Interleukin (IL)-17A and IL-22-producing neutrophils in psoriatic skin

Dear Editor, The proinflammatory cytokine interleukin (IL)‐17 is recognized as playing an important role in the pathogenesis of psoriasis, an inflammatory skin disease; the relevance of the IL‐23/IL‐17 axis is underlined by the recent clinical efficacy of treatments targeting this axis. The IL‐17‐producing T‐cell subset Th17 has been widely studied in and closely linked to immune‐mediated human diseases. More recently, we have learned that IL‐17 is produced not only by adaptive immune cells, including Th17 cells, CD8+ T cells, and γδ+ T cells, but also by basophils, mast cells and neutrophils in the skin. Neutrophils are prominent in lesional psoriatic skin and can accumulate in the stratum spinosum as pustules of Kogoj and in the stratum corneum in Munro microabscesses. Although not represented in all psoriatic biopsies, these are histologically pathognomonic characteristics of psoriatic skin and strongly point towards a role of neutrophils in psoriasis that remains enigmatic. IL‐17A is secreted as a homodimer and binds to its receptor, IL‐17R, on keratinocytes, leading to increased production of the cytokines tumour necrosis factor‐α, IL‐1β, IL‐8 and IL‐6.4,5 Stimulation of keratinocytes with IL‐17 also upregulates gene expression of human β‐defensin 2 and other antimicrobial peptides, and chemokine CCL20.6,7 Confirming our unpublished observations, human and murine studies have shown that innate immune cells are important producers of IL‐17 in psoriasis. The aim of this study was to broaden our understanding of the pathogenesis of psoriasis by investigating the prevalence of IL‐17‐producing neutrophils in psoriatic skin compared with their IL‐17‐producing counterparts from the adaptive immune system. We also wanted to explore the interaction between keratinocytes and neutrophils in vitro with emphasis on the IL‐23/IL‐17 axis. Briefly (detailed methods are given in File S1; see Supporting Information), double immunofluorescence labelling studies were performed on paraffin sections from 4‐mm punch biopsies collected from 25 individuals, after obtaining informed, written consent, using antihuman IL‐17 (cat. no. AF‐317‐NA; R&D Systems, Abingdon, U.K.) in combination with antihuman CD4 (clone EPR6855, cat. no. 5636‐1; Epitomics, Burlingame, CA, U.S.A.), antihuman CD8 (clone C8/144B, cat. no. M7103; Dako, Glostrup, Denmark) or antimyeloperoxidase (clone 59A5, cat. no. NCL‐MYELO; Leica Biosystems, Nussloch, Germany). We found a high prevalence of CD4+ T cells in psoriatic skin, especially in the dermis. As expected, the prevalence of both CD4+ and CD8+ T cells was higher in psoriatic skin than in healthy skin (Fig. S1a; see Supporting Information). Myeloperoxidase‐positive cells, the majority comprising neutrophils, were found in psoriatic skin, albeit less prevalently than T cells in the dermis. A higher proportion of neutrophils were IL‐17 positive compared with CD4+ and CD8+ T cells in psoriatic skin (Fig. S1b; see Supporting Information). These results confirm that a substantial proportion of neutrophils produce IL‐17 in lesional psoriatic skin (Fig. S2; see Supporting Information). No neutrophils were observed in healthy skin; this too was as we expected. We additionally wanted to investigate whether coculture of primary keratinocytes from healthy donors could induce IL‐17 production in neutrophils. Keratinocytes have been shown to increase the lifespan of neutrophils in vitro and to prime them for production of superoxide anions.9 We also decided to study whether neutrophils could produce IL‐22, which has been shown to be produced by murine neutrophils but, to our knowledge, has not been associated with human neutrophils.10 IL‐22 is known to induce keratinocyte proliferation and maturation, features that are also associated with psoriasis pathology. Surprisingly, simply coincubating keratinocytes for 4 h with neutrophils could induce IL‐17 and IL‐22 production in neutrophils. Concurrently, a subset of neutrophils upregulate CD11b and downregulate CD62L, suggesting a state of activation (Fig. S2). As controls, we incubated peripheral blood mononuclear cells with keratinocytes and neutrophils with control medium; neither induced IL‐17 and IL‐22 production. Expression of IL‐17A and IL‐22 mRNA was measured by real‐time quantitative polymerase chain reaction, confirming increased expression of IL17A and IL22 genes in neutrophils cocultured with keratinocytes compared with directly isolated neutrophils or neutrophils in control medium (Fig. S2). CD11b mediates adherence to keratinocytes, partly through ICAM‐1.11 Blocking ICAM‐1 in coculture experiments with keratinocytes and neutrophils gave a 30% decrease in adherence to keratinocytes. Blocking CD11b reduces adherence even more, suggesting another binding counterpart.11 In our experiments and in the timeframe of these experiments, we observed that a subset of neutrophils did upregulate CD11b but in our analyses of the keratinocytes, no concurrent induction of ICAM1 expression was noted. Interferon gamma, used as a positive control, did induce ICAM1 expression, confirming previous findings of induction of ICAM1 expression on keratinocytes by activated T cells and their proinflammatory cytokines. In summary, this study suggests that neutrophils, long known to be a part of the histological characteristics of psoriasis, contribute to skin inflammation by producing IL‐17 and IL‐22. We do not know the extent of their influence on the pathology of psoriasis or in comparison with other IL‐17‐/IL‐22‐producing cell types but recent publications addressing dermatopathology in skin diseases such as hidradenitis suppurativa, Schnitzler syndrome and delayed‐pressure urticaria suggest a broader role of neutrophil‐produced IL‐17 in skin inflammation. Another aspect of IL‐17 production by neutrophils in psoriatic inflammation is that neutrophils, comprising more than 50% of leukocytes in the blood, might contribute to the cardiometabolic comorbidities associated with severe psoriasis. Increased frequency of circulating neutrophils has been observed in patients with psoriasis, concomitant with an activated state of these cells; thus they may provide a link between psoriasis and cardiovascular disease.12 Future studies will address the pathways and temporal aspects of activation of neutrophils by keratinocytes. Professor Niels Borregaard contributed to this work and approved the final manuscript, but not the revisions. We would like to dedicate this work to his memory. We thank Ahmed Gehad and Jessica Teague for technical advice and help. Funding sources: this work was supported by an unrestricted grant from Novartis. B.D.‐A. is supported by grants from the Novo Nordisk Foundation, the Lundbeck Foundation, the Højmosegård Grant, the Aage Bangs Foundation, the A.P. Møller Foundation for the Advancement of Medical Sciences, and Else and Mogens Wedell‐Wedellsborgs Foundation. None of the funding sources were involved in the study.