Toll-Like Receptor 2-Mediated Suppression of Colorectal Cancer Pathogenesis by Polysaccharide A From Bacteroides fragilis

The beneficial role of gut microbiota in intestinal diseases has been highlighted recently. Bacteroides fragilis found in the human gastrointestinal tract is a well-studied example of a beneficial bacterium that protects against intestinal inflammation. Polysaccharide A (PSA) from B. fragilis induces the production of interleukin (IL)-10 from immune cells via Toll-like receptor 2 (TLR2) signaling in animal colitis models. The direct effect of PSA on human colorectal cancer (CRC) cells has not been studied. Here, we report the effect of PSA from B. fragilis on CRC pathogenesis in SW620 and HT29 CRC cells and the molecular signaling underlying these effects. We demonstrated that PSA induced the production of the pro-inflammatory cytokine, IL-8, but not IL-10, in CRC cells. PSA inhibited CRC cell proliferation by controlling the cell cycle and impaired CRC cell migration and invasion by suppressing epithelial mesenchymal transition. Moreover, as in the case of other animal intestinal diseases, the protective role of PSA against CRC pathogenesis was also mediated by TLR2. Our results reveal that PSA from B. fragilis plays a protective role against CRC via TLR2 signaling

TCR-independent functions of Th17 cells mediated by the synergistic actions of cytokines of the IL-12 and IL-1 families.

The development of Th17 cells is accompanied by the acquisition of responsiveness to both IL-12 and IL-23, cytokines with established roles in the development and/or function of Th1 and Th17 cells, respectively. IL-12 signaling promotes antigen-dependent Th1 differentiation but, in combination with IL-18, allows the antigen-independent perpetuation of Th1 responses. On the other hand, while IL-23 is dispensable for initial commitment to the Th17 lineage, it promotes the pathogenic function of the Th17 cells. In this study, we have examined the overlap between Th1 and Th17 cells in their responsiveness to common pro-inflammatory cytokines and how this affects the antigen-independent cytokine responses of Th17 cells. We found that in addition to the IL-1 receptor, developing Th17 cells also up-regulate the IL-18 receptor. Consequently, in the presence of IL-1β or IL-18, and in the absence of TCR activation, Th17 cells produce Th17 lineage cytokines in a STAT3-dependent manner when stimulated with IL-23, and IFN© via a STAT4-dependent mechanism when stimulated with IL-12. Thus, building on previous findings of antigen-induced plasticity of Th17 cells, our results indicate that this potential of Th17 cells extends to their cytokine-dependent antigen-independent responses. Collectively, our data suggest a model whereby signaling via either IL-1β or IL-18 allows for bystander responses of Th17 cells to pathogens or pathogen products that differentially activate innate cell production of IL-12 or IL-23

Image_3_Toll-Like Receptor 2-Mediated Suppression of Colorectal Cancer Pathogenesis by Polysaccharide A From Bacteroides fragilis.tiff

<p>The beneficial role of gut microbiota in intestinal diseases has been highlighted recently. Bacteroides fragilis found in the human gastrointestinal tract is a well-studied example of a beneficial bacterium that protects against intestinal inflammation. Polysaccharide A (PSA) from B. fragilis induces the production of interleukin (IL)-10 from immune cells via Toll-like receptor 2 (TLR2) signaling in animal colitis models. The direct effect of PSA on human colorectal cancer (CRC) cells has not been studied. Here, we report the effect of PSA from B. fragilis on CRC pathogenesis in SW620 and HT29 CRC cells and the molecular signaling underlying these effects. We demonstrated that PSA induced the production of the pro-inflammatory cytokine, IL-8, but not IL-10, in CRC cells. PSA inhibited CRC cell proliferation by controlling the cell cycle and impaired CRC cell migration and invasion by suppressing epithelial mesenchymal transition. Moreover, as in the case of other animal intestinal diseases, the protective role of PSA against CRC pathogenesis was also mediated by TLR2. Our results reveal that PSA from B. fragilis plays a protective role against CRC via TLR2 signaling.</p

Image_1_Toll-Like Receptor 2-Mediated Suppression of Colorectal Cancer Pathogenesis by Polysaccharide A From Bacteroides fragilis.tiff

<p>The beneficial role of gut microbiota in intestinal diseases has been highlighted recently. Bacteroides fragilis found in the human gastrointestinal tract is a well-studied example of a beneficial bacterium that protects against intestinal inflammation. Polysaccharide A (PSA) from B. fragilis induces the production of interleukin (IL)-10 from immune cells via Toll-like receptor 2 (TLR2) signaling in animal colitis models. The direct effect of PSA on human colorectal cancer (CRC) cells has not been studied. Here, we report the effect of PSA from B. fragilis on CRC pathogenesis in SW620 and HT29 CRC cells and the molecular signaling underlying these effects. We demonstrated that PSA induced the production of the pro-inflammatory cytokine, IL-8, but not IL-10, in CRC cells. PSA inhibited CRC cell proliferation by controlling the cell cycle and impaired CRC cell migration and invasion by suppressing epithelial mesenchymal transition. Moreover, as in the case of other animal intestinal diseases, the protective role of PSA against CRC pathogenesis was also mediated by TLR2. Our results reveal that PSA from B. fragilis plays a protective role against CRC via TLR2 signaling.</p

IL-12 synergizes with IL-1β or IL-18 to induce the transition of Th17 cells to IFNγ-expressing progeny independent of TCR stimulation.

