Identification of biomarkers involved in the resolution phase of inflammation: a translational study of Specialized Pro-resolving Mediators role in Rheumatoid Arthritis

OBJECTIVES: Rheumatoid Arthritis (RA) is a chronic autoimmune disease in which uncontrolled inflammation lead by cells from innate and adaptive immune system leads to tissue damage and disability. To date, the wider pharmacological armamentarium significantly increased the chance of disease control and sustained clinical remission achievement in RA. However, little is known about the mechanisms involved in the resolution phase of inflammation in rheumatic diseases as well as the possible role of Specialized Pro-resolving Mediators (SPMs) as putative pathogenetic and/or therapeutic targets. The aim of this translation study was to dissect whether SPMs and their receptors ERV1, ALX/FPR2 and BLT1 might act as soluble or tissue biomarkers in RA useful for patient stratification across disease phases in clinical practice, improving the therapy management. Moreover, the secondary outcome was wider aiming to increase our knowledge about RA pathophysiology of remission status in. METHODS: 68 patients with RA (27 naïve-to-treatment, 23 DMARDs-not-responder and 18 in sustained clinical and ultrasound remission respectively) were enrolled in the study and underwent PB drawing and ultrasound-guided ST biopsy (n=48). 13 patients with undifferentiated peripheral inflammatory arthritis (UPIA) and 9 with osteoarthritis (OA) were enrolled as comparison groups. Demographic, clinical, immunological and ultrasonographic features were collected for each patient. Determination of serum cytokines and chemokines concentrations (IL-1beta, TNF-alpha, IL-6, IFN-gamma, IL-12p70, IL-10, IL-4, IL-2, Chemerin and GAS6) were performed by ELISA. Furthermore, SPMs and Arachidonic Acid (AA) derived pro-inflammatory molecules determinations in snap frozen synovial tissue biopsies from RA patients in different disease phases (active and remission respectively) were performed by LC-MS/MS. Expression of ERV1, ALX/FPR2 and BLT1 in CD45+CD3+ and CD45+CD19+ was assessed by FACS on PB and on synovial tissue-derived cell suspensions. Moreover, ERV1, ALX/FPR2 and BLT1 expression was assessed by FACS on NK cells (CD45+CD3-CD19-CD56+), neutrophils and monocytes (CD45+CD14+) from PB and macrophages (CD45+CD11b+CD64+) from ST only respectively. Synovitis degree was determined using a H&E based semiquantitative score. Some ST samples were used for quantification of ERV1, ALX/FPR2 and BLT1 genes expression by RT-PCR. RESULTS: Synovial tissue inflammation in terms of semiquantitive score and the cytokine milieu in peripheral blood directly mirror the disease Activity status in RA. RT-PCR on ST samples revealed that ST from RA in high disease activity was enriched of SPM receptors when compared to RA in sustained remission and OA (ERV1: 4.4 vs 1.1 (p= 0.012) and 1.2 (p= 0.005); ALX/FPR2: 4.9 vs 1.5 (p= 0.0006) and 0.8 (p= 0.003); BLT1: 5.9 vs 1.6 (p= 0.016) and 1.1 (p= 0.002) respectively). In particular, C-Reactive Protein (CRP) serum levels, directly correlated with BLT1 expression on PB-derived CD45+CD14+ cells (r=0.27; p=0.023) of RA regardless to the disease phase. Conversely, ST of RA in sustained remission was depleted of BLT1 in CD45+CD3+ cells compared to other conditions (OA p=0.017; UPIA p=0.002; naïve-to-treatment RA p=0.01). LC-MS/MS analysis revealed that synovial tissue of RA in sustained remission the ratio between SPM and AA-derived pro-inflammatory molecules is significantly increased when compared to synovial tissue of RA patients with high disease activity (101.3 vs 2153.00 (84.06-3333.00) respectively) CONCLUSIONS: SPM receptors expression in PB and ST compartments are reciprocally related to disease activity across disease phases in RA suggesting a putative active modulatory role in maintaining the remission phase.