Accepted Article Received Date : 04-May-2016 Revised Date : 31-Oct-2016 Accepted Date : 05-Jan-2017 Article type : Letter to the Editors Granzyme A potentiates chemokine production in IL-17 stimulated keratinocytes Stanley Cheuk1, Elisa Martini1, Kerstin Bergh1, David Chang1, Bence Rethi2, Mona Ståhle1, Liv Eidsmo1 Dermatology and Venereology Unit, Department of Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Solna Stockholm, Sweden Rheumatology Unit, Department of Medicine, Solna, Karolinska Institutet and Karolinska University Hospital, Solna Stockholm, Sweden Correspondence: Dr Stanley Cheuk and Dr Liv Eidsmo, Dermatology and Venereology Unit, Department of Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden E-mail address: stanley.cheuk@ki.se, liv.eidsmo@ki.se Key words: Psoriasis, Serine proteases, T cells, Inflammation, Cytotoxic granules Abstract Title: Granzyme A potentiates chemokine production in IL-17 stimulated keratinocytes Plaque psoriasis presents with focal skin inflammation, partially maintained by IL-17 mediated interactions between infiltrating epidermal T cells and activated keratinocytes Here we show that the majority of lesional epidermal CD8 T cells express Granzyme A, alone or in combination with IL-17 To assess pro-inflammatory properties of Granzyme A, primary human keratinocytes were stimulated with Granzyme A in the presence or absence of IL-17 Out of 33 analysed keratinocyte derived inflammatory This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record Please cite this article as doi: 10.1111/exd.13284 This article is protected by copyright All rights reserved Accepted Article mediators, Granzyme A potentiated IL-17 induced secretion of CXCL1, CXCL12 and CCL4 Intriguingly, all three chemokines are implicated in psoriasis pathogenesis and are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues Our results indicates that Granzyme A produced by lesional CD8 T cells have the capacity to specifically increase the chemokine production from inflamed keratinocytes; thereby, sustaining the inflammation in psoriasis lesion by amplifying the chemotactic inflammatory loop Background Granzyme(Gzm)s, a family of granule serine proteases that is stored in cytotoxic lymphocytes, have the capacity to induce cellular cytotoxicity in the presence of perforin (1) In psoriasis, where massive T cell-infiltration occurs in affected skin (2, 3), gene expression of GZMA and GZMB is upregulated in lesional skin alongside with genes related to the Th17/IL-23 axis (4, 5) We have previously reported elevated gene expression of GZMA, GZMB and PRF (Perforin) in CD8 T cells sorted from psoriasis lesions as compared to healthy skin (6) Despite increased frequency of GzmB and perforin expressing cells in lesional psoriasis compared to normal skin, atopic (AD) and allergic contact dermatitis (ACD) lesions harbor significantly more granzyme-expressing T cells as compared to psoriasis (7, s1) Moreover, apoptotic cells are scarce in psoriasis (s3) and keratinocytes from psoriasis lesions show resistance to apoptosis (s2), indicating alternative functionality of Gzms in psoriasis compared to AD and ACD Noncytolytic activity of Gzms is reported and in particular GzmA induce expression of proinflammatory cytokines IL-1β, IL-6 or IL-8 in primary monocytes (8), fibroblasts or epithelial cell lines (s4) Interestingly, GzmA deficient mice display no defects in cellular cytotoxicity but impaired LPS- (8) or bacterial-induced septic shock (9), further supporting its proinflammatory role (s4) Questions addressed We postulated that granzymes act as a pro-inflammatory mediator in psoriasis Granzyme and perforin expression in CD8 T cells from psoriasis lesions was assessed and primary keratinocytes were stimulated by GzmA and IL-17 to assess secretion of inflammatory mediators This article is protected by copyright All rights reserved Accepted Article Experimental design See supplementary information Results Granzyme A expressing CD8 T cells accumulate in psoriasis lesions Intracellular expression of GzmA, GzmB and perforin was assessed in T cells extracted from active psoriasis lesions and healthy skin (Figure 1) A small proportion of GzmB+ or perforin+ lesional CD8 T cells (Figure 1A) indicated limited gzm-mediated cellular cytotoxicity in psoriasis in accordance with earlier studies (s2-s3) In contrast, the majority of CD8 T cells from psoriasis lesions expressed GzmA Confocal microscopy confirmed intracellular GzmA+ granules in epidermal T cells (Figure 1b-c) Granzyme A potentiates chemokine production in IL-17 stimulated keratinocytes In psoriasis, CD8 T cells producing IL-17A accumulate in epidermis (6, s6) in close contact with keratinocytes that respond to IL-17 simulation with production of innate cytokines and chemokines (3) In the limited number of patients included in this study, no correlation between the frequency of GzmA+ T cells and lesional leukocyte infiltration or disease severity could be determined (data not shown) However, epidermal GzmA expressing CD8 T cells produced the psoriasis-associated cytokines IFNγ and IL-17A upon stimulation (Figure 1e,d) To investigate if GzmA potentiate psoriasis-related cytokine or chemokine production, primary human keratinocytes derived from healthy skin was incubated with endotoxin-free GzmA (Figure S1) in subcytotoxic concentration (8, s5) in the presence or absence of IL-17A GzmA neither promoted proliferation nor affected the IL-17 induced keratinocyte differentiation status as measured by gene expression of