Differential synovial tissue biomarkers among psoriatic arthritis and rheumatoid factor/anti-citrulline antibody-negative rheumatoid arthritis

Eighty-nine patients were enrolled in the study [34 oligo-polyarticular PsA patients (12 naïve and 22 inadequately responder to methotrexate (MTX-IR) respectively) and 55 RA patients (27 naïve and 28 MTX-IR respectively)] fulfilling the classification criteria for PsA or RA [6, 7]. At baseline, demographical, clinical, and inflammatory parameters were collected for each patient. All RA patients were confirmed as being seronegative (Ab^neg) for IgA- and IgM-RF (Orgentec Diagnostika, Bouty, UK) and ACPA (Axis Shield Diagnostics, Bouty, UK) using commercial ELISA at study entry. Each naïve PsA and Ab^neg RA patient was then treated with methotrexate (up to 20 mg/week) and followed every 3 months for at least 6 months to assess the rate of achievement of Minimal Disease Activity (MDA) or DAS remission for PsA and Ab^neg RA patients respectively [8, 9]. MTX-IR PsA and Ab^neg RA patients were treated according to the current recommendations [10, 11]. The study protocol was approved by the local Ethical Committee, and all subjects provided signed informed consent.

At study entry, each PsA and Ab^neg RA patient underwent ultrasound-guided synovial tissue biopsy of the knee. Joint inflammatory activity was tested for each patient using power Doppler as previously described [12]. Each tissue was tested through immunohistochemistry for the presence of CD68 (macrophages), CD21 (follicular dendritic cells), CD3 (T lymphocytes), CD20 (B lymphocytes), and CD31 (endothelial cells) staining following the already published protocol [12]. Other tissue sections were stained for plasma cell and mast cell detection, using CD138 mouse antihuman monoclonal antibody (clone MI15) or CD117 mouse antihuman monoclonal antibody (clone EP10) (all from Leica Biosystem, Newcastle, UK) by immunostainer BOND MAX III (Leica). Slides were examined by two independent evaluators using a light microscope (Leica DM 2000), and all tissues were evaluated using a numerical score based on the number of CD68⁺, CD21⁺, CD3⁺, CD20⁺, CD117⁺, and CD138⁺ cells (two different fields in each section), with a score of 0 indicating no positive cells, 1 indicating < 10% positive cells, 2 indicating 10–50% positive cells, and 3 indicating > 50% positive cells. CD31⁺ vessel count was done as mean of the values from three different fields in each section [12]. The inter-rater agreement coefficient was assessed for each single IHC marker (see Additional file 2: Table S1).

Statistical analysis was performed using SPSS V. 20.0 (SPSS. Chicago, IL, USA) and Prism software (GraphPad, San Diego, CA, USA). Categorical and quantitative variables were described as frequencies, percentage, and mean ± SD. Data on demographic and clinical features were compared between patients by the non-parametric Mann-Whitney U test or χ² test, as appropriate. Spearman’s rank correlation test was used for correlation in all analyses. ROC analysis was performed to identify the best cut-off value for IHC scores associated with the highest rate of MDA or DAS remission achievement in PsA and Ab^neg RA patients respectively. A value of p ≤ 0.05 was considered statistically significant.

Demographic and clinical characteristics of PsA and Ab^neg RA cohorts (naïve and MTX-IR respectively) enrolled in the study are summarized in Table 1. Comparing the different study cohorts, there were no significant differences according to age and gender (p > 0.05). PsA and Ab^neg RA patients naïve to treatment showed significant shorter disease duration (0.80 ± 0.24 years for naive PsA and 0.84 ± 0.95 Ab^neg RA patients respectively) compared to MTX-IR PsA and Ab^neg RA patients (5.83 ± 3.68 years for MTX-IR PsA, p < 0.001; 5.68 ± 5.28 for MTX-IR Ab^neg RA patients, p = 0.002 respectively). Considering the clinical parameters, there were no significant differences comparing PsA and Ab^neg RA patients based on swollen and tender joint counts or Disease Activity Scores in naïve and in MTX-IR subgroups (Table 1). However, considering inflammatory markers, ESR plasma levels were significantly higher in naïve Ab^neg RA patients (44.00 ± 25.40 mm/first hour) than MTX-IR Ab^neg RA patients (29.60 ± 36.94 mm/first hour; p = 0.03), whereas no differences were found for CRP plasma levels comparing naïve and MTX-IR PsA subgroups (Table 1).

