Fig 1: IL22 reduces Lgr5+stem cell numbers in vivo. (A) Mice were treated with IL22 (1 μg/day for 7 consecutive days). Tissues were collected on day 8. Isolated jejunal crypts were grown as enteroids in ENR medium without added IL22. Bright-field images after 2 days of culture are shown. Images are representative of 3 independent experiments. Bar, 100 μm. (B and C) Enteroid cross-sectional area (n = 8) and number (n = 8) were quantified. *P < .05, **P < .01. (D) Quantitative RT-PCR of relative mRNA expression in isolated jejunal epithelium. n = 3–7 in the representative experiment shown. *P < .05. (E and F) Lgr5-EGFP (detected using anti-GFP) and Olfm4 immunostains of jejunal tissue sections from IL22- or saline-treated mice. Numbers of positive cells per well-oriented crypt are shown. Each point (n = 12) represents a separate field from a total of 5–7 mice per condition. Bar, 100 μm. Bar, 20 μm (insets). **P < .01. (G and H) Lgr5-EGFP and Olfm4 immunostains of ileal tissue sections from IL22- or saline-treated mice. Numbers of positive cells per well-oriented crypt are shown. Each point (n = 12) represents a separate field from a total of 5–7 mice per condition. Bar, 100 μm. Bar, 20 μm (insets). **P < .01.
Fig 2: IL22 reduces Lgr5+ISC numbers and expression of active ISC markers in vitro. (A) Jejunal enteroids from Lgr5-EGFP-IRES-CreERT2 mice were cultured in ENR without or with IL22 for 3 days (5 ng/mL, PeproTech) and stained with Hoechst 33342 (blue). Images are representative of more than 3 independent experiments. Bar, 200 μm. (B) Flow cytometric analyses of Lgr5-EGFP-IRES-CreERT2 enteroids cultured in ENR without (left) or with (right) IL22 (5 ng/mL, PeproTech) for 3 days. Fractions of Lgr5+ cells within the designated windows are shown. P < .05. Data are representative of more than 3 independent experiments. (C and D) Quantitative RT-PCR analysis of relative mRNA expression in jejunal enteroids cultured in ENR without (blue symbols) or with (black symbols) IL22 (5 ng/mL, PeproTech; n = 6–7 in the representative experiments shown). *P < .05, **P < .01.
Fig 3: IL-22 correlates with miR-21-3p and to a minor extent with miR-21-5p induction. (A) RT-qPCR quantification on the relative expression of miR-21-5p compared to miR-16-5p levels in HaCaT cells and NHEKs stimulated with 50 ng/mL IL-22 for 24 h. (B) RT-qPCR quantification of the relative expression of miR-21-3p compared to miR-16-5p levels in HaCaT cells and NHEKs stimulated with 50 ng/mL IL-22 for 24 h. (C) Pearson correlations between relative the expression of miR-21-5p and IL-22 in human skin biopsies from healthy (n = 10) and psoriatic individuals (n = 9). (D) Pearson correlations between the relative expression of miR-21-3p and IL-22 in human skin biopsies from healthy (n = 10) and psoriatic individuals (n = 9). Data information: results are presenteds as mean values ± SEM. Data are representative of three independent experiments done in triplicates. The statistical comparison between groups was performed by using a two-tailed Student’s t-test: * p < 0.05, ** p < 0.01.
Fig 4: Requirements of STAT3 and NF-κB signaling for the induction of miR-21-3p in IL-22-stimulated keratinocytes. (A) Schematic representation of the miR-21 promoter gene with the putative binding sites for STAT3 (S1, S2, and S3) and NF-κB (k1, k2, k3, and k4) transcription factors. (B) The luciferase activity of NF-κB in HaCaT cells transfected with NF-κB luciferase reporter construct was measured after 24 h of 50 ng/mL IL-22 stimulation. (C) IL-22 induction of the STAT3 pathway in HaCaT cells was evaluated using a cell-based ELISA kit. (D) Representation of the fold change in miR-21 (5p and 3p) relative expression, assessed via RT-qPCR. The fold change corresponds to the ratio of 50 ng/mL IL-22 over 0 ng/mL IL-22-treated HaCaT cells and silenced with si-Ctrl, si-p65, si-STAT3, or si-p65/STAT3 for 24 h. Data information: results are presented as mean values ± SEM. Data for B and C are representative of two independent experiments and for D are representative of three independent experiments performed in triplicate. The statistical comparison between groups was performed using the two-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 5: Functional assay using the RNAi-inducible luciferase expression system (RILES) to monitor miR-21 activity in HaCaT cells. (A) Schematic representation of the RILES construction. Briefly, the firefly luciferase reporter gene is repressed by a translational repressor (CymR—in red). The latter is under the control of the miR-21 cassette (miR-21.5pT or miR-21.3pT cassette, black histogram). Thus, the binding of miR-21-5p or miR-21-3p to the miR-21.5pT and miR-21.3pT cassette, respectively, results in CymR degradation that is translated by luciferase expression. Changes in luciferase levels reflect whether or not a miRNA can induce CymR mRNA degradation or translation arrest, which indicate miRNA functionality. An empty miR cassette, pRILES, was used as a control plasmid. (B,C) miR-21-5p and miR-21-3p expression induction assessments using RILES technology in HaCaT cells stimulated with 0, 20, 50, and 100 ng/mL of IL-22 for 24 h. Data are presented as relative luciferase units (RLU) normalized to the total amount of protein (mg). Data information: results are presented as mean values ± SEM. Data are representative of three independent experiments performed in triplicate. The statistical comparison between groups was performed by using two-tailed Student’s t-test: * p < 0.05.
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