P75NTR IN THE MODULATION OF INFLAMMATORY RESPONSE MEDIATED BY SYNOVIAL FIBROBLASTS

Background: Our previous studies1 showed high expression levels of p75NTR, the nerve growth factor (NGF) receptor, in mononuclear cells (MNCs) obtained from blood and synovial fluids of patients with juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA). p75NTR binds with high affinity proNGF, the immature form of NGF whose concentration, as we recently demonstrated2, is extremely high in the synovial fluids of arthritis patients. In ex vivo experiments we demonstrated that recombinant proNGF increases inflammatory cytokine production in patient MNCs, an effect that was abolished using p75NTR inhibitors2. We also found that synovial fibroblasts (SFs) represent the main source of proNGF in the inflamed synoviae. At present it is not known whether proNGF can influence the activity of SFs that are key effector cells in synovia inflammation producing inflammatory mediators that regulate chondrocytes and osteoblasts activation. Objectives: To investigate the mechanisms regulating p75NTR expression in synovial fibroblasts of arthritis patients and to evaluate the effects of its inhibition on the inflammatory response. Methods: SFs from arthritis patients were used to study the activity of proNGF/p75NTR axis. SFs from osteoarthritis patiens (OA) and skin fibroblasts from healthy donors (HD) were used as controls. p75NTR, NGF, and cytokine transcripts were evaluated by quantitative PCR (qPCR). Protein expression of p75NTR, NGF, and proNGF were analyzed by Western Blot. ELISAs were used to evaluate NGF, proNGF and cytokine concentration in supernatants and synovial fluids. p75NTR was inhibited using LM11A-31, a synthetic inhibitor that blocks the binding between p75NTR and its specific ligand proNGF. Results: mRNA and protein expression of p75NTR were up-regulated in arthritis SFs compared to OA SFs and skin fibroblasts. In vitro stimulation with recombinant cytokines (IL-1β, TNF, IL-6), TLR-ligands (such as LPS), and JIA synovial fluids, containing a mixture of inflammatory mediators, strongly increased p75NTR mRNA expression in arthritis SFs. Interestingly, after stimulation we observed that also OA SFs and HD fibroblasts up-regulated p75NTR transcript, suggesting that p75NTR levels are modulated by the inflammatory mediators. SFs of arthritis patients spontaneously produced proNGF and its release was further enhanced by all the above-mentioned inflammatory stimuli, albeit with different ability. Thus patient SFs can both produce proNGF and bind it through p75NTR. The inhibition of proNGF binding to p75NTR using LM11A-31 in arthritis SFs, activated with inflammatory cytokines or with synovial fluids, significantly reduced the expression and release of pro-inflammatory cytokine. Conclusion: In addition to inducing p75NTR up-regulation, inflammatory stimuli increase the release of proNGF in arthritis SFs. Autocrine proNGF binds to p75NTR and further enhances pro-inflammatory cytokine production, creating a vicious circle that amplifies the inflammatory response. Blocking the binding of endogenous proNGF to its receptor p75NTR strongly reduces the production of inflammatory mediators and prospects the use of p75NTR inhibitors as a new therapeutic approach to chronic arthritis. References [1] Prencipe, et al., Nerve Growth Factor Down regulates Inflammatory Response in Human Monocytes through TrkA, J Immunol. 2014 [2] Minnone, et al., ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis, RMD Open. 2017 Disclosure of Interests: Luciapia Farina: None declared, Gaetana Minnone : None declared, Marzia Soligo : None declared, Luigi Manni : None declared, Gian Marco Moneta: None declared, Ivan Caiello: None declared, Luigi Manzo : None declared, Fabrizio De Benedetti Grant/research support from: Abbvie, SOBI, Novimmune, Roche, Novartis, Sanofi, Pfizer, Luisa Bracci-Laudiero: None declared