The MicroRNA miR-223 Constrains Colitis-associated Tumorigenesis by Limiting Myeloid Cell Infiltration and Chemokine Expression

Abstract

Aberrant intestinal inflammation plays a critical role in the development of colitis-associated colorectal cancer (CAC), yet the mechanisms controlling tumor development by the myeloid immune compartment are not fully understood. Although altered microRNA expression is observed in CAC, it is also unclear how myeloid-specific microRNAs impact the inflammatory process that underpins the continuum from ulcerative colitis to tumorigenesis. In this study, we report that miR-223 acts to limit myeloid-driven inflammation in the azoxymethane (AOM)-dextran sodium sulfate (DSS) model of CAC in mice. In this model, miR-223-/y mice present with significantly larger tumors with an enhanced proliferative signature. Immunoprofiling showed that miR-223-/y mice have significantly increased colonic myeloid immune infiltrate (neutrophils, monocytes, and macrophages) following AOM-DSS. This was accompanied by an increased inflammatory chemokine and cytokine signature for monocytes and neutrophils. Bone marrow chimera studies demonstrate that myeloid-expressed miR-223 is responsible for the enhanced tumor proliferation and inflammatory response. RNA sequencing identified several pathways that could be contributing to the development of CAC in miR-223-/y mice, including the IL-6/IL-17a cytokine family and STAT3 signaling. Lastly, neutrophil depletion with an anti-GR1 Ab (Ly6G/Ly6C) during the initial phase of the AOM-DSS model reduced the tumor burden in miR-223-/y mice. Collectively, our data indicate that miR-223 is an important regulator of mucosal inflammation and acts to constrain the progression from ulcerative colitis to CAC by limiting myeloid-associated inflammation.

Figure 1.. Increased tumor size and proliferative capacity in miR-223^−/y mice following AOM-DSS.

Schematic overview and timeline of the AOM-DSS study. A. number of macroscopic polyps per colon. Following H&E staining B. the number of tumors per section, C. quantification of dysplasia (μm²) per section, D. quantification of tumor size (μm²) per section was determined at the time of necropsy following treatment with AOM-DSS. E. Tumor grading according to size from H&E micrographs in WT or miR-223^−/y mice treated with chronic AOM/DSS F. Representative 20x H&E micrograph of a tumor from WT or miR-223^−/Y mice treated with chronic AOM/DSS. All data is expressed as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 versus the indicated counterpart (Unpaired, one-tailed students t-test). C + D: WT AOM-DSS: 16 tumors and 2 dysplastic regions counted from 17 mice; miR-223^−/Y AOM-DSS: 27 tumors and 9 dysplastic regions counted across 7 mice. Data is presented from 2 independent experiments. G. Western immunoblot assessment of PCNA, cyclin-D1, c-Myc, p-AKT^(Ser473), p-S6^(Ser235/236) and β-actin in colon biopsies (n=3 mice/group). H. Quantification of western immunoblots for PCNA, Cyclin D1 and c-Myc and p-AKT. All data is expressed as mean ± SEM; *, P<0.05; **, P < 0.01; ***, P<0.001 versus the indicated counterpart (One-way ANOVA). n=3 mice/group.

Figure 2.. Macrophage, neutrophils, and monocytes infiltrate into the colon of miR-223^−/y mice treated with chronic AOM-DSS.

A. Representative 20x micrographs of unaffected tissue and tumor of WT and miR-223^−/y mice treated with chronic AOM-DSS after immunohistochemical staining for Ly6G B. Quantification of Ly6G immunostaining. C. Representative 20x micrographs of unaffected tissue and tumor of WT and miR-223^−/y mice treated with chronic AOM/DSS after immunohistochemical staining for CD68 D. Quantification of CD68 immunostaining. E. Representative 20x micrographs of unaffected tissue and tumor of WT and miR-223^−/y mice treated with chronic AOM-DSS after immunohistochemical staining for CD206 F. Quantification of CD206 immunostaining. E. Representative 20x micrographs of healthy tissue and tumors WT and miR-223^−/y mice treated with chronic AOM-DSS after immunohistochemical staining for CCR2 F. Quantification of CCR2 immunostaining. All data is expressed as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 versus the indicated counterpart (One-way ANOVA). A+B. WT AOM-DSS n=16; miR-223^−/Y AOM-DSS n=4; C+D. WT AOM-DSS n=14; miR-223^−/Y AOM-DSS n=4; E+F. WT AOM-DSS n=11; miR-223^−/Y AOM-DSS n=4; G+H. WT AOM-DSS n=12; miR-223^−/Y AOM-DSS n=4; with 3 fields of view quantified per animal.

