Inter-Regulation of Th17 Cytokines and the IL-36 Cytokines In Vitro and In Vivo: Implications in Psoriasis Pathogenesis  Yijun Carrier, Hak-Ling Ma, Hilda E. Ramon, Lee Napierata, Clayton Small, Margot O'Toole, Deborah A. Young, Lynette A. Fouser, Cheryl Nickerson-Nutter, Mary Collins, Kyri Dunussi-Joannopoulos, Quintus G. Medley  Journal of Investigative Dermatology  Volume 131, Issue 12, Pages 2428-2437 (December 2011) DOI: 10.1038/jid.2011.234 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Regulation of IL-36 cytokines by IL-22 in mouse psoriasis-like skin. Tissue lysates from scid mice that received either naive T cells or saline (control) (a) and that co-received antibodies (isotype control or anti-IL-22) and naive T cells (c) were analyzed for the mRNA expression of mouse Il1f6, Il1f8, Il1f9, and Il1rl2 by quantitative PCR. In addition, ear tissues with or without psoriatic-like plaques were analyzed for the expression of IL-36α protein by western blot (b). (d) Ear tissues from naive BALB mice that received saline (control) or rmIL-22 were also subjected to the same gene expression analysis. One of two (a, total n=8, *P<0.005), one of three (c, n=5 per group, *P<0.01), and one of two (d, n=4 per group, **P<0.1) independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology 2011 131, 2428-2437DOI: (10.1038/jid.2011.234) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Induction of IL-36 gene expression in human primary keratinocytes (KC) in vitro. KC were incubated with (a) 0.32, 1.6, 8, 40, or 200ngml−1 of IL-22, (b, c) 200ngml−1 of IL-22 in combination with IL-17A, (d) IL-22 with tumor necrosis factor (TNF)-α, (e) IL-12 and IFN-γ, (f) Th1 or Th17 cytokines alone or in combination, as indicated, and (g) 1μgml−1 of IL-36 in the presence of IL-17A or TNF-α, all for 24hours. Relative expression levels of indicated genes are reported as mean±SD of triplicates of a representative donor. (c) IL-36γ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins detected by western blot analysis. The protein level of IL-36γ was ∼10ng in 15μg of cell extract as judged using recombinant IL-36γ as standards. Shown is one of four (a), six (b), two (c), six (d), three (e, f), and two (g) experiments with a total of eight donors. Journal of Investigative Dermatology 2011 131, 2428-2437DOI: (10.1038/jid.2011.234) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Self- or cross-regulation of gene expression of IL-36 cytokines in keratinocytes (KC) in vitro. Gene expression of human IL1F6, IL1F8, and IL1F9 in KC after 24hours of incubation with 0, 0.3, 1.1, 3.3, and 10μgml−1 of IL-36α, IL-36β, and IL-36γ in the absence or presence of 20ngml−1 of IL-17A (a), or after incubation with 1μgml−1 of IL-36 alone or in combination with 20ngml−1 of tumor necrosis factor (TNF)-α (b, c). Data are reported as mean±SD of triplicates of one representative donor out of five donors. Journal of Investigative Dermatology 2011 131, 2428-2437DOI: (10.1038/jid.2011.234) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Functions of IL-36 cytokines in keratinocytes (KC) in vitro. Induction of tumor necrosis factor (TNF)-α (a), IL-8 (b), and IL-6 (c) were detected in KC as mRNA transcripts (2hours) or as proteins in the supernatant (6hours) after incubation with either IL-36 cytokines alone or in combination with IL-17A or TNF-α. TNF-α protein was not measured in TNF-α-treated cultures. Gene expression levels of human DEF4 (d, 48hours), S100a7 (e, 48hours), KRT16 (f, 24hours), and KRT17 (g, 24hours) are reported as mean±SD of triplicates of one representative donor out of five donors. Journal of Investigative Dermatology 2011 131, 2428-2437DOI: (10.1038/jid.2011.234) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Correlation between IL-36 and Th17 cytokine gene expression in the human psoriasis skin lesions. (a) Transcript levels of human IL1F6, IL1F8, IL1F9, and IL1RL2 in non-lesional (non-L) or lesional skin biopsy pairs from human psoriasis patients (n=11); *P< 0.02. (b) Comparison of human IL1F6, IL1F8, and IL1F9 with IL22 and IL17a transcripts (units on x and y axis are in relative units normalized to transcripts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in the lesional skin biopsies. Statistical correlations were determined according to Pearson's value correlation test, and differences between pairs of cytokines were determined by two-tailed Student's t-test. Journal of Investigative Dermatology 2011 131, 2428-2437DOI: (10.1038/jid.2011.234) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions