IL-22RA2 Is a SMAD7 Target Mediating the Alleviation of Dermatitis and Psoriatic Phenotypes in Mice

Abstract

Long-term management of inflammatory skin diseases is challenging because of side effects from repeated use of systemic treatments or topical corticosteroids. This study sought to identify the mechanisms and developmental therapeutics for these diseases using genetic models and pharmacological approaches. We found that mice overexpressing SMAD7 in keratinocytes but not mice overexpressing the N-terminal domain of SMAD7 (i.e., N-SMAD7) were resistant to imiquimod-induced T helper 1/17- and T helper 2-type inflammation. We generated a Tat-PYC-SMAD7 (truncated SMAD7 protein encompassing C-terminal SMAD7 and PY motif fused with cell-penetrating Tat peptide). Topically applied Tat-PYC-SMAD7 to inflamed skin entered cells upon contact and attenuated imiquimod-, 2,4-dinitrofluorobenzene-, and tape-stripping-induced inflammation. RNA-sequencing analyses of mouse skin exposed to these insults showed that in addition to inhibiting TGFβ/NF-κB, SMAD7 blunted IL-22/signal transducer and activator of transcription 3 activation and associated pathogenesis, which is due to SMAD7 transcriptionally upregulating IL-22 antagonist IL-22RA2. Mechanistically, SMAD7 facilitated nuclear translocation and DNA binding of C/EBPβ to IL22RA2 promoter for IL22RA2 transactivation. Consistent with the observations in mice mentioned earlier, transcript levels of IL22RA2 were increased in human atopic dermatitis and psoriasis lesions with clinical remission. Our study identified the anti-inflammation functional domain of SMAD7 and suggests the mechanism and feasibility for developing SMAD7-based biologics as a topical therapy for skin inflammatory disorders.

Figure 1.. K5.Smad7 mice are resistant to IMQ-induced psoriasis-like skin dermatitis.

(a) Experimental design for IMQ-induced psoriatic lesions in K5.Smad7 and WT skin. (b) Representative gross images and corresponding H&E skin sections of IMQ-induced lesions on experimental day 6 from WT and K5.Smad7 mice. (c) PASI scores of IMQ-induced skin phenotypes of the WT littermate and K5.Smad7 mice on days 1–6 during the IMQ application. (d) The average epidermal thickness and the number of microabscesses in H&E. Dotted lines: epidermal-dermal boundary. Bars = 100 μm. Skin samples from WT (n = 8) and K5.Smad7 (n = 13) mice were analyzed using two-way ANOVA or two-tailed unpaired t-test for statistics. Data are representative of at least three independent experiments with 3–6 samples per group. Data represent mean ± SEM. IMQ, imiquimod; WT, wild type.

Figure 2.. Tat-PYC-SMAD7 treatment ameliorates IMQ-, DNFB-, and Tape stripping-induced skin inflammation.

(a, f, g) Experiment design for Tat-PYC-SMAD7 treatment tests on three skin inflammation models. (b) IF staining of HA-tagged Tat-PYC-SMAD7 2 h after topical application of the 0.5 μg protein on day 6 in the skin, with higher-power frames on the right. (c, j, k) Representative gross images and corresponding H&E skin sections of skin lesions. (d) PASI scores of IMQ-induced skin phenotypes. (e) Quantification of average epidermal thickness (left) and the number of mice (right) in c. Quantification of the average thickness (h) for AD skin and (i) for TS skin. Bars = 25 μm for b and 100 μm for the rest images. Data were assessed using two-way or one-way ANOVA or chi-square test. Data are representative of three independent experiments with five samples per group. Dotted lines: epidermal-dermal boundary. Data represent mean ± SEM. AD, atopic dermatitis; HA, hemagglutinin; IF, immunofluorescent; IMQ, imiquimod; TS, tape stripping.

Figure 3.. IL-22RA2 is a putative target responsible for PYC-SMAD7-derived IL-22 signaling suppression.