Each enrolled patient underwent US-guided ST biopsy, and IHC for CD68⁺, CD21⁺, CD20⁺, and CD3⁺ was performed. PsA patients showed similar IHC CD68⁺ cell score in the lining (1.95 ± 0.90) and sublining (1.81 ± 0.95) compared to Ab^neg RA patients (1.96 ± 0.75 for lining CD68⁺ cells, p = 0.94; 1.43 ± 0.76 for sublining CD68⁺ cells, p = 0.11 respectively) regardless of the treatment scheme (Fig. 1a–d). Furthermore, IHC analysis revealed that PsA and Ab^neg RA patients had comparable levels of CD21⁺ cells (0.72 ± 0.79 vs 0.60 ± 0.95; p = 0.18 respectively) regardless of the treatment scheme (Fig. 1a–d).

Stratifying the study cohorts according to the treatment scheme, naïve PsA showed similar IHC scores for lining (2.00 ± 0.85) and sublining CD68⁺ cells (1.71 ± 0.96) compared to naïve Ab^neg RA patients (1.96 ± 0.79 for lining CD68⁺ cells, p = 1.00; 1.44 ± 0.80 for sublining CD68⁺ cells, p = 0.39 respectively). Moreover, naïve PsA showed similar IHC scores for CD21⁺ cells (0.75 ± 0.94) compared to naïve Ab^neg RA patients (0.52 ± 0.91; p = 0.43) (Fig. 1g), showing a direct correlation between ESR plasma levels and lining CD68⁺ cells IHC score (r = 0.66; p = 0.04). Similarly, MTX-IR PsA patients showed comparable IHC scores for lining (1.92 ± 0.95) and sublining CD68⁺ cells (1.86 ± 0.96) than MTX-IR Ab^neg RA patients (2.00 ± 0.72 for lining CD68⁺ cells, p = 0.79; 1.46 ± 0.78 for sublining CD68⁺ cells, p = 0.25) (Fig. 1e–h). Finally, MTX-IR PsA showed similar IHC scores for CD21⁺ cells (0.71 ± 0.73) compared to MTX-IR Ab^neg RA patients (0.68 ± 1.00; p = 0.91) (Fig. 1g).

Analyzing the microanatomical organization of the synovial tissue infiltrates, 15 (44.1%) PsA patients compared to 24 (43.6%, p = 0.51) Ab^neg RA patients showed follicular synovitis regardless of the treatment regimen. Moreover, there was no difference in the rate of follicular synovitis stratifying patients according to the therapeutic regimen (41.7% of naïve PsA patients with follicular synovitis compared to 45.5% of MTX-IR PsA patients with similar synovitis pattern, p = 0.79; 44.4% of naïve Ab^neg RA patients with follicular synovitis compared to 42.8% of MTX-IR Ab^neg RA patients with similar synovitis pattern, p = 0.51).

CD20 IHC revealed that PsA and Ab^neg RA patients had similar levels of CD20⁺ cells (1.08 ± 0.73 in PsA vs 1.44 ± 0.89 in Ab^neg RA, p = 0.07) (Fig. 2a–d) and after stratification based on the treatment regimen, PsA patients showed similar IHC CD20⁺ cell score than Ab^neg RA (1.00 ± 0.85 in naive PsA vs 1.44 ± 0.92 in naive Ab^neg RA, p = 0.16; 1.14 ± 0.67 in MTX-IR PsA vs 1.45 ± 0.87 MTX-IR Ab^neg RA, p = 0.18) (Fig. 2e).

Analyzing synovial CD3⁺ cell distribution, IHC showed that PsA and Ab^neg RA patients had similar levels of synovial CD3⁺ cells (1.71 ± 0.86 in PsA and 1.54 ± 0.86 in Ab^neg RA; p = 0.45 respectively) (Fig. 2a–d). Moreover, PsA and Ab^neg RA patients did not differ in terms of synovial CD3⁺ cells stratifying patients based on the treatment scheme (1.45 ± 0.83 in naive PsA vs 1.57 ± 0.88 in naïve Ab^neg RA, p = 0.70; 1.84 ± 0.86 in MTX-IR PsA vs 1.52 ± 0.87 MTX-IR Ab^neg RA, p = 0.19) (Fig. 2f).

