Association of CCL2 with systemic inflammation in Schnitzler’s syndrome Karoline Krause, Robert Sabat, Ellen Witte-Händel, Anne Schulze, Viktoria Puhl, Marcus Maurer, and Kerstin Wolk   Charité – Universitätsmedizin Berlin, Germany Dept. of Dermatology and Allergy Institute of Medical Immunology Berlin-Brandenburg Centre for Regenerative Therapies British Journal of Dermatology. DOI: 10.111/bjd.17334

Karoline Krause, M.D. Kerstin Wolk, Ph.D.

Introduction What’s already known? Schnitzler’s syndrome (SchS) is a rare, acquired autoinflammatory disease, clinically characterized by urticarial exanthema, arthralgia and osterosclerosis, and episodes of fever The pathogenesis involves an over-activation of the IL-1 system The exact pathogenetic pathways are substantially unknown and biomarkers for assessing the inflammatory activity are missing in SchS

Objective This study aimed at the identification and characterization of pathogenetic players in SchS

Methods Collection of clinical, laboratory and demographic data of patients with SchS (n=17) Quantification of inflammatory parameters including cytokines, chemokines and antimicrobial proteins in the blood of these SchS patients and, for comparison, healthy subjects and patients with psoriasis and hidradenitis suppurativa using ELISA Analysis of CCL2 (a chemoattractant for monocytic and further mononuclear immune cells) expression in cytokine-stimulated primary cells [immune cells (peripheral blood mononuclear cells), keratino-cytes, fibroblasts, endothelial cells] by RT-qPCR and ELISA

Results CCL2 serum levels are markedly elevated and linked to disease activity in SchS IL-1b IL-6 IL-17 IL-19 1.5 30 1.0 12 P=0.003 P=0.024 P=0.098 P=0.461 0.8 1.0 20 8 0.6 serum level [pg/ml] 0.4 0.5 10 4 0.2 0.0 1.00 1.00 IL-22 TNF-a CCL2 b-def. 2 20 3 600 3000 0.75 0.75 P=0.683 P=0.003 P=0.003 P=0.954 serum level [ng/ml] CCL2 15 serum level [ng/ml] CCL2 2 0.50 400 2000 0.50 serum level [pg/ml] 10 0.25 1 200 1000 0.25 5 rs = 0.61, P=0.040 rs=0.53, P=0.064 0.00 0.00 5 10 15 1 2 5 4 3 healthy Schnitzler’s syndrome disease activity [PGA] bone / muscle pain [activity grade] CCL2 800 P=0.000 P=0.366 P=0.785 Serum levels quantified by ELISA; rs: Spearman’s correlation coefficient. healthy 600 Schnitzler’s syndrome serum level [pg/ml] 400 psoriasis vulgaris hidradenitis suppurativa 200

Results Mononuclear immune cells and fibroblasts produce CCL2 induced by IL-1ß and TNF-α CCL2 0.8 0.8 0.8 0.8 IL-1b: CCL2 quantification in cells and culture supernatants of 24h cytokine-stimulated primary immune cells (PBMC), endothelial cells (EC), fibroblasts (Fb) and keratinocytes (KC) using RT-qPCR and ELISA. 0.6 0.6 0.6 0.6 0 ng/ml 1 ng/ml secreted protein [ng/ml] 0.4 0.4 0.4 0.4 10 ng/ml 0.2 0.2 0.2 0.2 100 ng/ml 0.0 0.0 0.0 0.0 PBMC EC Fb KC CCL2, PBMC CCL2, Fb CCL2, Fb CCL2, Fb 6000 6000 10 400 300 4000 4000 1 IL-1b only secreted protein [% control] mRNA [relative to HPRT] secreted protein [% of IL-1b] 200 IL-1b + indicated cytokine(s) 2000 2000 0.1 100 0.0 0.0 0.01 TNF-a IL-1b IL-17 TNF-a + IL-17 - IL-12 IL-23 IL-36b TNF-a IFN-g GM-CSF - IL-1b IL-17A IL-19 IL-22 IL-24 IL-36b TNF-a IFN-g - IL-1b IL-17A TNF-a

Results CCL2 levels are associated with IL-1ß and TNF-α levels and anti-IL-1ß therapy response in SchS CCL2 Disease activity 1.00 1.00 0.6 0.0 0.2 1.0 0.8 0.4 0.6 0.0 0.2 1.0 0.8 0.4 15 5 25 20 10 25 P=0.004 P=0.004 20 0,75 0.75 serum level [ng/ml] CCL2 serum level [ng/ml] CCL2 15 0,50 0.50 serum level [ng/ml] PGA 10 0.25 0.25 5 rs=0.48, P=0.025 rs=0.64, P=0.010 0.00 0.00 0.001 0.01 0.1 1 10 100 0.1 1 10 TNF-a [pg/ml] 0 wks 1 wk 0 wks 1 wk 0 wks 1 wk 0 wks 1 wk IL-1b [pg/ml] ≥ 100 wks ≥ 100 wks time of patient treatment with canakinumab Serum levels quantified by ELISA; rs: Spearman’s correlation coefficient.

Discussion Quantifying CCL2 blood levels may allow the objective estimation of inflammatory disease activity and may help with the therapy decision in SchS patients CCL2 may be a key element of the pathogenetic cascades in SchS, especially those important for osterosclerosis

Discussion Proposed role of CCL2 in SchS

Conclusions What does this study add? The chemokine CCL2 is significantly upregulated and is linked to global disease activity and treatment response in SchS patients CCL2 is especially correlated with bone pain in these patients The cellular sources of CCL2 include mononuclear immune cells and fibroblasts IL-1β and TNF-α, whose blood levels correlate with CCL2 levels in SchS patients, are important CCL2 inducers

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