Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Cytokine signaling and pain modulation in Rheumatoid Arthritis

Introduction. Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disorder characterized by synovial inflammation leading to bone damage. Upon inflammatory stimuli, monocytes (Mo) migrate in synovial tissue differentiating into macrophages (Mφ) that participate to synovial fibroblasts (FLS) activation. Despite the therapeutic options, non-response is a challenge in those patients who experience persistently high disease activity. Among the causes of treatment failure persistent pain is another responsible cause. Aim. to investigate two possible mechanisms of treatment failure: Mo phenotype and pain pathway on synovial FLS. Methods. PART 1: I enrolled patients affected by RA starting treatment with baricitinib. At baseline, and after 4, 12 and 24 weeks of treatment we evaluated the disease activity (DAS28CRP) dividing the cohort in two groups (responders-R and non-responders-NR) according to the reduction of DAS28CRP of at least 1.2 points. Peripheral blood mononuclear cells (PBMC) were collected and analyzed at basal and after cytokine stimulation (IL2, IFN-alfa, IL6). Flow cytometry was used to characterize monocytes, T reg, T CD8+ and T CD4+ and the phosphorylation of STATs was studied. PART 2: the effect of the treatment with baricitinib on pain was analysed in a cohort of patients with RA; serum levels of the neurotransmitter CGRP were dosed before and after treatment. Synovial tissue and FLS from RA patients were studied for the expression of pain receptors (TRPV1 and TRPA1), and for SMOC2 production, a molecule capable of changing the phenotype of fibroblasts in a more aggressive one, and for the production of inflammatory cytokines and activation molecules. Results. PART 1: a reduction of the frequency of monocytes in 7 R-patients compared to 7 NR-patients, after 4 weeks of treatment with baricitinib (W4) with a significant reduction of the cytokine-induced phosphorylation of STAT1 were noted in R-patients, compared to NR. At all tested time points, classical monocytes represented the majority of monocytes, and their frequency declined from T0 to T1 in R patients. We confirmed in classical monocytes a pSTAT1 modulation by IFNα. Then, we verified the presence of a pre-existing IFN signature, analyzing the expression of ISG15, an IFNα negative regulator, and two ISGs, PKR and MXA. All three were upregulated after in vitro culture with IFNα, in Mo from both R and NR patients, but with higher levels in R patients at baseline. PART 2: during the third year I had the opportunity of studying another possible mechanism of treatment failure, refractory pain. Immune cells, including Mo-derived synovial Mφ - and nociceptors influence each other: once activated by inflammatory stimuli (IL6, IL1), nociceptors release neuropeptides (such as calcitonin gene-related peptide – CGRP) which in turn induce mononuclear cells to produce proinflammatory cytokines. Analyzing the cohort of patients enrolled in the previous two years I confirmed that elevated pain levels were detectable in those patients that we defined as NR. NR patients presented higher serum levels of CGRP, a pain mediator, compared to R, after one month of baricitinib. Moreover, I had the opportunity to work on the synovial tissue of early RA patients at Queen Mary University, London. During these six months abroad, I confirmed that FLS in RA participate to pain stimuli expressing pain receptors on the membrane (TRPV1, TRPA1) and that the innervation of synovial tissue can be variable and upregulated, as demonstrated by the staining I performed with PGP9.5 and TH. Conclusions. These results show that monocytes frequency might change accordingly to clinical response to baricitinib and that early STAT1 phosphorylation might represent a biomarker of response. In NR, persistent pain may be related to inflammatory cytokines locally regulating neurotransmitters and their receptors suggesting that pain can contribute to treatment failure.