CD117 IHC showed that PsA patients are characterized by higher IHC scores for CD117⁺ cells (1.25 ± 0.61) compared to Ab^neg RA patients (0.62 ± 0.46, p < 0.001) regardless of the treatment scheme (Fig. 3a–d). Interestingly, stratifying the study cohorts according to the treatment scheme, naïve PsA patients showed higher CD117⁺ cell IHC scores (1.29 ± 0.65) compared to naïve Ab^neg RA patients (0.59 ± 0.44; p = 0.0004). Similarly, MTX-IR PsA patients showed higher CD117⁺ IHC score (1.23 ± 0.61) compared to MTX-IR Ab^neg RA patients (0.64 ± 0.48, p = 0.001) (Fig. 3e).

Conversely, analyzing the distribution of CD138⁺ cells within the synovial tissue, Ab^neg RA patients showed higher IHC scores for CD138⁺ cells (1.58 ± 1.09) compared to PsA patients (0.72 ± 0.71; p < 0.001) independently of the treatment scheme (Fig. 3f–i). Considering the different study population subgroups, naive Ab^neg RA patients were characterized by higher CD138⁺ cell IHC score (1.65 ± 1.00) compared to naive PsA patients (0.96 ± 0.84; p = 0.04). Similarly, MTX-IR Ab^neg RA patients had higher CD138⁺ cell IHC score (1.51 ± 1.19) compared to MTX-IR PsA patients (0.59 ± 0.61, p = 0.002) (Fig. 3j). As shown in Fig. 3k, the combination of low IHC score for CD117⁺ (IHC score = 1) and high IHC score for CD138⁺ cells (IHC score = 3) significantly differentiates PsA than Ab^neg RA synovitis [OR (95% CI), 34.04 (1.535–2.398); p = 0.0002].

To assess the microvasculature at the synovial tissue level, each tissue was tested for the presence of CD31⁺ blood vessels. As shown in Fig. 4a–e, naïve PsA patients showed higher number of synovial CD31⁺ vessels (36.63 ± 11.02) compared to MTX-IR PsA patients (23.65 ± 11.67; p = 0.01). Similarly, naïve Ab^neg RA patients showed a higher number of synovial CD31⁺ vessels (35.43 ± 7.23) than MTX-IR Ab^neg RA patients (22.14 ± 5.32; p < 0.001). No significant differences in terms of CD31⁺ vessel count were found comparing PsA and Ab^neg RA patients stratified based on the same treatment category (Fig. 4e).

After study enrollment, each naïve patient started methotrexate treatment and was followed for at least 6 months on outpatient setting and treatment response rate was recorded at each clinical assessment (MDA for PsA and DAS remission for Ab^neg RA patients respectively). After 6 months follow-up from methotrexate beginning, 6 (50.0%) PsA patients achieved MDA, while 11 (40.7%) Ab^neg RA patients achieved DAS remission. As shown in Table 2, naïve PsA patients who reached the MDA status at 6 months follow-up had lower CD3⁺ cell IHC score (0.91 ± 0.33) before methotrexate beginning, compared to naïve PsA patients who did not achieve the clinical endpoint at 6 months (1.99 ± 0.84; p = 0.02). Conversely, naïve Ab^neg RA patients who reached the DAS remission status at 6 months follow-up had lower sublining CD68⁺ cell IHC score (0.92 ± 0.58) before methotrexate beginning, compared to naïve Ab^neg RA patients who did not achieved the clinical endpoint at 6 months (2.13 ± 0.55; p < 0.001) (Table 2). To define the best cut-off value for CD3⁺ cell scores and CD68⁺ cell sublining scores in naive PsA and Ab^neg RA respectively, ROC analysis was performed for each parameter (Additional file 1: Figure S1a-b). In particular, naive PsA patients who reached a MDA status after 6 months follow-up had more likely baseline CD3⁺ cell scores< 1.25 (83.3%) than naive PsA patients who did not reach MDA status (16.7%, p = 0.02). Moreover, naive Ab^neg RA patients who reached DAS remission after 6 months follow-up had more likely baseline CD68⁺ cell sublining scores < 2.25 (65.0%) than naive Ab^neg RA patients who did not reach DAS remission (0.0%, p = 0.03). In relation to MTX-IR patients, there was no significant difference in MDA and DAS remission status achievement in PsA and Ab^neg RA patients respectively, even stratifying patients based on the therapeutic strategy after MTX failure (c-DMARD combination or addition of b-DMARDs) (40.0% MTX-IR PsA patients achieved MDA after c-DMARD combination vs 64.7% MTX-IR PsA patients achieved MDA after b-DMARD addition, p = 0.32; 44.4% MTX-IR Ab^neg RA patients achieved MDA after c-DMARD combination vs 50.0% MTX-IR Ab^neg RA patients achieved MDA after b-DMARD addition, p = 0.78).