SAT0032\u2005ACTIVATION OF TH LYMPHOCYTES ALTERS THE PATTERN EXPRESSION AND CELLULAR LOCATION OF VIP RECEPTORS IN HEALTHY DONORS AND EARLY ARTHRITIS PATIENTS

Abstract

Background: CD4+ T helper (Th) cells are decisive in the struggle against pathogens and in the maintenance of immune homeostasis. Their activation and differentiation can take many forms and generate immune memory in which the extracellular environment plays a critical role in both homeostatic and pathological states. VIP is one of the best-studied neuropeptides with anti-inflammatory and immunomodulatory actions in normal and pathological conditions (1,2,3). There are evidences that VIP may have therapeutic effects for human Rheumatoid arthritis and might be used as a prognostic marker for this disease (3,4). However, there are no deepen studies related to the expression pattern, cellular location, signalling pathways and functionality of its receptors (VPAC1 and VPAC2) during the activation of Th cells. Further in-depth studies of their behavior can contribute to the design of new therapies based on their activation and/or blockade. Objectives: To describe the expression pattern, cellular location and functional role of VIP receptors, VPAC1 and VPAC2, during the activation of human memory Th cells in healthy conditions and in early arthritis (EA). Methods: Human memory Th cells were isolated from healthy donors (HD) and EA patients. Two experimental conditions, resting state and in vitro activation, were used. Cells were activated (anti-CD3/CD28) and cultured in presence or absence of VIP or specific VPAC receptors agonists. Cellular location of VPAC receptors was examined by western-blot and confocal microscopy, and their mRNA levels were analyzed by semi-qPCR. Cytokines, transcription factors and cell markers related to pathogenic profile of Th cells were analyzed by semi-qPCR, ELISA and flow cytometry. To study the signalling pathways, pull-down assays (Rap1), western-blot (CREB) and ELISA (cAMP) were performed. Expression of different chemokines were analyzed by flow cytometry whereas cell migration assays were performed using transwell membranes. Results: The protein expression pattern of VPAC1 did not change with the activation of Th lymphocytes, whereas VPAC2 was up-regulated. In resting cells, VPAC1 receptor was located on the plasma membrane and nucleus, while it only appeared in the nucleus in activated cells. VPAC2 was always found in plasma membrane location. VIP receptors signalled through a PKA-dependent pathway in both conditions, and also through a PKA-independent pathway in activated cells. VIP receptors agonists decreased the pathogenic profile during the activation of Th lymphocytes. Similar results were observed in Th lymphocytes from EA patients. Conclusion: VIP is an important immunomodulator of CD4+ T cells in normal and pathological conditions. Our data revealed a change in the expression pattern and cellular location of its receptors (VPAC) during the activation of these cells, giving a wider range of possibilities to dissect its cellular and molecular mechanisms, that would provide new therapies or targets as activity markers in inflammatory/autoinmune diseases. References [1] Gomariz RP, et al., Ann NY Acad Sci USA. 2006; 1070: 51-74 [2] Jimeno R, et al. J Leuk Biol. 2015; 98(2): 257-269. [3] Villanueva-Romero R, et al. J Immunol Res. 2018; 6043710. [4] Martínez C, et al. Plos One. 2014; 9(1): e85248 Acknowledgement: This work was supported by ISCIII, Spain, co-financed by FEDER (EU): RETICS program, RIER (RD16/0012/0008,0006 and 0011) and the projects (PI12/00758, PI14/00477 and PI17/0027). Disclosure of Interests: None declared