RA and TLR2 signals suppress pro-inflammatory cytokines from T cells.

<p>Cytokine production from purified splenic CD4⁺ T cells stimulated with anti-CD3ε and (A) Pam2CysK4, (B) cecal lysate, or (C) indicated TLR agonists in the presence or absence RA, TGF- or RA/TGF- β. Data are the mean ± SEM of 3–4 independent experiments. *, p < 0.05, **, p < 0.01 using Student’s t-test.</p

RA potentiates cytokine responses in both WT and TLR2KO DC.

<p>(A) WT splenic DC were cultured with 100 ng/ml of TLR2 ligand Pam2CysK4 and (B) WT and TLR2KO splenic DC were cultured with 10 μg/ml cecal lysate in the presence or absence of RA, TGF- β or RA/TGF- β. Supernatants were analyzed after 24 hours for cytokine production by ELISA. (C) Comparison of IL-10 IL- production from CD103⁺ LP DC and CD103⁺ SP DC. (D) Transcript levels of <i>aldh1a2</i> in spleen and LP of naïve WT and TLR2KO mice determined by quantitative PCR. For (A-C), data are the mean ± SEM of three independent experiments, for (D) 5 individual mice were analyzed. *, p < 0.05, **, p < 0.01 using Student’s t-test.</p

RA treatment suppresses IFN-γ during DSS in WT mice but enhances their secretion in TLR2KO mice.

<p>(A) Mucosal scrapings from mice receiving the DSS and water were harvested on day 10 and analyzed by ELISA for cytokine levels. (B) Quantitative RT-PCR for transcription factors associated with T helper subsets were also performed on colonic lamina propria samples taken at day 14. Data shown are the fold increase of Vehicle- and RA-treated mice compared to naive controls. Data are the mean ± SEM of 5–8 mice per group pooled from two independent experiments. (C) Expression of Foxp3 and IL-10 in colonic LP CD4⁺ T cells (n = 3 per group), one representative facs plot is shown. *, p < 0.05, **, p < 0.01 using Student’s t-test.</p

RA potentiates colonic injury and inflammation in the absence of TLR2 signaling.

<p>WT and TLR2KO mice were given 2.5% DSS for seven days to induce colonic damage and then placed on normal drinking water for seven days to allow for tissue repair. Upon first presence of fecal occult blood positivity for each, mice were supplemented with RA or vehicle control, which continued for the rest of the disease course. Weight loss (A) and fecal occult blood (B) (n = 10–14 mice per group) were monitored daily. (C) H&E staining of colonic sections of mice at day 14 (after 7 days of DSS followed by 7 days of water). (D) Histology scoring of H&E-stained colonic slides (n = 4 mice per group). (E) The percent of surface area containing ulcerated/denuded epithelium quantified using J Image software. (F) Colon lengths from mice at day 10 (n = 5–6 mice per group). *, <i>p</i> < 0.05, **, p < 0.01 using Students t-test.</p