P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy.

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.

Khalafalla MG ，Woods LT ，Camden JM ，Khan AA ，Limesand KH ，Petris MJ ，Erb L ，Weisman GA ... -  《-》

The P2X7 receptor-NLRP3 inflammasome complex predicts the development of non-Hodgkin's lymphoma in Sjogren's syndrome: a prospective, observational, single-centre study.

P2X7 receptor (P2X7R), trigger of acute inflammatory responses via the NLRP3 inflammasome, is hyperfunctioning in patients with Sjögren's syndrome (SS), where it stimulates IL-18 production. Some patients with SS develop a mucosa-associated lymphoid tissue non-Hodgkin's lymphoma (MALT-NHL). To prospectively evaluate the involvement and the putative prognostic role of this inflammatory pathway in the development of MALT-NHL. A total of 147 women with SS have been prospectively followed for a mean of 52 months, relating the expression and function of the P2X7R-inflammasome axis in salivary glands and circulating lymphomonocytes to the prognosis and the degree of the disease. At baseline, gene expression of P2X7R and of the inflammasome components NLRP3, caspase-1 and IL-18 increased according to the presence of germinative centres and was higher in autoantibody-positive individuals and strongly higher in those developing a MALT-NHL over the follow-up. Glandular expression of IL-18 was threefold higher in MALT-NHL than in controls or in the other patients with SS. P2X7R did not colocalize with generic markers of inflammatory infiltrate, like CD20, being selectively expressed by epithelial cells. P2X4R, sharing functional characteristics with P2X7R, did not differ in SS and controls. The increased P2X7R gene and protein expression was tissue specific, no difference being observed in peripheral lymphomonocytes between SS with MALT-NHL and SS not developing MALT-NHL. We propose the P2X7R-inflammasome axis as a novel potential pathway involved in both SS exocrinopathy and lymphomagenesis, reinforcing the hypothesis of a key role of IL-18, via its increased P2X7R-mediated production, in the pathogenesis of lymphoproliferative malignancies, and opening novel opportunities for the early diagnosis of lymphoproliferative complications and the development of potential targeted therapies.

Baldini C ，Santini E ，Rossi C ，Donati V ，Solini A ... -  《-》

Activation of P2X7 receptor and NLRP3 inflammasome assembly in hippocampal glial cells mediates chronic stress-induced depressive-like behaviors.

Yue N ，Huang H ，Zhu X ，Han Q ，Wang Y ，Li B ，Liu Q ，Wu G ，Zhang Y ，Yu J ... -  《Journal of Neuroinflammation》

The P2X7 receptor-inflammasome complex has a role in modulating the inflammatory response in primary Sjögren's syndrome.

Innate and adaptive immunity may contribute to gland dysfunction in patients with primary Sjögren's syndrome (pSS). The P2X7 receptor (P2X7 R)-NLRP3 inflammasome complex modulates the release of the inflammatory cytokines IL-1β and IL-18. The presence of P2X7 R in salivary glands suggests an interesting scenario for the initiation and amplification of the innate immune response in pSS. Therefore, the aim of this study was to assess the role of the P2X7 R-NLRP3 inflammasome in pSS. Twenty-one consecutive patients with pSS according to the American-European Consensus Group criteria and 15 patients with sicca syndrome (i.e. without Sjögren's syndrome, non-SS) were enrolled in this study, together with six control (CTL) subjects. Expression of the P2X7R-NLRP3 platform and IL-18 was determined by real-time PCR and western blotting in gland specimens and peripheral lymphomonocytes; data were related to patients\x92 clinical, serological and histopathological characteristics. The presence of IL-18 was determined in gland and saliva samples. P2X7 R expression was significantly higher in salivary glands from individuals with pSS than in those from non-SS and CTL subjects. Accordingly, the gene expression levels of the inflammasome components NLRP3, ASC and caspase-1 were significantly higher in pSS gland specimens, and this was paralleled by an increased expression of mature IL-18 in pSS saliva samples. The expression of both the P2X7 R and the inflammasome components was a marker of disease-related glandular involvement, being increased in patients with anti-Ro/SSA positivity and correlated with focus score. The results of this study suggest an involvement of the P2X7 R-inflammasome-caspase-1-IL-18 axis in the development of pSS exocrinopathy. This finding provides the basis for studying the complex mechanisms underlying pSS, as well as for developing novel potential therapeutic strategies.

Baldini C ，Rossi C ，Ferro F ，Santini E ，Seccia V ，Donati V ，Solini A ... -  《-》

P2X(7) receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the NLRP3/IL-1β pathway.

Mounting evidence supports the hypothesis that inflammation modulates sympathetic sprouting after myocardial infarction (MI). The myeloid P2X7 signal has been shown to activate the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, a master regulator of inflammation. We investigated whether P2X7 signal participated in the pathogenesis of sympathetic reinnervation after MI, and whether NLRP3/interleukin-1β (IL-1β) axis is involved in the process. We explored the relationship between P2X7 receptor (P2X7 R) and IL-1β in the heart tissue of lipopolysaccharide (LPS)-primed naive rats. 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (BzATP), a P2X7 R agonist, induced caspase-1 activation and mature IL-1β release, which was further neutralized by a NLRP3 inhibitor (16673-34-0). MI was induced by coronary artery ligation. Following infarction, a marked increase in P2X7 R was localized within infiltrated macrophages and observed in parallel with an up-regulation of NLRP3 inflammasome levels and the release of IL-1β in the left ventricle. The administration of A-740003 (a P2X7 R antagonist) significantly prevented the NLRP3/IL-1β increase. A-740003 and/or Anakinra (an IL-1 receptor antagonist) significantly reduced macrophage infiltration as well as macrophage-based IL-1β and NGF (nerve growth factor) production and eventually blunted sympathetic hyperinnervation, as assessed by the immunofluorescence of tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP 43). Moreover, the use of Anakinra partly attenuated sympathetic sprouting. This indicated that the effect of P2X7 on neural remodelling was mediated at least partially by IL-1β. The arrhythmia score of programmed electric stimulation was in accordance with the degree of sympathetic hyperinnervation. In vitro studies showed that BzATP up-regulated secretion of nerve growth factor (NGF) in M1 macrophages via IL-1β. Together, these data indicate that P2X7 R contributes to neural and cardiac remodelling, at least partly mediated by NLRP3/IL-1β axis. Therapeutic interventions targeting P2X7 signal may be a novel approach to ameliorate arrhythmia following MI.

Yin J ，Wang Y ，Hu H ，Li X ，Xue M ，Cheng W ，Wang Y ，Li X ，Yang N ，Shi Y ，Yan S ... -  《-》