Figure 3.. miR-223^−/y mice have increased chemokine and cytokine signature for monocytes and neutrophils following AOM-DSS.

A. Relative mRNA expression of miR-223–3p. B. Relative mRNA expression of the pro-inflammatory macrophage markers nos2, tnf, il-1β and S100a8. C. Relative mRNA expression of the anti-inflammatory macrophage markers arg1, chi3l3, retnla, il-4, and irf4. D. Relative mRNA expression of the monocyte chemokines ccl2 and ccl7; neutrophil chemokines cxcl1 and cxcl2. E. Relative mRNA expression of the myeloid growth factors m-csf1, il-34 and gm-csf. All data is expressed as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 versus the indicated counterpart (One-way anova). A. n=5–6 mice/group, B-E. n=3–6 mice/group.

Figure 4.. miR-223^−/y mice present with an upregulation of pathways associated with poor prognosis in CAC.

A. Heat map of the Pearson correlation between all samples analysed via RNA sequencing. B. Venn diagram illustrating the overlap of mRNA shared between treatment groups. C. Volcano plot of differentially expressed mRNA between miR-223^−/y AOM-DSS vs WT AOM-DSS. D. Heat map of the top up-regulated differentially expressed mRNA associated with Inflammatory Bowel Disease between miR-223^−/y AOM-DSS vs WT AOM-DSS. E. Heat map of the most down regulated differentially expressed mRNA associated with Inflammatory Bowel Disease between miR-223^−/y AOM-DSS vs WT AOM-DSS. F. Enrichment plot for Inflammatory Bowel Disease (MMU05321) from GSEA analysis (NES: 1.209; FDR q-val: 0.473). G. Heat map of CRC associated mRNA that are up-regulated in miR-223^−/y AOM-DSS vs WT AOM-DSS. H. Heat map of CRC associated mRNA that are down-regulated in miR-223^−/y AOM-DSS vs WT AOM-DSS. I. Enrichment plot for Colorectal Cancer (MMU05210) from GSEA analysis (NES: 1.343; FDR q-val: 0.281) J. Enrichment plots for p53 signalling (MMU04115) (NES: 1.404; FDR q-val: 0.239), Apoptosis (MMU04210) (NES: 1.404; FDR q-val: 0.212), DNA damage bypass (R_MMU_73893) (NES: 1.368; FDR q-val: 0.308) and Regulation of PTEN stability and activity (R_MMU_8948751) (NES: 1.391; FDR q-val: 0.402) from GSEA analysis. (NES, normalized enrichment score; FDR, false discovery rate). n=3 mice/group.

Figure 5.. Bone marrow chimera identifies hematopoietic deficiency of miR-223 enhances susceptibility and inflammatory signature to AOM-DSS.

Schematic overview and timeline of the bone marrow chimera AOM-DSS study. A. number of macroscopic polyps per colon. Following H&E staining B. representative image of colon polyps. C. number of tumors per section, D. quantification of dysplasia (μm²) per section, E. quantification of tumor size (μm²) per section was determined at the time of necropsy following treatment with AOM-DSS. F. Tumor grading according to size from H&E micrographs in WT or miR-223^−/y mice treated with chronic AOM/DSS. RT-PCR was used to measure relative mRNA expression of the following markers from colonic tissues of indicated genotypes; G. pro-inflammatory markers, nos2, tnf, il-1b, il-6. H. anti-inflammatory markers arg1, chi3l3, retnla, il-4, irf4. I. monocyte chemokines ccl2 and ccl7 and neutrophil chemokines cxcl1 and cxcl2. All data is expressed as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 versus the indicated counterpart (one-way ANOVA). C-F; WT→WT: 4 tumors and 3 dysplastic regions from 5 mice; WT→miR-223^−/y: 1 tumor and 3 dysplastic regions from 7 mice; miR-223^−/y→WT: 19 tumors and 7 dysplastic regions from 5 mice; miR-223^−/y→miR-223^−/y: 10 tumors and 7 dysplastic regions from 4 mice. G-I. n=5–6 mice/group.