<p>Bulk Th1 cells (A) were activated as shown for 48 hours and the concentration of IFNγ in culture supernatants was determined by ELISA (B). Naïve CD4 T cells from <i>Il17f/Thy1.1</i> reporter mice were differentiated into Th17 cells and the Thy1.1hi fraction was enriched by FACS (C). Cells were then treated with indicated cytokine combination for 48 hours and ELISA was performed to measure the concentration of IFNγ in culture supernatants (D). To determine the role of STAT-4, naïve CD4 T cells from WT or <i>Stat4-/-</i> mice were cultured under Th17 polarizing conditions and their differentiation confirmed by intracellular cytokine staining (E). The number of IL-17A+ T cells was normalized based on intracellular cytokine staining then re-stimulated with indicated cytokine(s) for 48 hours and ELISA was performed to measure IFNγ concentration. Error bars represent means ± s.d. of triplicate determinations. Flow cytometry plots are gated on CD4+ cells and the numbers represent the percentages of cells in each quadrant. Data are representative of at least three independent experiments (*p<0.05, **p<0.01, **p<0.001 and **p<0.0001).</p

IL-23 signaling during Th17 differentiation enhances TCR-independent production of IL-17A by committed Th17 effectors in STAT3-dependent manner.

<p>(A) Naïve CD4 T cells were cultured for 3 rounds under Th17 polarizing conditions with WT or <i>p40-/-</i> splenic feeder cells in the absence or presence of exogenous IL-23. Cells were stained intracellularly for IL-17A and IFNγ after PMA/ionomycin activation for 5 h in the presence of monensin. (B) CD4 T cells from the cultures in (A) were purified and the re-stimulated at a density of 1x106 IL-17A+ cells/ml (based on the frequencies shown in (A) with indicated cytokine(s) for 48 hours. ELISA was performed to measure IL-17A concentration in culture supernatants. (C) Th17 cells, polarized as in (A), were re-stimulated with titrated doses of IL-1β or IL-18 and a constant dose of IL-23 for 48 hours. ELISA was performed to measure IL-17A concentration of the culture supernatant. (E) Th17 cells polarized for 3 rounds were purified and Stat3-specific siRNA (1.5 or 3.0 μg) or Allstar negative control (3.0 μg) was administered. Aliquots of cultured cells were examined daily by western blot for expression of STAT3. After 72 hours, Th17 cells that had been treated with 3.0 μg of STAT3 siRNA were left untreated or were treated for 48 hours with the indicated cytokines and the concentration of IL-17A in culture supernatants was determined by ELISA. Error bars on all graphs represent means ± s.d. of triplicate determinations. All data are representative of at least three independent experiments (*p<0.05, **p<0.01 and **p<0.001).</p

IL-23 acts synergistically with IL-1β or IL-18 to induce production of Th17-associated cytokines by committed Th17 cells.

<p>(A) Naïve CD4 T cells were cultured with irradiated <i>p40-/-</i> splenic feeder cells under Th17-polarizing condition for 3 rounds of 7 days each. A portion of the cells was then stimulated with PMA/ionomycin in the presence of monensin and stained intracellularly for IL-17A and IFNγ to confirm their differentiation into Th17 cells (left). Of the remaining cells, 1x106 cells/well were re-stimulated with indicated cytokine(s) for 12 h with monensin added for the final 5 hours, and examined for intracellular expression of IL-17A and IFNγ. (B) 2x105 highly polarized FACS-sorted Thy1.1+ (IL-17F+) cells were either left unstimulated, re-stimulated with anti-CD3 and anti-CD28, or with the indicated cytokines for 48 hours. ELISA was performed to quantify IL-17A, IL-17F, and IL-22 in culture supernatants. (C) CD4 T cells were isolated from MLNs of either WT (grey bars) or colitic IL-10-deficient mice (black bars) and stimulated with PMA plus ionomycin for 5 h in the presence of monensin before being stained intracellularly for IL-17A and IFNγ. (D) Total MLN CD4 T cells (5x105 cells/well) from WT or <i>Il10-/-</i> CD4 T cells were stimulated with indicated cytokine(s) for 48 hours and the concentration of IL-17A in culture supernatants was determined by ELISA. Error bars represent means ± s.d. of triplicate determinations. Data are representative of at least three independent experiments (*p<0.05, **p<0.01, **p<0.001 and **p<0.0001).</p

Acquisition of cytokine responsiveness by developing Th17 cells.

<p>Naïve T cells were activated in the presence of IL-12/23p40-deficient feeder cells under Th17-polarizing conditions in the presence or absence of exogenous IL-23, or under Th1-polarizing conditions. On day 5, entire cell populations were examined for expression of (A) <i>Il1r</i>, (B) <i>Il18r</i>, (C) <i>Il12rβ2</i>, (D) <i>Il12rβ1</i>, and (E) <i>Il23r</i> mRNA by real-time PCR, or examined by flow cytometry for surface expression of (F) IL-1R and (G) IL-18Rα. (H) CD4 T cells were activated under Th17-polarizing conditions with plate-bound anti-CD3 and soluble anti-CD28 and assessed after 24, 48, and 72 hours for expression of <i>Il1r</i>, and <i>Il18r</i>. PCR results are pooled from at least three independent experiments and were normalized to 18S rRNA and are expressed as fold differences (log2) relative to naïve CD4 T cells. (*p<0.05, **p<0.005, ***p<0.001).</p