Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation - PubMed (original) (raw)

Keratinocyte overexpression of IL-17C promotes psoriasiform skin inflammation

Andrew Johnston et al. J Immunol. 2013.

Abstract

IL-17C is a functionally distinct member of the IL-17 family that binds IL-17 receptor E/A to promote innate defense in epithelial cells and regulate Th17 cell differentiation. We demonstrate that IL-17C (not IL-17A) is the most abundant IL-17 isoform in lesional psoriasis skin (1058 versus 8 pg/ml; p < 0.006) and localizes to keratinocytes (KCs), endothelial cells (ECs), and leukocytes. ECs stimulated with IL-17C produce increased TNF-α and KCs stimulated with IL-17C/TNF-α produce similar inflammatory gene response patterns as those elicited by IL-17A/TNF-α, including increases in IL-17C, TNF-α, IL-8, IL-1α/β, IL-1F5, IL-1F9, IL-6, IL-19, CCL20, S100A7/A8/A9, DEFB4, lipocalin 2, and peptidase inhibitor 3 (p < 0.05), indicating a positive proinflammatory feedback loop between the epidermis and ECs. Psoriasis patients treated with etanercept rapidly decrease cutaneous IL-17C levels, suggesting IL-17C/TNF-α-mediated inflammatory signaling is critical for psoriasis pathogenesis. Mice genetically engineered to overexpress IL-17C in KCs develop well-demarcated areas of erythematous, flakey involved skin adjacent to areas of normal-appearing uninvolved skin despite increased IL-17C expression in both areas (p < 0.05). Uninvolved skin displays increased angiogenesis and elevated S100A8/A9 expression (p < 0.05) but no epidermal hyperplasia, whereas involved skin exhibits robust epidermal hyperplasia, increased angiogenesis and leukocyte infiltration, and upregulated TNF-α, IL-1α/β, IL-17A/F, IL-23p19, vascular endothelial growth factor, IL-6, and CCL20 (p < 0.05), suggesting that IL-17C, when coupled with other proinflammatory signals, initiates the development of psoriasiform dermatitis. This skin phenotype was significantly improved following 8 wk of TNF-α inhibition. These findings identify a role for IL-17C in skin inflammation and suggest a pathogenic function for the elevated IL-17C observed in lesional psoriasis skin.

PubMed Disclaimer

Figures

Figure 1

Figure 1. IL-17C RNA and protein are increased in lesional psoriasis skin

Real-time quantitative PCR analyses of IL-17 ligand and receptor family members in control patient (NN) and psoriasis patient uninvolved (PN) and involved lesional (PP) skin (A). ELISA analyses of IL-17A and IL-17C demonstrates significant increases in both cytokines, but IL-17C protein expression is ~125-fold higher than IL-17A in lesional skin (B). IL-17C and IL-17A immunohistochemistry in control and psoriasis skin shows IL-17C protein localized to epidermal, vasculature and dermal areas and IL-17A localized to very few cells in the dermal papillae(C). Cutaneous IL-17C mRNA significantly decreases in responding patients 72 hours following etanercept treatment (D). * P<0.05 compared to NN in (A) or Day 0 of treatment in (D); ** P<0.05 compared to PN in (A) or as indicated in (B).

Figure 2

Figure 2. IL-17C and TNFα induce characteristic psoriasis-related transcriptome genes in both additive and synergistic manners

Human dermal microvascular endothelial cells stimulated with IL-17C (200ng/ml) for 6 hours increase mRNA expression of TNFα and IL-6 (A). Representative western blots of cell lysates confirm the increase in TNFα protein (B). Real-time quantitative PCR analyses of key psoriasis-transcriptome genes in human primary epidermal keratinocytes stimulated with IL-17C (200ng/ml), TNFα (2ng/ml) or both as indicated for 24 hrs (C). In vitro experimental data are representative of at least three independent experiments. * P<0.05 compared to control.

Figure 3

Figure 3. K5-IL-17C transgenic mice develop a psoriasiform skin phenotype

A tetracycline-repressible binary mouse molecular genetics approach similar to that previously described (23) was utilized to genetically overexpress IL-17C in a keratinocyte-specific manner using the K5 promoter (A). Mice spontaneously develop regions of affected (involved) and unaffected (uninvolved) skin with involved skin characterized by hyperkeratosis (B). Real-time quantitative PCR analyses of IL-17C gene expression of control mouse and uninvolved and involved skin of K5-IL-17C mice shows significant increases in IL-17C gene expression in K5-IL-17C mice (C). Representative western blot demonstrates increases in IL-17C protein in uninvolved and involved K5-IL-17C skin (D). Hematoxylin & eosin-stained dorsal skin of control mouse skin and K5-IL-17C mouse uninvolved and involved skin and epidermal thickness quantitation (E). Ki67 and loricrin immunostained dorsal skin demonstrates increases in cell proliferation and loss of terminal differentiation between control and K5-IL-17C mouse skin (F). Solid lines in D highlight the epidermis; arrow is pointing at a parakeratotic scale; arrowhead is pointing at a mitotic body; black asterisks are placed on dermal blood vessels; white asterisks are placed on foci of inflammatory infiltrate. * P<0.05 compared to control mouse. Scale bar is 100µm.

