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    2020-08-30

    r> Enhancement of Th17 inflammatory responses could not be explained by the lack of direct action of IL-1 on myeloid cells,
    Figure 2. IL-1R in Colonic Epithelial Cells Plays a Direct Pro-Tumorigenic Role Independent of Inflammation
    CRC development in 6-month-old Il1r D/F-CPC-APC (epithelial deficiency) and Il1r D/WT (control) mice.
    (A) Representative macroscopic images of tumor-bearing colons.
    (B) Microphotographs of H&E stained colons of indicated genotypes.
    (C) CRC tumors were assessed macroscopically upon necropsy of mice with epithelial IL-1R deficiency and controls, N R 21, ***p < 0.0001.
    (E) Representative images of Ki-67 staining in tumors at 3 months of age.
    (G) CRC tumors of 3-month-old Il1r D/F-CPC-APC and Il1r D/WT control mice, N = 8 *p < 0.01. (H–J) CRC analysis in Il1rf/f-CDX2ERT-Apcf/f and Il1rf/WT control mice, injected with tamoxifen, and allowed to develop tumors for 5–6 weeks. (H) Representative images of CRC bearing colons. (I) Tumor analysis N R 5, *p < 0.05. (J) Western Blot analysis of nuclear lysates of intestinal epithelial tumor Bay 11-7821BAY 11-7082 (IECs) from indicated Il1r-CDX2ERT-Apcf/f mice for NF-kB (p65) expression. Loading was normalized to histone H3. Each lane shows tumors from individual mice. Data are mean ± SEM. Representative of 3 independent experiments. See also Figure S3.
    Figure 3. IL-1R Signaling in T Cells Promotes IL-17A Dependent TEI and CRC
    CPC-APC mice were transplanted with BM from CD4Cre+Il1rf/f or control mice and allowed to develop CRC for 4.5 months, representative of 3 experiments with 6–8 mice each
    (A) Macroscopic images of CPC-APC mice transplanted with CD4Cre+-Il1rf/f or control BM.
    (B) H&E stained CRC bearing colonic rolls.
    (E) Multiplex ELISA analysis of tumor culture supernatants (24 hr) for IL-17A and IL-22 proteins.
    (F) Representative FACS plot (N = 5) of LPL tumor fraction from RorcGFP-CPC-APC reporter mice treated with Anakinra or PBS (control) for 3 days. (G) Representative IF images (N R 5) of CRC tumors stained for Ki-67.
    (H and I) IHC staining of paraffin-embedded CRC tumors for p-STAT3.
    as myeloid cells did not directly contribute to IL-17A production (Figure S1D). We therefore reasoned that IL-1R1 deficiency in myeloid cells triggered additional stimuli promoting IL-17A pro-duction. As various intestinal microbial species are essential for induction of IL-17A (Ivanov et al., 2009; Wu et al., 2009), we tested whether alterations in CRC-associated microbes were responsible for the heightened CRC. qRT-PCR for generic bacterial 16S rRNA revealed an increased bacteria presence in 
    tumors from mice lacking IL-1R1 on myeloid cells (Figure 4G) with specific increase in bacteria included Bay 11-7821BAY 11-7082 E. coli, Enterobacter-oides, and SFB (Figure 4G). We further tested whether short term IL-1 blockade with Anakinra (IL-1RA) would induce the same bacterial perturbations. Anakinra treatment for 12 days did increase bacterial infilitration of CRC tumors, particularly with E. coli (Figure S5C), but did not alter CRC tumorigenesis (Figures S5D and S5E).
    Figure 4. IL-1R Signaling in Myeloid Cells Is a Negative Regulator of Bacterial Invasion and TEI
    CDX2ERT-Apcf/f mice were transplanted with BM from CD11bCre+Il1rf/f (myeloid IL-1R deletion) or CD11bCre+Il1rf/w (heterozygous, control) mice, allowed to reconstitute BM for 2 months, injected with tamoxifen and analyzed in 6 weeks.
    (A) Representative images of CRC bearing colons from CDX2ERT-Apcf/f mice transplanted with indicated BM.
    (B) Representative microphotographs of H&E stained colon sections.
    (D) IF Ki-67 staining of paraffin-embedded CRC sections from indicated genotypes, representative of N R 5.
    (legend continued on next page)
    Tumor Associated Bacteria Are Essential for Driving Enhanced CRC in the Absence of IL-1 Signaling in Myeloid Cells
    To establish a functional and causative relationship between myeloid IL-1R1 inactivation, increased bacterial presence, inflammation and CRC, we treated CD11bCre+Il1r1f/f /CDX2ERT-Apcf/f and control mice with broad spectrum anti-biotics for 4 weeks, starting after the epithelial transformation (Figure 4H). Ablation of intestinal and tumor associated micro-biota resulted in a drastic reduction of CRC tumorigenesis in both control and myeloid IL-1R deficient mice (Figure 4I and 4K). Bacterial 16S RNA, SFB 16S RNA, and Il17a mRNA in tu-mors from myeloid IL-1R1-deficient mice treated with antibiotics were strongly reduced compared to the untreated group (Fig-ure 4K). Importantly, difference in CRC was no longer observed between antibiotic-treated control and myeloid IL-1R1 deficient animals (Figures 4J and 4K).