(a) Overlapping *DEGs across transcriptomes of IMQ, AD, and TS skin by Tat-PYC-SMAD7 treatment. (b) Enriched KEGG pathways for overlapping DEGs. (c) Volcano plots for transcriptomes of inflamed skin samples, with Il22ra2 as a common DEG upregulated by Tat-PYC-SMAD7 treatment. (d) mRNA fold change by qPCR and IL22RA2 levels by ELISA using lysates of lesional skin samples. (e) Enrichment plot and (f) heatmap for IL-22 signaling-upregulated genes. (g) IL-22 levels detected by ELISA and (h) western blot for IL-22 signaling markers using protein lysis of lesional skin samples. Samples were qualified using two-tailed unpaired t- or Mann-Whitney U test. Data are representative of three independent experiments with five samples per group. Data represent mean ± SEM. AD, atopic dermatitis; DEG, differentially expressed gene; FC, fold change; IMQ, imiquimod; KEGG, Kyoto Encyclopedia of Genes and Genomes; pSTAT3, phosphorylated signal transducer and activator of transcription 3; STAT3, signal transducer and activator of transcription 3; TS, tape stripping.

Figure 4.. Tat-PYC-SMAD7 dampens IL-22 signaling and relevant pathological events.

(a) Representative images and (b) quantification of IHC staining for IL-22 signaling markers pSTAT3, S100A8, Ki-67, and CD31 in skin sections of IMQ, AD, and TS samples. (c) Levels of IL-24, IL-33, and CXCL1 by ELISA from IMQ, AD, and TS skin lysis. (d) Representative images and (e) quantification of MFI staining on sections of IMQ, AD, and TS skin lesions. Dotted lines: epidermal-dermal boundary. White dot frames indicate areas in higher magnification to the right of each image. Bars = 25 μm for all slides. Samples from each group were qualified using two-tailed unpaired t or Mann-Whitney U test for statistics. Data are representative of three independent experiments with 4–5 samples per group. Data represent mean ± SEM. AD, atopic dermatitis; IHC, immunohistochemistry; MFI, multiplex fluorescent immunohistochemistry; IMQ, imiquimod; pSTAT3, phosphorylated signal transducer and activator of transcription 3; TS, tape stripping.

Figure 5.. IL-22RA2 inhibition blocks the anti-inflammatory effects of SMAD7.

(a) Representative gross images and corresponding H&E skin sections of IMQ-induced lesions with in vivo delivery of either control or Il22ra2-directed siRNA. (b) PASI scores of IMQ-induced skin phenotypes of WT and K5.Smad7 mice during IMQ application. (c) Average epidermal thickness and number of microabscesses in H&E. (d) Western blot using protein lysis of lesional skin samples. Representative images (e) and (f) quantification of IHC and MFI staining on sections of skin lesions. Dotted lines: epidermal-dermal boundary. Bars = 100 μm for all slides. A total of 3–4 skin samples from each group were qualified using two-way ANOVA or one-way ANOVA for statistics. Data are representative of at least two independent experiments with 3–4 samples per group. Data represent mean ± SEM. IHC, immunohistochemistry; IMQ, imiquimod; pSTAT3, phosphorylated signal transducer and activator of transcription 3; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; WT, wild type.

Figure 6.. PYC-SMAD7 promotes C/EBPβ DNA binding conferring IL22RA2 transcription expression.

(a) IF visualizing IL-22RA2-expressing cells in Tat-PYC-SMAD7-treated skin. (b) IF visualizing HA-tag in PYC-SMAD7-HA transfected HaCaT cells and WT HaCaT cells. (c) Western blot using cell lysate from HaCaT and PYC-SMAD7-HA transfected HaCaT cells. (d) mRNA change by qPCR and protein expression by western blot and (e) IL-22RA2 levels by ELISA from HaCaT and PYC-SMAD7-HA transfected HaCaT cells transfected with CEBPB or Ctrl siRNA for 48 h. (f) IF showing the nucleus/cytoplasmic presences of IL-22RA2, C/EBPβ, and HA. (g) ChIP assay and (h) ChIP-qPCR for binding to ~580 base pair site of IL-22RA2 promoter. (i) IL22RA2 gene intensity of microarray data from GEO database. Data were qualified using two-tailed unpaired or paired t-test or two-way or one-way ANOVA for statistics. Data are representative of two or three independent experiments. Data represent mean ± SEM. PYC-HA-HaCaT or PYC-HaCaT: PYC-SMAD7-HA transfected HaCaT cells. ChIP, chromatin immunoprecipitation; Ctrl, control; GEO, Gene Omnibus Expression; h, hour; HA, hemagglutinin; IF, immunofluorescent; siRNA, small interfering RNA; TS, tape stripping.