Adenomatous polyps are driven by microbe-instigated focal inflammation and are controlled by IL-10-producing T cells

Abstract

Interleukin (IL)-10 is elevated in cancer and is thought to contribute to immune tolerance and tumor growth. Defying these expectations, the adoptive transfer of IL-10-expressing T cells to mice with polyposis attenuates microbial-induced inflammation and suppresses polyposis. To gain better insights into how IL-10 impacts polyposis, we genetically ablated IL-10 in T cells in APC(Δ468) mice and compared the effects of treatment with broad-spectrum antibiotics. We found that T cells and regulatory T cells (Treg) were a major cellular source of IL-10 in both the healthy and polyp-bearing colon. Notably, T cell-specific ablation of IL-10 produced pathologies that were identical to mice with a systemic deficiency in IL-10, in both cases increasing the numbers and growth of colon polyps. Eosinophils were found to densely infiltrate colon polyps, which were enriched similarly for microbiota associated previously with colon cancer. In mice receiving broad-spectrum antibiotics, we observed reductions in microbiota, inflammation, and polyposis. Together, our findings establish that colon polyposis is driven by high densities of microbes that accumulate within polyps and trigger local inflammatory responses. Inflammation, local microbe densities, and polyp growth are suppressed by IL-10 derived specifically from T cells and Tregs.

Conflict of interest statement

Disclosures

The authors disclose no potential conflicts of interest.

Figures

Figure 1. IL-10 is produced by T-cells in polyposis

(A) Thy1.2 B6 (N=3) and APCΔ468 (N=4) mice were reconstituted with IL-10Thy1.1 x Foxp3GFP reporter bone marrow. Histogram depicts frequency (%) of IL-10+ staining cells as a percent of total cells (DAPI). At least 5 representative regions counted per mouse. (B) Thy1.2 B6 and APCΔ468 mice were reconstituted with IL-10Thy1.1 x Foxp3GFP reporter bone marrow. Histogram depicts frequency (%) as a percent of total nuclear cells (DAPI) of total CD4+, as well as CD4+Foxp3+, and CD4+Foxp3− cells. Shaded regions of each histogram represent frequency of IL-10-expressing cells in the different T-cell subsets. At least 5 representative regions counted per mouse. (C) Representative Foxp3GFP and CD4 co-staining in reconstituted B6 and APCΔ468 mice. CD4 visualized by a Cy5-conjugated secondary antibody. (D) Representative Foxp3GFP and IL-10 co-staining in reconstituted B6 and APCΔ468 mice. IL-10 visualized by an anti-Thy1.1-biotin primary antibody and streptavidin-AlexaFluor-594 conjugated flurochrome. (E) Pie charts depict proportion of IL-10 expressed in either the CD4+Foxp3+ or CD4+Foxp3− compartment in B6 or APCΔ468 mice.

Figure 2. Colonic mitotic activity & polyp load are exacerbated in IL-10-deficiency

(A) Upper histogram depicts quantification of Ki67 staining within healthy appearing tissue of the colon B6, CD4CreIL-10fl/fl, IL-10−/−, APCΔ468, CD4CreIL-10fl/flAPCΔ468, and IL-10−/−xAPCΔ468 mice. Data represent the ratio of the height of Ki67-staining cells/total height of each villi per 200X view. Lower histogram (mean ± SEM) depicts colon length of mice in millimeters (mm). (B) Representative Ki67 staining of colons. Black bar represents average height of Ki67+ cells within the villi. (C) Colonic polyp counts of the various mouse strains. (D) Photographs of distal colon segments from APCΔ468, CD4CreIL-10fl/flAPCΔ468, and IL-10−/− mice. (E) Upper panels show representative 100X colonic polyp from an APCΔ468, CD4CreIL-10fl/flAPCΔ468, and IL-10−/−xAPCΔ468 mice. Double-headed arrows depict width of expanded stromal regions. Lower panels show representative 200X micrographs of colonic polyps from APCΔ468, IL-10−/−xAPCΔ468 and CD4CreIL-10fl/flAPCΔ468 mice, showing crypts at the basal surface of the polyp. Inlay images at 400X, basal crypts of the three mouse strains.