Figure 4

Figure 4. K5-IL-17C mouse skin has increases in dermal angiogenesis and VEGF

Mouse endothelial cell antigen (MECA)-immunostained dorsal skin of control mouse skin and K5-IL-17C mouse uninvolved and involved skin (A). VEGF ELISA and immunohistochemistry demonstrate increases in VEGF protein in uninvolved and involved K5-IL-17C skin compared to littermate control skin (B). qRT-PCR of primary human KCs and ECs stimulated with IL-17C (200ng/ml) demonstrates significant increases in VEGF mRNA. * P<0.05 compared to control mouse. ** P<0.05 compared to uninvolved K5-IL-17C skin (B). * P<0.05 compared to control stimulated (C). Scale bar as indicated.

Figure 5

Figure 5. Characterization of the immune cell infiltrate in K5-IL-17C mouse skin

Representative photographs of immunohistochemistry and cellular quantitation of CD4-, CD8-, CD11c- and F4/80- positive cells in control mouse dorsal skin and K5-IL-17C uninvolved and involved dorsal skin. * P<0.05 compared to control mouse. ** P<0.05 compared to uninvolved K5-IL-17C skin. Scale bar is 100µm.

Figure 6

Figure 6. Psoriasis-related innate defense and cytokines/chemokines are increased in K5-IL-17C skin

Real-time quantitative PCR analyses of key innate defense molecules and psoriasis-related pro-inflammatory cytokines/chemokines demonstrates significant increases in S100A8/S100A9 and IL-17F in K5-IL-17C uninvolved mouse skin compared to control mouse skin and significant increases in these and other key pro-inflammatory molecules in involved skin compared to littermate control animals (A). Representative western blots confirming increases in pro-inflammatory cytokines and innate defense molecules (B). ELISA analyses of TNFα, IL-6 and IL-17A confirm the increases in gene expression observed between control and K5-IL-17C animals (C). Representative protein expression array confirms increases in IL-1α and IL-1β protein in involved K5-IL-17C and control mouse skin and identifies increases in other known psoriasis-related molecules. Asterisks represent reference spots (D). *P<0.05 compared to control littermates; ** P<0.05 compared to K5-IL-17C uninvolved skin or as indicated.

Figure 7

Figure 7. Systemic inhibition of TNFα in K5-IL-17C mice leads to improvement in disease severity

Photographs of the same severely affected K5-IL-17C mouse before and after 8 weeks of TNFα antibody treatment (A). Representative images of hematoxylin & eosin-stained and CD4- and CD8-immunostained dorsal skin of K5-IL-17C involved skin treated with either anti-TNFα antibodies or IgG and quantitation of epidermal thickness and CD4+ and CD8+ cell numbers. The hatched line represents control mouse levels. * P<0.05 compared to K5-IL-17C involved + IgG (B). Real-time quantitative PCR analyses of key innate defense molecules and psoriasis-related pro-inflammatory cytokines/chemokines demonstrates decreases in IFNγ, IL-6 and IL-1β in involved skin between K5-IL-17C+ IgG and K5-IL-17C+ TNFα treated mice. *P<0.05 compared to control mice (C).

References

    1. Iwakura Y, Ishigame H, Saijo S, Nakae S. Functional specialization of interleukin-17 family members. Immunity. 2011;34:149–162. - PubMed
    1. Li H, Chen J, Huang A, Stinson J, Heldens S, Foster J, Dowd P, Gurney AL, Wood WI. Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family. Proc Natl Acad Sci U S A. 2000;97:773–778. - PMC - PubMed
    1. Bordon Y. Cytokines, IL-17C joins the family firm. Nat Rev Immunol. 2011;11:805. - PubMed
    1. Johansen C, Riis JL, Gedebjerg A, Kragballe K, Iversen L. Tumor necrosis factor alpha-mediated induction of interleukin 17C in human keratinocytes is controlled by nuclear factor kappaB. The Journal of biological chemistry. 2011;286:25487–25494. - PMC - PubMed
    1. Ramirez-Carrozzi V, Sambandam A, Luis E, Lin Z, Jeet S, Lesch J, Hackney J, Kim J, Zhou M, Lai J, Modrusan Z, Sai T, Lee W, Xu M, Caplazi P, Diehl L, de Voss J, Balazs M, Gonzalez L, Jr, Singh H, Ouyang W, Pappu R. IL-17C regulates the innate immune function of epithelial cells in an autocrine manner. Nat Immunol. 2011;12:1159–1166. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources