POS1148 MECHANISMS OF PAIN MODULATION BY JAK INHIBITION: UPADACITINIB REGULATES PAIN-RELATED PATHWAYS AND BDNF EXPRESSION IN MICROGLIAL CELLS
Authors: L. Navarini, M. Vomero, E. Corberi, O. Berardicurti, G. Imperatori, D. Currado, L. Kun, F. Trunfio, F. Saracino, L. Lamberti, A. Marino, L. La Barbera, P. Krashia, M. D’amelio, R. Giacomelli
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Chronic pain is one of the most critical symptoms reported by patients with Rheumatoid Arthritis, and even when joint inflammation improves, disabling residual pain may persist in a significant number of patients. During chronic inflammation, microglial cells, characterized by TMEM119 expression, acquire an activated phenotype characterized by the production of different pro-inflammatory cytokines and pain-related molecules, leading to a neuroinflammation process. Among these mediators, the brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is pivotal in generating abnormal pain perception, leading to nociplastic pain (1). IL-6, a Janus Kinase 1 (JAK1)-dependent pro-inflammatory cytokine, seems critical for the production of BDNF (2). Treatment with Upadacitinib, a JAK1 inhibitor, has been proven effective in improving disease activity and relieving pain quickly (3); nonetheless, the biological mechanisms underlying its efficacy in pain perception still need further investigation. <h3>Objectives:</h3> This study aims to investigate whether and how Upadacitinib may affect pain-related and neuroinflammation-related molecules expression in monocyte-derived microglia, specifically regarding BDNF. <h3>Methods:</h3> Microglia were differentiated from monocytes by treatment with NGF, MCP-1, IL-34, GM-CSF, and M-CSF for 14 days and further activated toward a pro-inflammatory phenotype using LPS and IFN-gamma, as summarized in Figure 1A. For all the experiments, microglia were exposed to six conditions in a culture medium for 48 hours: A1 untreated, A2 IL-6, A3 IL-6+Upadacitinib 0.1 uM, B1 IL-6+Upadacitinib 1 uM, B2 Upadacitinib 0.1 uM, and B3 Upadacitinib 1 uM. TMEM119 and BDNF expression in microglia was evaluated by immunofluorescence. Intracellular BDNF levels were analyzed by flow cytometry and Western Blot. In all the culture conditions, microglia underwent RNA-sequencing analysis. RNA from microglia was extracted using all prep DNA/RNA/miRNA universal kits (Qiagen, Germany), and bulk RNA sequencing was carried out using the Illumina system. Heatmaps were generated in RStudio statistical software (R version 3.3.0+; Boston, MA, USA) with the NMF package. All the other statistical analyses have been performed using GraphPad Prism v.10 (Boston, MA, USA). <h3>Results:</h3> TMEM199, a microglia marker, was expressed at baseline condition (Figure 1B) and BDNF (Figure 1C). Flow cytometry analysis showed that the treatment of Upadacitinib, both at the concentration of 0.1 uM and 1 uM, alone and in combination with IL-6, reduced BDNF levels in microglial cells compared to the untreated (A1) and IL-6 (A2) conditions (Figure 1D, * p&lt;0.05). Western blot analysis confirmed that microglia treated with the combination of IL-6 and Upadacitinib (both 0.1 uM and 1 uM) showed a decreased production of BDNF compared to the microglia treated with IL-6 alone (p&lt;0.0001). Transcriptomic analysis showed that Upadacitinib is able to reduce gene expression of JAK/STAT signaling (Figure 1E). Moreover, the neuroinflammation-related pathways are affected by Upadacitinib treatment toward a less pro-inflammatory phenotype (Figure 1F). Furthermore, Upadacitinib treatment, alone or in combination with IL-6, induces a modulation of acute and chronic pain-related genes. <h3>Conclusion:</h3> Our results show that Upadacitinib is crucial in modulating gene expression in microglia, characterized by decreased JAK/STAT, neuroinflammatory, acute, and chronic pain-related pathways. Moreover, in this cell type, Upadacitinib reduces the production of BDNF, a molecule involved in pain perception and nociplastic mechanisms. <h3>REFERENCES:</h3> [1] Atta AA et al, Inflammopharmacology 2023;31:1053-67 [2] Schulte-Herbrüggen O et al, J Neuroimmunol 2005;160:204-9 [3] Bergman M et al, Arthritis Res Ther 2022;24:155 <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> Luca Navarini Sanofi, GSK, AbbVie, Novartis, AbbVie, MSD, Italfarmaco, UCB, Novartis, Janssen-Cilag, GSK, Eli-Lilly, Sanofi, Marta Vomero: None declared, Erika Corberi: None declared, Onorina Berardicurti: None declared, Giulia Imperatori: None declared, Damiano Currado: None declared, Lyubomyra Kun: None declared, Francesca Trunfio: None declared, Francesca Saracino: None declared, Ludovica Lamberti: None declared, Annalisa Marino: None declared, Livia La Barbera: None declared, Paraskevi Krashia: None declared, Marcello D'Amelio: None declared, Roberto Giacomelli: None declared