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  • Bay 11-7821BAY 11-7082 To ascertain whether increased IL A I

    2020-08-30

    To ascertain whether increased IL-17A/IL-22 response in tu-mors in mice lacking IL-1R1 in myeloid Bay 11-7821BAY 11-7082 was driven in part by increased IL-1 action in T cells and increased IL-17A produc-tion, we created CD11bCre-CD4Cre-Il1r1f/f ‘‘double Cre’’ mice and then generated CRC-bearing BM chimeras. Additional ablation of IL-1R signaling in T cells reduced the heightened tumorigenesis observed in myeloid Il1r1 / mice and reduced the production of pro-tumorigenic IL-17A (Figures S5F and S5G). Moreover, in independent azoxymethane/dextrane sulfate sodium (AOM/DSS) induced model of colitis-associated cancer CD4Cre+Il1r1f/f mice displayed lower tumorigenicity, while CD11bCre+Il1r1f/f mice showed higher tumorigenicity in comparison with controls (data not shown).
    To independently confirm protective role of IL-1R1 in myeloid cells, we next generated LysMCreCre/WT (referred to as LysMCre+) and LysMCre- Il1r1f/f (control) mice and used their BM to create chimeric CDX2ERT-Apcf/f mice, to delete IL-1R1 using an overlapping Cre-deleter. LysMCre, similarly to CD11bCre, excised Il1r1 in monocytes, macrophages, and neu-trophils (Figures S5A and S5B). LysMCre+Il1r1f/f->CDX2ERT-Apcf/f mice also developed more CRC (Figures 5A and 5B). The difference was even more notable when myeloid IL-1R1-deficient mice and controls were housed separately as co-
    housing typically allows microbiota equalization (Figures 5A and 5B). LysMCre+Il1r1f/f->CDX2ERT-Apcf/f mice also demon-
    strated increased mRNA expression of Il17a, Il22, and Ptgs2 (Figure 5C), as well as bacterial 16S rRNA, E. coli, and SFB in tu-mors (Figure 5D). Moreover, Yo-Yo 1 DNA staining of whole-mount colons revealed an increase in bacteria located on the im-mediate surface of the tumor (Figure 5E).
    As CRC load in genetically identical animals was affected by their housing status and bacteria attached to the tumors of myeloid Il1r1 / mice, we next characterized the microbial alter- 
    ation induced by the absence of IL-1R in myeloid cells. We per-formed Illumina MiSeq 16S rRNA sequencing on DNA samples from the colonic contents of naive mice, co-housed and sepa-rated tumor bearing mice, as well as from the epithelial ‘‘shakes’’ (mechanical isolation of epithelium-adherent bacteria) of normal and tumor tissue from control and LysMCre+Il1r1f/f mice. Prin-cipal component analysis (PCA) demonstrated that myeloid specific IL-1R ablation did not lead to global changes in fecal mi-crobiota in naive mice (Figure 5F). This was largely confirmed by 16S qRT-PCR of naive colonic tissue (Figure S5H). CRC forma-tion did induce differences in fecal microbiota between wild type and myeloid Il1r1 / mice, but that difference was not evident if mice were co-housed (Figure 5F). On the other hand, significant differences were detected in the microbiota composition of bac-terial adherent to the tumor tissue, but not to the normal tissue in LysMCre+ mice (Figures 5F and 5G). PERMANOVA analysis es-tablished that the only differences between LysMCre+Il1r1f/f mice and controls mice was in tumor-adherent bacteria and in fecal microbiota, when mice were housed separately (Figure 5G). Among increased bacteria we found Escherichia-Shigella genera, represented by E. coli in the tumor-adherent fraction of myeloid cell specific Il1r1 / mice (Figures 5H and 5I), also de-tected by qRT-PCR analysis of tumor tissue (Figures 4G and 5D). It was likely that differences in fecal but not in tumor-asso-ciated microbiota could be mitigated by co-housing. Together, these suggested that myeloid IL-1R signaling repressed bacte-rial invasion and tumor-associated inflammation.
    Neutrophil Specific IL-1R Signaling Controlled Bacterial Invasion and Tumor Elicited Inflammation
    To address whether IL-1R expression by monocytes/ macrophages or by neutrophils control CRC, we first targeted
    IL-1R1 in monocytes and macrophages by injecting BM from CX3CR1CreCre/WT(Cre+)Il1rf/f or CX3CR1CreWT/WT(Cre-)Il1rf/f
    control mice into CDX2ERT-Apc recipients. We did not detect any differences in tumor number or size (Figures S6A and S6B). Next, using Ly6GCre strain, we inactivated IL-1R1 specif-ically in neutrophils. Similar to total myeloid Il1r / , mice lacking IL-1R1 only in neutrophils demonstrated an increase in CRC when IL-1R-deficient and control mice were co-housed. Even stronger acceleration of CRC was detected when mice were housed separate (Figures 6A and 6B). Il17a, Il22, and Ptgs2 expression in tumors, as well as Il1a and Il1b expression in monocytes was increased in the absence of neutrophil-specific IL-1R signaling (Figures 6C and 6D) as was protein expression of both IL-17A and IL-22 (Figures 6E–6G). We also detected an association of bacteria with CRC tumors (Figures 6H and 6I) and an increased presence of distinct bacteria, including