Novel cytokine and chemokine markers of hidradenitis suppurativa reflect chronic inflammation and itch

To the Editor, Hidradenitis suppurativa (HS) is an auto‐inflammatory skin disease characterized by recurrent, chronic painful and pruritic inflammatory nodules, abscesses and sinus tracts in predominantly the axillary, inguinal, and gluteal areas. A key element of the HS pathophysiology is occlusion of the follicular infundibulum and subsequent cyst formation, followed by rupture of the cyst inducing an intense inflammatory response. Accordingly, identification of inflammatory markers is important for the clinical stratification of HS and may help refining treatment choices. To date, no studies have investigated inflammatory protein levels in the serum/plasma and skin in parallel in a cohort of HS patients. Therefore, the primary aim of this study was to simultaneously detect important cytokines and chemokines in, respectively, the plasma and lesional skin of patients with HS at a single time point. Blood and skin samples from 20 patients with a dermatologist‐ verified diagnosis of HS and 10 healthy controls (Data S1) were prospectively collected in the Department of Dermatology of the Erasmus University Medical Center and Sint Franciscus Hospital in Rotterdam, the Netherlands. Skin samples of HS patients suffering of Hurley I to III disease severity were taken from actively inflamed, non‐fluctuating, indurated, erythematous lesions, or plaques recurring on fixed locations. The research protocol was approved by the local Institutional Review Board (reference MEC‐2013‐337/NL45264.078.13). All participants provided written informed consent. Punch biopsies of 4 mm in diameter were obtained and immediately snap‐frozen in liquid nitrogen. Venous blood was collected in vacuum EDTA tubes under sterile conditions, and after separation of the plasma samples were aliquoted and stored at −80°C until analysis. Samples were analyzed using the Meso Scale Discovery (MSD) V‐PLEX Human Cytokine 30‐plex kit (K15054D; Meso Scale Discovery, Gaithersburg, MD, USA) according to the manufacturers’ instructions (Data S1). Moreover, three chemokines, which have not previously been reported to be overexpressed in HS patients, were additionally analyzed by immunohistochemistry (Data S1). Plasma protein concentrations were expressed as picogram (pg) per milliliter (mL), whereas skin protein levels were normalized for milligram (mg) tissue dry weight (pg/mg). In case, a protein level was below the detection limit, the lowest limit of quantification (LLOQ) was used for further calculations. If more than 50% of the samples per analyzed protein in either the HS or the healthy control group had values below the LLOQ, values were substituted by two categories: detectable vs non‐detectable, that is above or below the LLOQ, respectively. For the primary objective, either the Mann‐ Whitney U test or Fisher's exact test was used to assess the null‐ hypothesis that there was no difference in the levels of individual markers between control and HS samples. Secondly, correlations between protein levels of plasma and lesional HS skin were calculated (Data S1). Statistical analyses were conducted using SPSS Statistics 24.0 (IBM Corporation, Armonk, NY, USA). A two‐sided P value below 0.05 was considered significant. This level was corrected by a false discovery rate using the Benjamini‐Hochberg test for multiple comparisons. In plasma, 20 of 30 (66.7%) analytes were detected. In the skin, 25 of 26 (96.2%) proteins were detected, while four proteins (IL‐4, IL‐7, VEGF, GM‐CSF) were not analyzed because they have not been validated for skin‐derived samples. In plasma, CCL‐26 was detected significantly more often in HS patients (16 of 20) compared with healthy controls (2 of 10), P = 0.004 (Table 1). Accordingly, the median CCL‐ 26 level in HS patients was 24.9 pg/mL, interquartile range 19.1−37.0 (Figure S1). In contrast, plasma CXCL‐10 levels were significantly lower in HS patients, P = 0.003. In lesional skin, IL‐16 (P < 0.001), IL‐ 17A (P < 0.001), CXCL‐8 (P = 0.001), plus IL‐8 HA (P = 0.011), representing very high CXCL‐8 concentrations, IL‐12/23p40 (P = 0.007), CCL‐4 (P = 0.011), CXCL‐10 (P = 0.011) showed higher levels in HS patients compared with healthy controls (Table 2, Figure S2). The elevated CCL‐4 and CXCL‐10 protein levels in HS lesions were confirmed by immunohistochemistry (Figure S3). A strong staining of CCL‐26 was observed in lesional skin, despite the fact that CCL‐26 protein was not detected in lesional HS skin by the MSD assay (Table 2, Figure S3). Only weak correlations were observed between protein levels in HS plasma and lesional skin (Data S1, Table S1). Chemokine CCL‐26 (also known as eotaxin‐3) is a newly identified inflammatory marker in HS patients. Significant elevation of this chemokine in the serum has previously been reported in atopic dermatitis and cutaneous T‐cell lymphoma, which are characterized by the infiltration of eosinophils, basophils, and specific subpopulations of T cells, and all, like HS, diseases characterized by high pruritus scores. Interestingly, CCL‐26 was found in Abbreviations: CCL, C-C motif ligand; CRP, C-reactive protein; CXCL, C-X-C motif ligand; EDTA, ethylenediaminetetraacetic acid; GM-CSF, granulocyte-macrophage colonystimulating factor; HS, hidradenitis suppurativa; IL, interleukin; LLOQ, lowest limit of quantification; MMP, matrix metalloproteinase; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.