Fig 1: Suppressive effect of calcitriol on RANKL expression in Th cells in an inflammatory environment. A, mRNA level of RANKL in Th cells (determined by qRT-PCR) following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). B, Protein level of RANKL in Th cells (determined by Western blotting) following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group; left panel) and semi-quantitative analysis of the protein expression level (normalized to the level of actin) in terms of the relative grey density (right panel). C, Representative flow cytometry plots of IL-17+/RANKL+ Th cells following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). D, Quantification of the proportion of IL-17+/RANKL+ Th cells (assessed by flow cytometry). E, Representative immunofluorescence images of IL-17+/RANKL+ Th cells (cell nucleus, blue fluorescence; IL-17 protein, green fluorescence; RANKL protein, red fluorescence; scale bar: 100 µm). F, Quantification of the proportion of IL-17+/RANKL+ Th cells (calculated from an immunofluorescence assay). The data are shown as the mean ± SD; *P < .05 and **P < .01 represent significant differences between the indicated columns
Fig 2: Calcitriol facilitated the polarization of Th cells towards the Th2 phenotype while inhibiting the polarization of Th cells towards the Th17 phenotype in an inflammatory environment. A, CFSE-based flow cytometry was used to assess the proliferation of Th2 cells (IL-4 labelled) in the indirect co-incubation (transwell) system. B, CFSE-based flow cytometry was used to assess the proliferation of Th17 cells (IL-17 labelled) in the indirect co-incubation (transwell) system. C, Representative flow cytometry plots of CD4+/IL-4+ Th2 and CD4+/IL-17+ Th17 cells following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). D, Quantification of the proportions of CD4+/IL-4+ Th2 and CD4+/IL-17+ Th17 cells (calculated by flow cytometry) and the Th2/Th17 ratio. E, mRNA levels of Th2 polarization-related markers (GATA3, STAT5 and IL-4) in Th cells following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). F, Protein levels of Th2 polarization-related markers (p-GATA3, STAT5 and IL-4) in Th cells (determined by Western blotting) following incubations in various conditions (left panel) and semi-quantitative analysis of the protein expression levels (normalized to the level of actin) in terms of the relative grey density (right panel). G, The mRNA levels of Th17 polarization-related markers (STAT3, ROR?T and IL-17) in Th cells following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). H, The protein levels of Th17 polarization-related markers (p-STAT3, ROR?T and IL-17) in Th cells (determined by Western blotting) following incubations in various conditions (left panel) and semi-quantitative analysis of the protein expression levels (normalized to the level of actin) in terms of the relative grey density (right panel). The data are shown as the mean ± SD; *P < .05 and **P < .01 represent significant differences between the indicated columns
Fig 3: Staining human neutrophils by anti-IL-17B (AF1248) antibodies (Abs). (A) Immunofluorescence (top panels) and immunohistochemistry (lower panels) staining of two FFPE cases of human pustular psoriasis using anti-IL-17B (AF1248) and CD66b Abs (as labeled). Top panels show DAPI, FITC channel, and merge to recognize neutrophil shape; lower panels show different magnification of IHC and double IHC to characterize IL-17A+ cells with the neutrophil marker CD66b. (B) Cytospins of neutrophils incubated without (top panel) or with 5 µM R848 (bottom panel) for 3 h. Original magnification 200× [first row in (A) and left image in third row, scale bar 100 µm] and 400× [second row in (A), center/right images in third row in (A), as well as in (B), scale bar 50 µm]. Images of the second row in (A) represent magnifications of images in first row. (C) AF1248 immunoblot of lysates from neutrophils either freshly isolated (T0, from two donors) or incubated for 3 h with or without 2 µg/ml IL-6 plus 0.2 µg/ml IL-23 (low), 20 µg/ml IL-6 plus 2 µg/ml IL-23 (high), or 5 µM R848. Recombinant human IL-17B (rhIL-17B) was used as positive control. Panels (B,C) display representative experiments out of two independent ones with similar results.
Fig 4: H3K4me1 or H3K27Ac levels at the IL-17A, IL-17F, and SOCS3 genomic loci of Th17 cell lines and resting/IL-6 plus IL-23-activated neutrophils. Enrichment levels of H3K4me1 (left panels) and H3K27Ac (right panels) at the IL-17A (A), IL-17F (B), and SOCS3 (C) genomic loci by chromatin immunoprecipitation (ChIP) analysis in human Th17 cell lines and neutrophils incubated for 1 h with or without 20 µg/ml IL-6 plus 2 µg/ml IL-23. (A–C) Schemes illustrating the positions of the designed primer pairs amplifying promoter and potential enhancer regions of IL-17A, IL-17F, and SOCS3 for ChIP analysis are depicted at the top of each panel. Coimmunoprecipitated DNA samples were expressed as percent of the total input. Panels in (A–C) depict a representative experiment out of two independent ones with similar results. Error bars represent SEs calculated from triplicate qPCR reactions.
Fig 5: Chromatin immunoprecipitation (ChIP)-Seq profiles of H3K4me1 and H3K27Ac at the IL17A and IL17F loci in human neutrophils and Th17 cell lines. Representative snapshots depicting H3K4me1 and H3K27Ac ChIP-seqs at the IL17A and IL17F genomic loci in freshly isolated human neutrophils or, as retrieved from NIH Epigenomics Roadmap Initiative (72), in phorbol mysistate acetate/ionomycin-stimulated Th17 cell lines.
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