Fig 2: PY109 induces IL-22 in human T cells from healthy donors and patients with IBD.CD4+ T cells were purified from PBMCs of healthy participants (H.P.) and patients with IBD. Cells were activated with anti-CD3 and anti-CD28 antibodies in the presence of Th17-polarizing cytokines (TGF-ß1, IL-1ß, and IL-6) with or without 5 µM of PY109 for 5 days. Cells were re-stimulated with PMA, ionomycin, and brefeldin A and RNA expression of AHR (A), CYP1A1 (B), IL22 (C), and IL17A (D) analyzed by qPCR using 18S as an internal control. (E) Representative scatterplots of IL-22 and IL-17A staining from one donor. Flow cytometry quantification of IL-22 (F) and IL-17A (G). Dot plots represent 7 to 9 healthy donors and 10 to 12 patients with IBD. *P < 0.05, PY109 versus control (paired t test).

Fig 3: Antibiotics in adult mice ameliorate psoriasis by reduction of IL-17 and IL-22 in T cells.Young adult mice (4-weeks-old) were exposed to antibiotics in drinking water (adultATB) for four weeks, and last six consecutive days followed by imiquimod treatment. (a) Representative photographs of back skin of control and adultATB mice followed by imiquimod treatment. Photos were taken at day 6. (b) H&E stained skin sections of control and adultATB mice after imiquimod treatment. Scale bar, 200 µm. (c) Thickness of skin measured by digimatic caliper at day 6 in control and imiquimod treated mice. Data showed represents average of at least two measurements. Increased skin infiltration in control compared adultATB mice based on staining of skin CD45+ cells. (d) Flow cytometric analysis of single-positive CD3+TCR?dlow+IL-17+ cells or CD3+TCR?d-IL-17+ cells in control (n=6–7) and adultATB (n=6–7) mice after imiquimod treatment. (e) Flow cytometric analysis of single-positive CD3+TCR?dlow+IL-22+ cells or CD3+TCR?d-IL-22+ cells in control (n=6) and adultATB (n=6) mice after imiquimod treatment. (f) Frequency of skin CD3+TCRß+Foxp3+ cells in the skin of control (n=5) and adultATB (n=7) mice after imiquimod treatment. (g) Diversity of the microbiota (Observed OTUs, Shannon Index) in adultATB and control mice. Primary samples were collected per cage (Pooled samples from 3 independent experiments) and analysed using 16S rRNA sequencing of region V4. (h) Relative abundance at phylum level between control and adultATB mice. Each column represents pooled samples from individual experiments. (i) Summarized phylum level analysis from three individual experiments showing difference between control and adultATB mice. (j) Relative abundance at family level between control and adultATB mice. Each column represents pooled samples from individual experiments. (k) Summarized family level analysis from three individual experiments showing difference between control and adultATB mice. Results are shown as mean±s.e.m., significance in (c–f) was determined by unpaired two-tailed Student's t-test (*P<0.05; **P<0.01; ***P<0.001), significance in (i,k) was determined using the non-parametric Kruskal–Wallis test (*P<0.05).

Fig 4: Imiquimod increases IL-17 and IL-22 in both ?d and aß T cells in skin.Skin and ear of wild type C57BL/6 mice were treated by topical application of imiquimod cream (Fougera, n=4) or control cream (Vaseline, n=3) for 6 consecutive days. (a) Weight loss of imiquimod and control cream treated mice monitored daily. (b) Photographs of imiquimod and control cream treated skin and ear; photos were taken at day 6. H&E stained ear and back skin sections of imiquimod treated and control mice. Scale bar, 200 µm. (c) Thickness of skin measured by Digimatic Caliper at day 6 in control and imiquimod treated mice. Data showed represents average of at least two measurements. (d) Ear thickness of imiquimod and control cream treated mice monitored daily. Ear thickness was measured using Digimatic Caliper. (e) Quantitative PCR analysis of Th17 associated cytokines and Foxp3 in skin after 6 days of imiquimod and control cream treatment. (f) Representative flow cytometric analysis of TCR?d+V?4+IL-17+ (upper raw) and TCR?d+V?4+IL-22+ cells (lower row) in the skin of control (n=3) and imiquimod treated mice (n=4). (g) Representative flow cytometric analysis of single-positive TCRß+IL-17+ (upper raw) or TCRß+IL-22+ (lower raw) cells in the skin of control (n=3) and imiquimod treated mice (n=4). Data representative of more than three experiments, results are shown as mean±s.e.m., significance determined by unpaired two-tailed Student's t-test (*P<0.05; **P<0.01; ***P<0.001).

Fig 5: PY109 attenuates DSS-induced colitis in mice.Mice in colitis model were recorded for survival (A), body weight (B), and disease activity score (C). Data represent three independent trials with n = 7 per group per trial. (D) Colon lengths at day 11 (n = 12 to 13). (E) Hematoxylin and eosin staining (100×) and (F) histological score at day 11 (n = 8). (G and H) Colon goblet cells at day 11 (n = 7 to 8). (I) RNA sequencing (RNA-seq) analysis of colon from untreated mice (n = 4) and dextran sulfate sodium (DSS)–treated mice ± PY109 (n = 5) at day 11. Heatmap demonstrates colitis transcriptome counter-regulated by PY109. (J) Volcano plot of differentially expressed genes by PY109. Red, increased genes; green, decreased genes; blue, AHR target genes. (K) Heatmap of reported AHR target mediators and Il22 or Il17a regulated barrier genes with red denoting increase and green denoting decrease. (L and M) qPCR expression level of Il22 and Il17a in the whole colon at days 7 and 11 of the DSS model. (N to Q) Flow cytometry staining of LPMC. IL-22+ and IL-17A+ were stained in combination with T helper staining (CD3+CD4+; N to O) or ILC3 staining (CD3-RORrt+; P and Q). (R and S) Absolute number of ILC3 (R) or intraepithelial CD3+CD45+ TCR ?d+ cells (S). (T to V) qPCR of the epithelial fraction was performed for detection of antimicrobial peptides S100a8 (T), S100a9 (U), and RegIII? (V). qPCR data represent n = 4 to 8 per group. Flow cytometry data represent n = 4 to 10 per group. Data represent mean ± SEM, *P < 0.05 versus control (Student’s t test).