Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: SYNOVIAL MACROPHAGES IN PATIENTS WITH RHEUMATOID ARTHRITIS: PREDICTORS OF RESPONSE AND THEIR MODIFICATIONS TO TREATMENT

ABSTRACT Introduction: Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation. Predicting treatment response remains challenging both in treatment naïve patients and in Methotrexate (MTX) – failure. Recent research highlights the relevance of synovial biopsies (SB) to better understand immune cell dynamics, particularly the role of macrophage polarization, in assessing disease activity and treatment outcomes. Aim: This study aims to determine the predictive value of macrophage infiltration in synovial tissue for treatment outcomes in RA patients. Specifically, it explores how histological and immunohistochemical profiles from synovial biopsies correlate with disease remission and response to DMARDs in different subsets of disease, specifically Early Rheumatoid Arthritis (ERA) patients and Methotrexate failure patients treated with TNFi. In this second cohort, the study investigates macrophage immune profiles (M1/M2) pre-and -post-treatment. Methods: Two cohorts of RA patients were enrolled. The Early Rheumatoid Arthritis (ERA) cohort included patients naïve to DMARD treatment with disease duration < 12 months. They received a baseline pre-treatment SB. The MTX-Failure cohort included patients with persistent disease activity despite MTX therapy, eligible for bDMARDs [Tumor Necrosis Factor Inhibitor (TNFi)]. With a repeated biopsy protocol, they received a pre-TNFi SB with second tissue samples repeated after 12 weeks of treatment. SB were analyzed using histology and immunohistochemistry to quantify immune infiltrates. Histological features [Synovial Hyperplasia, Fibrinoid Necrosis (FN), Hypervascularization, and Inflammatory Infiltrate] and IHC (CD3, CD20, CD138, CD68) were each semi-quantitatively assessed on a 0-3 scale with 7 levels. Flow cytometry was used to differentiate macrophage subpopulations (M1/M2) in MTX-failure patients. Statistical analyses identified predictors of treatment response using univariate analysis and multivariate logistic regression. Results: In the ERA cohort, a strong association was observed between synovial CD68 and Fibrinoid Necrosis scores [r = 0.44 (0.27 - 0.56); p < 0.0001]. Patients were categorized as CD68NecrosisHIGH (CD68 + Necrosis ≥ 3) and CD68NecrosisLOW (CD68 + Necrosis < 3). CD68NecrosisHIGH exhibited higher pre-treatment disease activity [5.48 (1.6) versus 4.8 (1.7); p = 0.03] and a greater fall in DAS28 [1.99 (2.06) versus 1.1 (2.27), p = 0.03], SDAI [21.45 (IQR 23.3) versus 11.65 (IQR 17.5); p = 0.003] and CDAI [16 [14.9] versus 10.5 (20.1), p = 0.04]. CD68NecrosisHIGH patients had a higher EULAR Moderate/Good Response rate. In the MTX-Failure Cohort, histology was not sensitive enough to detect significant differences between responders and non-responders to TNFi. Flow cytometry demonstrated an increase of M2 macrophages (CD163+ and/or CD206 macrophages) for both atypical and typical monocytes (CD14 -/+) after 12 weeks of TNFi therapy. Conclusion Histology, and in particular Fibrinoid Necrosis and CD68 + in ERA synovial biopsies identify patients with higher disease activity and predict a better treatment response at three months. A model including synovial CD68 and fibrinoid necrosis with baseline clinical features predicts EULAR response at 3 months. The results were not confirmed in non-naive patients. However, the MTX-failure cohort was characterized by a restoration of the M1/M2 balance after 12 weeks of TNFi, reinforcing the evidence of a central role of macrophages.