P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes
Francesca D’Addio, Andrea Vergani, Luciano Potena, Anna Maestroni, Vera Usuelli, Moufida Ben Nasr, Roberto Bassi, Sara Tezza, Sergio Dellepiane, Basset El Essawy, Maria Iascone, Attilio Iacovoni, Laura Borgese, Kaifeng Liu, Gary Visner, Sirano Dhe-Paganon, Domenico Corradi, Reza Abdi, Randall C. Starling, Franco Folli, Gian Vincenzo Zuccotti, Mohamed H. Sayegh, Peter S. Heeger, Anil Chandraker, Francesco Grigioni, Paolo Fiorina
Francesca D’Addio, Andrea Vergani, Luciano Potena, Anna Maestroni, Vera Usuelli, Moufida Ben Nasr, Roberto Bassi, Sara Tezza, Sergio Dellepiane, Basset El Essawy, Maria Iascone, Attilio Iacovoni, Laura Borgese, Kaifeng Liu, Gary Visner, Sirano Dhe-Paganon, Domenico Corradi, Reza Abdi, Randall C. Starling, Franco Folli, Gian Vincenzo Zuccotti, Mohamed H. Sayegh, Peter S. Heeger, Anil Chandraker, Francesco Grigioni, Paolo Fiorina
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Research Article Immunology

P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

Abstract

Purinergic receptor-7 (P2X7R) signaling controls Th17 and Th1 generation/differentiation, while NOD-like receptor P3 (NLRP3) acts as a Th2 transcriptional factor. Here, we demonstrated the existence of a P2X7R/NLRP3 pathway in T cells that is dysregulated by a P2X7R intracellular region loss-of-function mutation, leading to NLRP3 displacement and to excessive Th17 generation due to abrogation of the NLRP3-mediated Th2 program. This ultimately resulted in poor outcomes in cardiac-transplanted patients carrying the mutant allele, who showed abnormal Th17 generation. Transient NLRP3 silencing in nonmutant T cells or overexpression in mutant T cells normalized the Th profile. Interestingly, IL-17 blockade reduced Th17 skewing of human T cells in vitro and abrogated the severe allograft vasculopathy and abnormal Th17 generation observed in preclinical models in which P2X7R was genetically deleted. This P2X7R intracellular region mutation thus impaired the modulatory effects of P2X7R on NLRP3 expression and function in T cells and led to NLRP3 dysregulation and Th17 skewing, delineating a high-risk group of cardiac-transplanted patients who may benefit from personalized therapy.

Authors

Francesca D’Addio, Andrea Vergani, Luciano Potena, Anna Maestroni, Vera Usuelli, Moufida Ben Nasr, Roberto Bassi, Sara Tezza, Sergio Dellepiane, Basset El Essawy, Maria Iascone, Attilio Iacovoni, Laura Borgese, Kaifeng Liu, Gary Visner, Sirano Dhe-Paganon, Domenico Corradi, Reza Abdi, Randall C. Starling, Franco Folli, Gian Vincenzo Zuccotti, Mohamed H. Sayegh, Peter S. Heeger, Anil Chandraker, Francesco Grigioni, Paolo Fiorina

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Figure 4

P2X7R loss of function in a preclinical model of heart transplantation reduces allograft survival, with increased vasculopathy and Th1/Th17–mediated responses.

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P2X7R loss of function in a preclinical model of heart transplantation r...
(A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).