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  • br Neutrophil Specific IL R Signaling Controlled


    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
    E. coli, inside CRC tumors (Figure 6J). No significant differences in bacteria adherent to the colonic epithelium in naive mice were
    (G) qRT-PCR for bacterial infiltration with primers specific for 16S rRNA from indicated bacteria, normalized to mouse housekeeping gene (RpL32), N = 18 *p < 0.05.
    (H) Scheme of experiment for antibiotic-mediated microbiota depletion. CD11bCre+Il1rf/f or control BM-chimeric CDX2ERT-Apcf/f mice were injected with tamoxifen and put on broad-spectrum Deferoxamine or regular water for 4 weeks.
    (I) Representative images of CRC bearing colons from mice treated or untreated with antibiotics.
    (J) Tumors upon necroscopy after 4 weeks of antibiotics treatment, N R 6.
    Figure 5. Myeloid IL-1R Signaling Prevents Tumor Associated Dysbiosis
    (A–I) CDX2ERT-Apcf/f mice were reconstituted with BM from LysMCre+Il1rf/f or Cre- (control) mice and CRC was induced with 2 injections of tamoxifen.
    (A) Macroscopic tumor multiplicity, size, and load analysis upon necropsy 6 weeks after last tamoxifen injection, for mice which are either co-housed or housed separately according to BM genotype. N R 5, p < 0.05.
    (B) Representative images of CRC bearing colons of BM transplanted mice
    (D) qRT-PCR analysis of CRC tumor lysates for specific bacterial content indicated; results are normalized to RpL32 expression N = 20, *p < 0.05.
    (E) IF images of adhesive bacteria (white arrows) associated with tumors from LysMCre+Il1rf/f or control mice. YoYo-1 and phalloidin staining of whole colon tissue from indicated mice. Representative of 3 independent experiments, each of at least 5 tumors.
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    detected (Figure S7A) Thus, increased infiltration of CRC tumors by bacteria in the absence of IL-1R signaling in neutrophils results in enhanced bacteria-driven inflammation and CRC tumorigenesis.
    IL-1R Signaling Controls Anti-Bacterial Functions of Neutrophils
    Neutrophil recruitment into the tumors was not affected by myeloid specific or neutrophil specific IL-1R ablation, while donor BM neutrophils exhibited profound deletion of exon 5 of the Il1r1 gene indicating efficient cell recruitment and cell spe-cific gene deletion (Figures S7B–S7D). (Figures 7A and 7B and S7B–S7D). We did not find any significant changes in the pres-ence of neutrophil extracellular traps (NET’s) in CRC tumors of LysMCre+Il1r1f/f and controls (Figure S7E). Also, ex vivo experi-ments did not detect significant differences in intracellular levels of bactericidal ROS in neutrophils (Figure S7F), although for these experiments intratumoral neutrophils undergo mechanical isolation possibly resulting in their activation. CD11b+Ly6G+ neu-trophils, enriched or sorted from spleens, bone marrow, thiogly-collate elicited peritoneal exudate or from CRC tumors, were able to kill various bacteria, including lab strain of E. coli and bulk fecal bacteria, upon IL-1b treatment. That ability was impaired when neutrophils were rendered IL-1R deficient (Fig-ures 7C–7E). We further found that the absence of functional IL-1R signaling led to a decreased ability of neutrophils to ingest zymosan particles or GFP-labeled Salmonella typhimurium bac-teria (Figures 7F–7I). Expression of antimicrobial peptides was also reduced in the absence of IL-1R in neutrophils (Figure 7J).