The P2X7 loss-of-function Glu496Ala polymorphism affects ex vivo cytokine release and protects against the cytotoxic effects of high ATP-levels

Anke Wesselius 0 Martijn JL Bours 0 Ilja CW Arts 0 Esther HE Theunisz Piet Geusens Pieter C Dagnelie 0 0 Department of Epidemiology, Maastricht University, School for Public Health and Primary Care (CAPHRI) , Peter Debyeplein 1, Maastricht, MD 6200 , The Netherlands Background: The P2X7 receptor plays an important role in cytokine release during the inflammatory response in vivo. Polymorphisms within the P2X7 receptor gene that lead to loss of receptor function may contribute to impaired cytokine release by immune cells. Therefore, we investigated whether a known loss-of-function polymorphism (Glu496Ala) in the P2X7 receptor gene leads to alterations in cytokine release in response to ATP. Results: An ex vivo whole blood model was used to induce an inflammatory reaction with the pro-inflammatory stimuli LPS and PHA (phytohemagglutinin). Blood from n=9 subjects with the Glu496Ala P2X7 SNP (P2X7MUT) and n=7 'wild-type' subjects (no P2X7 SNP; P2X7WT) was used. Addition of ATP (0.9-3 mM) to LPS/PHA-stimulated whole blood induced an increase in IL-1 release in P2X7MUT subjects, whereas decreased release was observed in P2X7WT subjects. Decreased levels of IL-6 and TNF- in response to ATP were shown in both P2X7MUT and P2X7WT subjects, which was less pronounced in P2X7MUT subjects. ATP at 3 mM also significantly decreased levels of lactate dehydrogenase (LDH) in P2X7MUT subjects compared to P2X7WT subjects. Conclusions: The presence of the non-synonymous Glu496Ala loss-of-function polymorphism within the P2X7 receptor gene is likely to be of importance in the release of cytokines during inflammation. Furthermore, this study suggests that carriers of the Glu496Ala loss-of-function polymorphism are protected against the cytotoxic effects of high ATP-levels. - Background The innate immune system constitutes the first line of defense against anything that compromises tissue homeostasis. Activation of innate immunity results in the induction of inflammation, which is essence, aims to restore the structural and functional integrity of tissues and organs. Adenosine 50-triphosphate (ATP) is thought to be one of the DAMPs playing a major role in the inflammation and cytokine storm. It is present in high concentrations within the cytoplasm of every cell and can be released after cell damage by a variety of injurious agents [1,2]. After release, ATP acts through binding to specific receptors known as purinergic P2 receptors (P2R), of which the P2X7 subtype is a potent mediator of cytokine processing and release [3]. The purinergic P2X7 receptor is a ligand-gated ion channel which has a wide tissue distribution, being expressed by virtually all cell types, including cells of the immune system, i.e. monocytes, macrophages, dendritic cells, and T cells [4]. Activation of this receptor by brief exposure to extracellular ATP opens a cation channel, which allows Ca2+ influx, as well as K+ efflux [5]. Longer exposure to ATP leads to dilatation of the P2X7 channel to a pore, which allows uptake of permeants up to the size of ethidium+ [6,7]. Activated P2X7 receptors are known to play an important role in regulating the inflammatory response in vivo (reviewed in [8]. Research indicates that activation of the P2X7 receptor causes massive release of the pro-inflammatory mediator IL-1. The P2X7-mediated release of IL-1 by immune cells is suggested to be regulated by various mechanisms including: a) cytotoxicity of IL-1 producing immune cells [9], b) K+ efflux [9], and c) activation of the inflammasome NALP3 via pannexin-1 (reviewed in [10]). In addition to IL-1, other pro-inflammatory mediators are also upregulated via P2X7 receptor, including IL-6, IL-18 and TNF- (reviewed in [11]). These data point to an important role of P2X7 receptor-mediated signaling in inflammation, and also suggest that polymorphisms within the P2X7 receptor gene that lead to loss of receptor function have the potential to impair cytokine release by immune cells in vivo. Several non-synonymous single nucleotide polymorphisms (SNP) have been characterized in the P2X7 receptor gene (reviewed in [12]). One such SNP concerns the nucleotide at position 1513, which changes a glutamic acid to an alanine acid at amino position 496 (Glu496Ala). Previous research in human monocytes, showed that the Glu496Ala polymorphism decreased the P2X7 receptor mediated K+ efflux, thereby delaying P2X7 receptor mediated release of IL-1 [13]. Furthermore, it was shown that subjects homozygous for the variant allele of the Glu496Ala polymorphism had reduced sensitivity to inflammation compared to wildtype subjects [14]. In the present study, we further tested the hypothesis that subjects homozygous for the Glu496Ala loss-of-function polymorphism produce lower levels of IL-1 in response to ATP. In addition to levels of IL-1, we also explored whether production of other inflammatory cytokines in response to ATP was altered in subjects carrying the Glu496Ala P2X7 receptor SNP. To test our hypotheses, we used an ex vivo inflammation model by stimulating whole blood with the potent inflammatory stimuli LPS and PHA (phytohemagglutinin). Previous research showed that this whole blood assay, in contrast to isolated cells or cell lines grown in medium, closely resembles the in vivo situation and forms an appropriate and reproducible culture condition to measure cytokine production ex vivo [15]. Results Study population Of the eligible 14 P2X7MUT subjects, a total of 9 P2X7MUT (aged 50-75 years; 2 men and 7 women) donated blood for this ex vivo experiment. Two out of these 9 P2X7MUT subjects showed no other SNPs in the P2X7 receptor gene. The other seven subjects were shown to have several other non-synonymous SNPs in the P2X7 receptor gene in addition to the Glu496AlaP2X7 receptor polymorphism (Table 1). Of the eligible 19 P2X7WT subjects, a total of 7 P2X7WT (aged 55-74 years; 1 man and 6 women) were willing to donate blood. Effects of LPS/PHA stimulation on cytokine release Stimulation of whole blood from P2X7MUT subjects with LPS + PHA for 24 hours induced a strong rise in levels of IL-1, TNF-, IL-6, IL-10 and IFN- (Table 2). No statistically significant differences in IL-1, TNF-, IL-6, IL-10 and IFN- levels in unstimulated whole blood in the absence of ATP were observed between P2X7MUT and P2X7WT subjects. In LPS/PHA-stimulated blood, levels of IL-1 and TNF- tended to be slightly higher and levels of IL-6, IL-10 and IFN- tended to be slightly lower in LPS/PHA-stimulated whole blood from P2X7MUT subjects, even though the differences were not statistically significant (Table 2). No effect of the P2X7 receptor on the production of the other measured cytokines (i.e. IL-2, IL-7, IL-8, IL-12, Table 2 Effect of LPS + PHA stimulation on the release of cytokines (Release of IL-1, TNF-, IL-6, IL-10 and IFN- in whole blood from wild type subjects (P2X7WT) and subjects homozygous for the Glu496Ala polymorphism (P2X7MUT) a)) Figure 1 Cytokine and L (...truncated)