IL ‐17A localizes in the exocytic compartment of mast cells in psoriatic skin

Dear Editor, Psoriasis is a frequent, chronic recurrent inflammatory skin disease that substantially affects patients’ quality of life. Interleukin (IL)‐17A is believed to play a major role in its pathogenesis, as strategies to block this cytokine proved to be particularly effective in the treatment of psoriasis.1 However, the cellular sources of IL‐17A in psoriasis are still debated. T helper (Th)17 cells have been suggested as major contributors,2 3 although this hypothesis has been recently challenged by the histological analysis of lesional psoriatic skin from several independent groups. In the epidermis, neutrophils – forming so‐called Munro's microabscesses – stain strongly positive for IL‐17A, while in the dermal compartment IL‐17A is found mainly in mast cells, less so in neutrophils and only rarely in T cells.3,4,5 The role of mast cells as a possible source of IL‐17A in vivo remains debated. IL‐17A transcripts were found in mast cells cultured upon dissection of psoriatic skin6 and CD34‐derived mast cells were shown to produce IL‐17A in a RORC‐dependent manner in vitro.7 However, tonsil‐derived mast cells and LAD2 mast cell lines were reported to lack IL‐17A transcription machinery and rather to capture exogenous IL‐17A in vitro.8 9 The discrepancy between these studies might reflect the use of mast cells derived from different tissues or technical artefacts introduced by the in vitro manipulation. Herein, by analysing the skin lesions ex vivo using immunofluorescence, we provide additional evidence showing that mast cells produce IL‐17A in psoriasis. Six patients, five men and one woman aged 38–71 years (mean 51), with chronic plaque‐type psoriasis and mean Psoriasis Area and Severity Index of 9, were included in the study. All patients were off topical treatments for ≥ 1 week, systemic treatments for ≥ 2 weeks and biologics for ≥ 4 weeks. In agreement with previous studies,4 analysis of biopsy material from lesional skin revealed that IL‐17A was mainly found in tryptase+ dermal mast cells (range 91–100%) (Fig. S1a, c; see Supporting Information) and myeloperoxidase+ neutrophils found in epidermal Munro's microabscesses (Fig. S1b left panel; see Supporting Information). Neutrophils sitting in the dermis and T cells represented only a minority of IL‐17A‐expressing cells, while no CD68+ macrophages were found to contain IL‐17A (Fig. S1b right panel, c and d; see Supporting Information). Mast cells were present in normal and nonlesional skin in lower frequency than in lesional psoriatic skin but, similar to the latter, the majority stained positive for IL‐17A (Fig. S1a; see Supporting Information). IL‐17A staining was specific, as demonstrated by abrogation of the signal when the IL‐17A antibody was preincubated with a 10× excess of recombinant IL‐17A cytokine (Fig. S1a; see Supporting Information). To establish whether mast cells produce rather than capture IL‐17A, we assessed the presence of IL‐17A protein in a compartment of exocytosis (indicating production) or endocytosis (indicating capture) via confocal microscopy in samples from normal or psoriatic skin. We found that IL‐17A partially colocalized with the endoplasmic reticulum (calnexin) and Golgi apparatus (Golgi SNAP receptor complex) (Fig. 1a), but not with markers of late endosomes (Rab7) or of endocytic vesicles (clathrin, caveolin I) (Fig. 1b; and data not shown), suggesting that mast cells actively synthesize IL‐17A in lesional psoriatic skin. Activation of mast cells results in both degranulation (release of prepackaged mediators) and de novo synthesis, a process especially regarding cytokines. The high level of intracellular IL‐17A staining observed in skin‐resident mast cells in both the normal and pathological condition (Fig. S1a; see Supporting Information), prompted us to hypothesize that IL‐17A is stored in mast cell granules rather than being de novo synthesized. Confirming this hypothesis, IL‐17A strongly colocalized with tryptase and LAMP‐1, molecules expressed in mast cell secretory granules and secretory lysosomes, respectively (Fig. 1c). Details of antibodies used in this study are provided in Table S1 (see Supporting Information). IL‐17A is produced by mast cells in psoriatic plaques. Expression of IL‐17A (red) in combination with calnexin (endoplasmic reticulum) and Golgi (green, a), clathrin (endocytic vesicle) and Rab7 (late endosome) (green, b) or LAMP‐1 (secretory lysosomes) and tryptase (secretory granules) (green, c), assessed by immunofluorescence analysis in one representative psoriatic lesional skin sample of four. Two sections per patient were analysed. Original magnification × 63. Scale bar = 2 μm. Shown is the three‐dimensional (3D) reconstruction of colocalization. Together, our data indicate that skin mast cells synthesize IL‐17A and accumulate it in secretory granules. Our data are thus consistent with a model in which mast cells represent a source of IL‐17A in psoriatic lesional skin. A better understanding of the function of mast cell‐derived IL‐17A in psoriasis, and the stimuli involved in its production/release, is needed to define alternative therapeutic strategies and to better understand the mechanisms of action of anti‐IL‐17A biologics. Of note, clinical improvement of psoriasis upon neutralization of IL‐17A is accompanied by disappearance of epidermal neutrophils but not of IL‐17A+ mast cells.10 Mast cells armed with IL‐17A in the skin might well have a role in the pathogenic events leading to relapse after treatment interruption. Finally, further work should also address depiction of the IL‐17‐mediated events orchestrated by mast cells possibly participating in host innate protective mechanisms. We thank Professor G. Kaya (Department of Dermatology and Venereology, University Hospitals of Geneva, Switzerland) and Dr B.S. Schreve (Department of Pathology and Immunology, University of Geneva, Switzerland) for help in collecting patient samples. We also thank Professor C. Chizzolini (Department of Immunology and Allergy, University Hospitals of Geneva, Switzerland) and Dr Ludovic Wrobel (Department of Dermatology and Venereology, University Hospitals of Geneva, Switzerland), for critical discussion. E‐mail: wolf-henning.boehncke@hcuge.ch Funding sources: this work was supported by grant 310030_152680 from the Swiss National Science Foundation (SNSF), the Ernest Boninchi Foundation, the De Reuter Foundation and the Société Académique de Genève. Conflicts of interest: W.‐H.B. received honoraria as advisor or invited speaker from Amgen, Lilly and Novartis. The other authors state no conflicts of interest. Fig S1. Psoriatic plaques contain large numbers of IL‐17A‐positive mast cells and neutrophils. Table S1. List of antibodies used in the study.