MKI67, IVL, KRT16 or KRT10 (Supplementary Figure 2A) A multiplex immunoassay was used to screen supernatants for secretion of 33 different inflammatory mediators (Supplementary Table) IL-17 induced secretion of the cytokines IL-6, IL-8 and IL-23 (Figure 2, Supplementary Table), whereas GzmA treatment alone did not induce secretion of inflammatory mediators Interestingly, the combination of GzmA and IL-17 significantly increased the release of the proinflammatory chemokines CXCL1, CXCL12, and CCL4 compared to IL-17 stimulation alone, but did not alter the levels of IL-17-induced cytokines IL-6, IL-8 and IL-23 (Figure This article is protected by copyright All rights reserved Accepted Article 2, Supplementary Table) Upregulation of CXCL1 in IL-17 stimulated keratinocyte by GzmA was blocked by the serine protease inhibitor 3,4-Dichloroisocoumarin (DCI) (Supplemental Figure 2B) IL-1α secretion was increased by GzmA alone or in combination with IL-17 as previously reported (9), but this difference did not reach statistical significance Conclusions Proinflammatory activity of GzmA (8, s5) has not been explored in the context of skin diseases Here, we show that CD8 T cells in psoriasis skin lesions display a dominant expression of GzmA over GzmB in the absence of perforin expression Signals recruiting GzmA+ T cells into the skin and potential triggers of GzmA expression in the context of chronic inflammation deserves future investigation Potentially Tc17 polarizing condition would favour development of GzmA+GzmB-Prf- CD8 T cells in the context of psoriasis We found that GzmA potentiated IL-17 induced secretion of three out of the 33 inflammatory mediators analyzed in keratinocytes Intriguingly, all three chemokines, CXCL1, CXCL12 and CCL4, are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues and have been implicated in psoriasis (s7s9) Our results indicates that GzmA produced by lesional CD8 T cells have the capacity to specifically increase chemokine secretion from inflamed keratinocytes; thereby, sustaining the focal inflammation in psoriasis lesions by amplifying a chemotactic inflammatory loop GzmA may additionally potentiate the effect of other proinflammatory mediators on keratinocytes or stroma-cells present in the skin Given the involvement of CD8 T cells in various inflammatory skin diseases (s10-s12), the potential proinflammatory function of Gzms in other skin diseases and the underlying mechanisms warrant further investigation Acknowledgement We thank all patients that participated in this study, Lennart Blomqvist, Irene Gallais and Emma Wadman for help with sample collection This article is protected by copyright All rights reserved Accepted Article Ethics statement – The study was performed according to the Declaration of Helsinki Principles and approved by the Stockholm Regional Committee of Ethics (approval numbers 2012/50-31/2) Written consent was collected from all donors SC, EM, KB, DC, BR performed the experiments SC, LE designed the research study BR, MS, LE contributed essential reagents or tools SC, EM, LE analysed the data SC and LE wrote the paper that was read and revised by all authors Funding Funding was obtained from Swedish Medical Council, Psoriasisfonden, Hudfonden and the Stockholm County Council SC was funded by Croucher Foundation, Hong Kong LE is a Ragnar Söderberg Fellow of Medicine and supported by the Stockholm County Council (clinical research appointment) REFERENCE Voskoboinik I, Whisstock J C, Trapani J A Perforin and granzymes: function, dysfunction and human pathology Nature Reviews Immunology 2015: 15: 388-400 Nestle F O, Kaplan D H, Barker J Psoriasis N Engl J Med 2009: 361: 496-509 Perera G K, Di Meglio P, Nestle F O Psoriasis Annu Rev Pathol 2012: 7: 385-422 Zaba L C, Suárez-Fariñas M, Fuentes-Duculan J, et al Effective treatment of psoriasis with etanercept is linked to suppression of IL-17 signaling, not immediate response TNF genes The Journal of allergy and clinical immunology 2009: 124: 10221010.e1021-1395 Suárez-Fariñas M, Fuentes-Duculan J, Lowes M A, et al Resolved psoriasis lesions retain expression of a subset of disease-related genes J Invest Dermatol 2011: 131: 391-400 Cheuk S, Wikén M, Blomqvist L, et al Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis The Journal of Immunology 2014: 192: 3111-3120 This article is protected by copyright All rights reserved Accepted Article Yawalkar N, Schmid S, Braathen L R, et al Perforin and granzyme B may contribute to skin inflammation in atopic dermatitis and psoriasis The British journal of dermatology 2001: 144: 1133-1139 Metkar S S, Menaa C, Pardo J, et al Human and mouse granzyme A induce a proinflammatory cytokine response Immunity 2008: 29: 720-733 Arias M A, Jimenez de Bagues M P, Aguilo N, et al Elucidating sources and roles of granzymes A and B during bacterial infection and sepsis Cell Rep 2014: 8: 420-429 Figure Legends Figure CD8 T cells from psoriasis lesion express granzyme A (a) Representative histograms of granzyme A (GzmA), granzyme B (GzmB) and perforin expression in CD8 T cells freshly prepared from lesional psoriasis epidermis, dermis or PBMC gated on CD45+CD3+gdTCR-CD8+ cells Blue histograms depict isotype controls (b) Representative confocal microscopy showing CD3 (green) and granzyme A (red) staining in psoriasis lesion Scale bar = 10μm (c) Proportion of granzyme A (left), granzyme B (middle) and perforin (right) in epidermal, dermal and peripheral blood CD8 T cells from psoriasis (Ps, n=8) or healthy control skin (C, n=9) Mann-Whitney test, ** p