Figure 3. Colonic infiltration of eosinophils and mast cells

(A) Quantification of colon mast cells by CAE (grey bars) and mMCP2 (open bars) for APCΔ468, IL-10−/−xAPCΔ468, and CD4CreIL-10fl/flAPCΔ468 mice. (B) Representative 400X micrographs of CAE staining (upper panels) or mMCP2 staining (lower panels) of APCΔ468, IL-10−/−xAPCΔ468, and CD4CreIL-10fl/flAPCΔ468 colonic polyps. Arrows point to mast cells. Inlay at 1000X. (C) Quantification of colon eosinophils by MBP staining in the submucosa (grey bars) and mucosa (open bars) for APCΔ468, IL-10−/−xAPCΔ468, and CD4CreIL-10fl/flAPCΔ468 mice. (D) Representative 400X micrographs of MBP staining in the submucosa (upper panels) or mucosa (lower panels) in APCΔ468, IL-10−/−xAPCΔ468, and CD4CreIL-10fl/flAPCΔ468 colonic polyps. Arrows point to eosinophils. Inlay at 1000X. (E) Quantification of eosinophils by MBP staining in CD4CreIL-10fl/flAPCΔ468 mice in the healthy, marginal, and polyp tissue in the colon. (F) Representative micrographs of CD4CreIL-10fl/flAPCΔ468 polyps stained with the eosinophil-specific marker MBP. Colonic polyps (at 50X) with inlay (at 400X) showing clusters of MBP+ cells.

Figure 4. Colonic microbiota is enriched in polyps as compared to healthy marginal tissues

(A) Scheme showing that B6 colonic tissue, as well as polyp and marginal tissue from polyp-bearing mice, was harvested and bacteria sequenced by MiSeq for relative bacterial abundance in the various tissues. Fold difference between polyp and margin, or polyp and healthy B6 tissues was calculated and graphed in (B–D). Relative fold change of microbes in the polyp versus marginal tissue of polyp-bearing mice, or polyp tissues versus B6 tissues, of the phyla (B) Bacteroidetes, (C) Firmicutes, and (D) Proteobacteria. N=4 mice (2 CD4CreIL-10fl/flAPCΔ468 and 2 APCΔ468 mice). Paired polyp and margin data points were used to calculate the relative fold-increase of polyp to margin, or margin to polyp. Significance was determined by performing a paired, one sample t-test with the theoretical mean set to a value of 0, with P<0.05 noted by *. Only values above 0.0020 (0.2%) relative abundance were analyzed.

Figure 5. Bacterial composition differs between B6, APCΔ468, and CD4CreIL-10fl/flAPCΔ468 colonic mucosa

PCoA plot of bacterial composition of healthy B6 colon (squares), and marginal-to-the-polyp healthy appearing colonic tissue from APCΔ468 (circles) and CD4CreIL-10fl/flAPCΔ468 (diamonds) mice. N=4 for each strain. PC3 on the y-axis with a 10.77-percent variation explained. PC1 on the x-axis with a 37.24-percent variation explained.

Figure 6. Antibiotic treatment alters microbiota and inflammation-associated colonic polyposis in IL-10-deficiency

(A) Scheme for mice undergoing antibiotic treatment. At 2.5-months of age, APCΔ468 and CD4CreIL-10fl/flAPCΔ468 mice received an injection of a 200ul cocktail of antibiotics by intra-peritoneal (i.p.) injection. This cocktail included kanamycin (4mg/ml), gentamycin (0.35mg/ml), colisitin (8500U/ml), metronidazole (2.12mg/ml), and vancomycin (0.45mg/ml). Mice were treated every other day for a total of 3 weeks, then they were allowed 1 week of rest prior to sacrifice and analysis. (B) Number of mast cells by CAE staining of APCΔ468 and CD4CreIL-10fl/flAPCΔ468 mice following antibiotic treatment (per polyp completely filling a 200X field). (C) Number of eosinophils by eosin staining of APCΔ468 and CD4CreIL-10fl/flAPCΔ468 mice following antibiotic treatment (per polyp completely filling a 400X field). (D) Number of polyps counted in the colon following antibiotic treatment of APCΔ468 and CD4CreIL-10fl/flAPCΔ468 mice. Relative abundance of Bacteroides (E) and Porphyromonas (F) in fecal pellets collected before (pre) and after (post) antibiotic treatment from polyp-bearing animals (N=3 APCΔ468 and N=4 CD4CreIL-10fl/flAPCΔ468 mice). Bar depicts average abundance score per data set. (G) Shannon diversities of microbial populations in fecal pellets collected before (pre) and after (post) antibiotic treatment from polyp-bearing animals (N=3 APCΔ468 and N=4 CD4CreIL-10fl/flAPCΔ468 mice). Histogram plot shows diversity index of seven different polyp-bearing mice. Bar depicts average abundance score per data set.