THU0016 ALTERED EXPRESSION AND FUNCTION OF P2X7 RECEPTOR IN PATIENTSAFFECTED BY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)
Authors: Anna Lisa Giuliani, Federica Furini, Alessandra Bortoluzzi, Marcello Govoni, Francesco DI Virgilio
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Extracellular ATP (eATP) is one of the most diffuse danger associated molecular patterns (DAMPs) released actively through specific mechanisms from intact cells, or passively from damaged or dying cells<sup>1</sup>. An implication for eATP has been found in SLE<sup>2</sup>. Among receptors for eATP, P2X7R is deeply involved in inflammatory and immune processes and its activation drives different intracellular pathways such as NLRP3-inflammasome activation, IL-1β maturation and release, IL-6 and TNF-α production, regulation of lymphocyte proliferation and cell apoptosis<sup>3</sup>. Previous studies pointed out a possible relationship between P2X7R signaling pathways and SLE pathogenesis<sup>4 5</sup>. A marked inflammatory condition characterize serositis, that are among the most common manifestations of SLE, and chloroquine is one of the main drugs employed. <h3>Objectives</h3> The aim of this study was to investigate P2X7R expression and activity in SLE. <h3>Methods</h3> 48 SLE patients, and 20 healthy control (HC) subjects were enrolled. Among SLE patients, 16 (SLE-S) presented, and 32 (SLE-NS) did not present history of serositis. All subjects gave written informed consent to peripheral venous blood withdrawal after approval by the local ethic committee. Plasma samples were used to measure IL-1β, IL-6 and TNF-α levels by ELISA. Mononuclear cells were isolated from blood samples by Ficoll gradient sedimentation and employed as follow: i) assessment of IL-1β, IL-6 and TNF-α release after stimulation with lipopolysaccharide (LPS) and/or Benzoyl ATP (BzATP); ii) evaluation of P2X7R mRNA expression by RT-PCR; iii) measurement of P2X7R activity as BzATP-induced increase of intracellular Ca<sup>2+</sup> concentration using the Fura2/AM probe. <h3>Results</h3> In SLE patients respect to HC, plasma IL-1β levels were unmodified whereas IL-6 was higher, resulting significantly increased in SLE-S. Monocytes isolated from SLE patients released lower quantities of IL-1β after stimulation with BzATP, whereas the release of both IL-6 and TNF-α was significantly augmented in SLE-NS respect to both HC and SLE-S subjects after all types of stimulation. RT-PCR showed reduced P2X7R and augmented NLRP3 mRNA expression in SLE patients. Accordingly, P2X7R activity was significantly reduced in all SLE patients and did not appear to be influenced by a chloroquine pre-treatment. <h3>Conclusion</h3> In SLE patients, compared to HC subjects, we found reduced P2X7R mRNA expression, increased NLRP3 mRNA, as a possible compensating mechanism, and correspondingly, significantly lower BzATP-induced intracellular Ca<sup>2+</sup> increase, without an apparent influence by chloroquine, one of the drugs most diffusely used for SLE treatment. The in vitro IL-1β release was reduced, whereas plasma IL-1β was unaltered, indicating an alternative source, other than monocytes, of this cytokine. Conversely, IL-6 and TNF-α levels were increased in vitro, and IL-6 was present in plasma at higher levels. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 and TNF-α as possible therapeutic targets. <h3>References</h3> [1] Giuliani, A.L. et al. Extracellular nucleotides and nucleosides as signalling molecules. Immunol Lett, doi:10.1016/j.imlet.2018.11.006 (2018). [2] Magna, M. &amp; Pisetsky, D.S. The Role of Cell Death in the Pathogenesis of SLE: Is Pyroptosis the Missing Link? Scand J Immunol 82, 218-224, (2015). [3] Di Virgilio, F. et al. The P2X7 Receptor in Infection and Inflammation. Immunity 47, 15-31, (2017). [4] Di Virgilio, F. &amp; Giuliani, A.L. Purinergic signalling in autoimmunity: A role for the P2X7R in systemic lupus erythematosus? Biomed J 39, 326-338, (2016). [5] Faliti, C.E. et al. P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus erythematosus. J Exp Med, (2019). <h3>Disclosure of Interests</h3> Anna Lisa Giuliani: None declared, Federica Furini: None declared, Alessandra Bortoluzzi: None declared, Marcello Govoni: None declared, Francesco Di Virgilio Consultant for: FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a UK-based biotech Company involved in the development of P2X7R-targeted therapeutics.