Healthy controls
Healthy subjects were recruited through a field campaign. The recruitment was prospective aiming for each 10 females and 10 males in 6 different decades (21–30, 31–40, 41–50, 51–60, 61–70, and 71–80 years) [25]. A detailed history taking and clinical examination was done in all subjects by skilled rheumatologists (AH/JR) to rule out tenderness, stiffness, swelling, and bony swelling. Subjects must have been free of present or past signs of rheumatic disease and of cancer, diabetes mellitus, cardiovascular disease (angina, myocardial infarction, stroke) as well as chronic renal, gastrointestinal, and hepatic disease. Subjects had to be tested negative for rheumatoid factor or anti-cyclic citrullinated protein antibodies (ACPA). The presence of psoriasis or a family history of psoriasis was also not allowed.
Patients
We recruited each 15 patients in the following forms of mono- and poly-symptomatic psoriatic disease: (1) isolated psoriatic skin disease (skin, S) (2) isolated enthesitis in patients with personal or family history of psoriasis (entheses, E), (3) isolated arthritis in patients with personal or family history of psoriasis (arthritis, A), (4) psoriatic skin disease with enthesitis (SE), (5) psoriatic skin disease with arthritis (SA), (6) arthritis and enthesitis in patients with personal or family history of psoriasis (EA), and (7) the full spectrum with psoriatic skin disease, arthritis, and enthesitis (SEA). Psoriatic skin disease (S) was defined as active plaque psoriasis confirmed by the dermatologist. Enthesitis (E) was defined as tenderness at least one entheseal site of the SPARCC score over at least 6 weeks in patients with active psoriasis (SE, SEA) or patients with personal or family history of psoriasis (E, EA). The presence of entheseal inflammation has to be confirmed by a positive Power Doppler signal at least one entheseal site. Arthritis (A) was defined as joint swelling and pain for at least 6 weeks in patients with active psoriasis (SA, SEA) or patients with personal or family history of psoriasis (A, EA). To reduce the level of complexity psoriatic patients with an axial disease were not included in this study. For practical reasons, we accepted very minor skin abnormalities (PASI up to 1) in the subgroups without skin disease. All healthy subjects and patients gave written informed consent. The ethical committee of the University Clinic of Erlangen approved the study.
Clinical assessments
Demographic (age, sex, body mass index, smoking status) and disease activity parameters for skin (psoriasis area severity index, PASI), entheseal disease (Spondyloarthritis Research Consortium of Canada, SPARCC), and joints disease (swollen joint count 66) were collected in all patients.
Serum analyses
High-sensitivity CRP was measured by turbimetric assay with an Optilyte Analyzer from The Binding Site (Birmingham, UK). In addition, the following markers were analyzed by enzyme-linked immune sorbent assay: (1) calprotectin (S100A8/S100A9 heterodimers; R&D Diagnostics, cat. no. DS8900; normal range 481–6540 ng/mL; intra-assay precision 5.6 ng/mL; inter-assay precision 6.1 ng/mL); (2) IL-22 (R&D Diagnostics, no. D2200; normal range 0–53.3 pg/mL; intra-assay precision 56.5 pg/mL; inter-assay precision 63.0 pg/mL); (3) IL-8 (R&D Diagnostics, no. HS800; normal range 2.8–16.5 pg/mL; intra-assay precision 5.5 pg/mL; inter-assay precision 5.0 pg/mL; (4) lipocalin 2 (R&D Diagnostics, no. DLCN20; normal range 2.8–16.5 pg/mL; intra-assay precision 1.14 ng/mL; inter-assay precision 1.05 ng/mL); (5) beta-defensin 2 (Alpha Diagnostic, catalog no. 100–250-BD-2; no information on precision and normal range provided by manufacturer), [6] IL-17 (R&D Diagnostics, no. HS170; normal range not detectable − 0.4 pg/mL; intra-assay precision 2.03 pg/mL; inter-assay precision 2.09 pg/mL), [7] IL-23 (R&D Diagnostics, no. D2300B; normal range not detectable − 40.5 pg/mL; intra-assay precision 180 pg/mL; inter-assay precision 203 pg/mL); (8) VEGF (R&D Diagnostics, no. DVE00; normal range 62–707 pg/mL; intra-assay precision 29.2 pg/mL; inter-assay precision 32.8 pg/mL), [9] LL37 (Cusabio, no. E14948; no information on precision and normal range provided by manufacturer), (10) pentraxin 3 (R&D Diagnostics, no. DPTX30B; normal range not detectable − 1.36 ng/mL pg/mL; intra-assay precision 2.12 ng/mL; inter-assay precision 2.05 ng/mL).
Measurements were done in a blinded way in duplicates by scientists not involved in the clinical patient assessment (MS, KN, YL) in a three-step approach: (i) first signal finding step, (ii) second validation step, and (iii) longitudinal step. In the signal finding step (which did not include routine CRP, 10 healthy controls and 10 patients with active polymorphic psoriatic arthritis (including skin, entheseal, and joint disease, SEA) were tested for all 10 parameters (see above) to find a difference between healthy controls and disease. Cut-offs for positive values were defined as the normal value plus 3 standard deviations. In the validation step, 105 controls and 105 disease samples (each 15 from the 7 aforementioned disease patterns) were tested for the 5 parameters (beta-defensin 2, lipocalin 2, IL-22, IL-8, calprotectin), which showed significant differences between controls and disease, as well as for CRP. And finally, in the longitudinal step, each 10 PsA patients (A, SA or SEA) receiving treatment with either TNF- or IL-17 inhibition were tested for the parameters before treatment and 3 months after initiation of treatment.
Statistical analysis
Data were collected, organized, and analyzed through SPSS software for statistics (IBM SPSS 21.0, IBM corporation®, Armonk, NY, USA). With respect to demographic and disease-specific characteristics, categorical variables are presented as numbers and percentages, continuous variables are provided as mean ± standard deviation (SD), if not stated otherwise. Assumptions of normally distributed continuous variables were tested using quantile-quantile plots, Kolmogorov-Smirnov, and Shapiro-Wilk test. For comparison of the above-mentioned serum parameters between healthy controls and the respective disease groups, unpaired Student’s t test was applied. For comparison of baseline pre-treatment values and post-treatment values, paired Student’s t test was used. P values ≤ 0.05 were considered statistically significant.