlates with poor prognosis in CRC patients, according to CRC ‘‘Immu-noscore’’ (Bindea et VX765 al., 2013; Tosolini et al., 2011). Here we found that IL-1 signaling in T cells was essential for the produc-tion of IL-17A and IL-22 cytokines in CRC tumors, consistent with what has been observed in other inflammatory conditions in mice and humans (Shaw et al., 2012; Zhou and Littman,
    (F–I) 16S RNA sequencing of fecal, normal tissue and tumor adhesive bacteria isolated from naive and tumor bearing LysMCre+Il1rf/f and control mice, which were either co-housed or separately housed.
    (F) Principal component analysis of different groups of bacteria.
    (H) Heatmap of altered (p < 0.05) bacteria in tumor-adhesive fraction of LysMCre+Il1rf/f mice, ‘‘red’’-overrepresented, ‘‘blue’’- underrepresented.
    (I) Specific representation of E. coli bacteria in different groups, N = 5. See also Figure S5.
    Figure 6. IL-1R Signaling in Neutrophils Is Essential for Control of Bacterial Invasion and Excessive Inflammation in CRC
    CRC analysis in CDX2ERT-Apcf/f mice transplanted with BM from Ly6GCre+Il1rf/f or Ly6GCre- controls.
    (A) Macroscopic tumor multiplicity, size, and load analysis upon necropsy 5–6 weeks after last tamoxifen injection. N = 7, *p < 0.04 ***p < 0.001; data for co-housed or separated by genotype mice is shown separately.
    (B) Representative images of CRC bearing colons.
    (H) Increased bacteria (arrows) associated with tumors. Yo-Yo-1 staining of whole tumor-bearing colon tissue, quantified in (I), N R 5.
    (J) qRT-PCR analysis of CRC tumor lysates for specific bacterial content with 16S rRNA specific primers; normalized to RpL32 expression, N = 17 *p < 0.05. Data are means ± SEM. Representative of 3 independent experiments. See also Figures S6 and S7.
    Figure 7. IL-1R Signaling in Neutrophils Controlled Anti-Bacterial Response
    (A and B) FACS analysis of tumor-infiltrating neutrophils from LysMCre+Il1rf/f or control BM transplanted CDX2ERT-Apcf/f mice. (A) Representative FACS plots and (B) quantification, N = 5.
    (C) Splenic neutrophils isolated from LysMCre+Il1rf/f or control BM transplanted tumor bearing mice were treated with IL-1b and incubated with fecal bacteria from tumor-bearing mice. Representative images of anaerobic (left) and aerobic (right) colonies on blood agar plates.
    (D and E) Neutrophils derived from BM and peritoneal cavities of LysMCre+Il1rf/f or control mice and neutrophils from spleen of tumor bearing mice (D), intra-tumoral neutrophils from CRC bearing mice with Ly6GCre+Il1rf/f or control BM (E) were stimulated with IL-1b and incubated with E. coli for 1 hour, followed by bacteria plating. Quantification of CFU on agar plates after serial dilutions. N R 3.
    (F) Representative confocal images of peritoneal neutrophils (‘‘red’’ Ly6G) ingesting Salmonella-GFP (‘‘green’’).
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    The novel observation of this work was that IL-1 signaling in myeloid cells is anti-tumorigenic, at least in the context of tumors growing at ‘‘microbial-rich’’ surfaces. IL-1R signaling in myeloid cells kept specific species/genera of tumor infiltrating microbes at bay and prevented local, tumor-specific dysbiosis and excessive amounts of pro-tumorigenic inflammatory cytokines. We propose that increased infiltration of ‘‘bad’’ (pro-tumorigenic) and a decrease in ‘‘good’’ (anti-tumorigenic) gut bacteria led to an increased bacterial recognition by myeloid cells and production of IL-17A, which induces IL-1b and IL-23, along with other tumor-promoting inflammatory entities, which drive CRC. 16S rRNA sequencing of the microbiome had shown that the observed dysbiosis did not occur in naive mice and was induced in a tumor-specific manner.