(D) DCs were either unstimulated or stimulated with 100?ng/mL LPS in the presence or absence of GSK-J4 for 16?h. which codify for the recombination activating gene 1, an enzyme fundamental for the development of T and B lymphocytes. Interestingly, although we did not observe any difference in the production of inflammatory or anti-inflammatory cytokines in the colonic mucosa between both experimental groups, the systemic administration of GSK-J4 induced a significant attenuation of the bodyweight loss in and promoters We next analysed whether GSK-J4 has an impact on the production of RA by GALT-DCs and thus an increase of IL-10 by CD4+ T cells. These analyses indicated that, indeed, GSK-J4 treatment promoted a robust increment in RALDH-activity in PRT062607 HCL MLN DCs. The results also suggest a higher RALDH-activity on those DCs infiltrating the cLP upon GSK-J4 treatment (Fig.?3A,B). Of note, PRT062607 HCL PRT062607 HCL this effect was observed at the peak of DSS-induced colitis (day 12), but not at an earlier time point (day 8; figure S2C,D). A similar effect potentiating RALDH-activity was observed in DCs isolated from MLN or spleen and treated ex vivo with GSK-J4 either in the absence or in the presence of an inflammatory stimulus (Fig.?4A, B). Since there are three isoforms of RALDH which display distinct substrate affinities and present differential expression in some cell types15C17, we next analysed the effect of Rabbit Polyclonal to BRI3B GSK-J4 on the expression of the different RALDH isoforms in DCs and compared this to the direct effect of RA. Interestingly, our results show that GSK-J4 induced and transcription, while it had a very week effect in the levels of transcripts. Conversely, the effect of RA was confined to transcription (Fig.?3C). Similar results were observed in the presence of LPS PRT062607 HCL (Fig.?4C). Taken together, these results suggest a complementary effect of GSK-J4 and RA, in promoting a tolerogenic potential in DCs. Open in a separate window Figure 3 GSK-J4 increases RALDH activity and expression in DCs by enriching the mark H3K4me3 and decreasing H3K27me3 on the and promoters. (A) Representative dot-plot of RALDH activity using Aldefluor assays in DCs isolated at day 12 from the colonic lamina propria (colon) and MLN of mice treated as described in Fig.?1A. Numbers represent the frequencies of cells in the corresponding quadrant. (B) Frequencies of Aldefluor+ CD11c+ cells from at least six animals per group. (C,D) Splenic CD11c+ DCs from C57BL/6 mice were treated with 25?nM GSK-J4 or 10?nM RA for 16?h. (C) RT-qPCR analysing (top panel), (middle panel), and (bottom panel) expression were performed on DCs. Relative expression levels were normalized using 18S RNA as control. (D) DCs were either unstimulated or stimulated with 100?ng/mL LPS in the presence or absence of GSK-J4 for 16?h. Chromatin Immunoprecipitation (ChIP) assays were carried out using specific antibodies to H3K4me3 (top panels), H3K27me3 (bottom panels). Association of H3K27me3 or H3K4me3 to the promoters of (left panels), (middle panels), and (right panels) was quantified by qPCR by using specific primers. PCR products were normalized to the input DNA and histone H3 levels. Values represent mean??SEM from six independent experiments. *(left panel), (middle panel), and (right panel) expression were performed on DCs. Relative expression levels were normalized using 18S RNA as control. Data represent mean??SEM from six independent experiments. *in DCs. For this purpose, we determined the extent of tri-methylation of histone H3 both at lysine 4 (H3K4me3) and lysine 27 (H3K27me3), which have been described to.