Figure 6.. IL-6 cytokine family and p-STAT3 signalling are increased in miR-223^−/y mice in the chronic AOM-DSS model of CAC.

A. Dot plot for Reactome pathways upregulated in miR-223^−/y AOM/DSS. B. Heat map of the differentially expressed mRNA in the cytokine signalling in the immune system pathway for miR-223^−/y AOM-DSS vs WT AOM-DSS (n=3/group). C. Heat map of the differentially expressed mRNA in the IL-6 signalling family for miR-223^−/y AOM-DSS vs WT AOM-DSS. D. Gene expression was analysed in colon tissue by RT-PCR for expression of il-6, il-11, lif, il-22 and il-17a. E. Western immunoblot assessment of p-stat3, stat3 and β-actin in colon biopsies. F. Representative 20x micrographs of unaffected and tumor tissue from WT and miR-223^−/y mice treated with chronic AOM-DSS after immunohistochemical staining for p-stat3. G. Quantification of p-stat3 staining. H. Representative 20x micrographs of unaffected and tumor tissue from WT and miR-223^−/y mice treated with chronic AOM-DSS after immunohistochemical staining for stat3. I. Quantification of stat3 staining. All data is expressed as mean ± SEM; *,P<0.05; **,P<0.01; ***,P<0.001 versus the indicated counterpart (One-way ANOVA). A, B, C, E, F: n=3 mice/group; D. n=3–6 mice/group; F&H: WT AOM-DSS n=5; miR-223^−/Y AOM-DSS n=4; G&I: WT AOM-DSS n=5; miR-223^−/Y AOM-DSS n=4 with 3 fields of view presented per animal.

Figure 7.. Antibody-mediated depletion of infiltrating Ly6G⁺ myeloid cells with RB6–8C5 reduced early tumorigenesis in miR-223^−/y mice in an acute model of AOM-DSS.

Schematic overview and timeline of the acute AOM-DSS study. miR-223^−/y mice were treated with either 20μg (i.p) anti-GR1 (Lyg6/Ly6C: RB6–8C5 clone) or an isotype control, every 3 days for 3 weeks (8 total injections). Following H&E staining A. number of tumors per section, quantification of dysplasia (μm²) per section and quantification of tumor size (μm²) per section was determined at the time of necropsy following treatment with AOM-DSS. B. Tumor grading according to size (μm²) was determined from H&E micrographs in miR-223^−/Y mice treated either IgG or anti-GR1 antibody with representative H&E micrographs. Flow cytometry was performed on enzyme digested colons from WT and miR-223^−/Y mice post treatment to assess myeloid immune subsets. From live cells (AquaVi negative), CD45⁺ singlets, C. Neutrophils were identified Ly6G⁺ CD11b⁺. D. monocytes as Ly6G⁻ CD11b⁺ Ly6C⁺ MHCII⁻ (inflammatory/infiltrating) or Ly6C⁺ MHCII⁺ (intermediate/differentiating). E. CD64⁺ MHCII⁺ tissue resident macrophage were further subdivided into CX3CR1⁺ CD11b⁻ or CX3CR1⁺ CD11b⁺. F. Dendritic cell subsets were identified as CD64⁻ MHCII⁺ CD11c⁺ and further subdivided into CX3CR1⁺ CD103⁻ or CX3CR1⁻ CD103⁺ (migratory). All data is expressed as mean ± SEM; *, P<0.05 versus the indicated counterpart (one-way ANOVA). A+B. Effects of anti-GR1 on the volumetric size of tumors in miR-223^−/y mice following an acute regime of AOM-DSS; IgG: 15 tumors and 7 dysplastic regions from 6 mice; anti-GR1: 6 tumors 1 dysplastic region from 5 mice. C-H. n=3–5 mice/group.