The P2X₇ receptor and intracellular pathogens: a continuing struggle

Abstract

The purinergic receptor, P2X₇, has recently emerged as an important component of the innate immune response against microbial infections. Ligation of P2X₇ by ATP can stimulate inflammasome activation and secretion of proinflammatory cytokines, but it can also lead directly to killing of intracellular pathogens in infected macrophages and epithelial cells. Thus, while some intracellular pathogens evade host defense responses by modulating with membrane trafficking or cell signaling in the infected cells, the host cells have also developed mechanisms for inhibiting infection. This review will focus on the effects of P2X₇ on control of infection by intracellular pathogens, microbial virulence factors that interfere with P2X₇ activity, and recent evidence linking polymorphisms in human P2X₇ with susceptibility to infection.

Introduction

Host organisms and their cells have evolved a large array of mechanisms for controlling infection. Simultaneously, many pathogens have also attempted to circumvent the defense mechanisms. Thus, pathogens can subvert macrophage antimicrobial function, manipulating intracellular signaling pathways [1–3], interfering with membrane trafficking or the cell cycle [4], and modifying host metabolism [5].

The ultimate effect of microbial invasion depends on tissues infected and the microbial strategies of survival, with different strategies of pathogen adaptation being associated with varying degrees of damage of host tissues [6, 7]. Conversely, the host has also evolved defense mechanisms for resisting infection. In this review we will discuss how pathogens and host immune systems have co-evolved to thwart each other’s attacks, focusing on the effects of purinergic signaling on infection and the immune response. We will thus consider effects of P2X₇ and the pathogen on fusion between host cell phagosomes and lysosomes, production of reactive oxygen species (ROS) by the host, and modulation of host cell apoptosis. Finally, we will discuss microbial enzymes that deplete the P2X₇ ligand and polymorphisms in human P2X₇ that influence the ability to control infection.

Interference with membrane trafficking

Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic, and phagocytic membrane-trafficking pathways. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by lysosomes [8]. Bacteria such as Salmonella and Mycobacterium arrest the maturation of the phagosome at specific stages of the phagolysosomal route [9, 10]. Intracellular survival of Chlamydia depends in part on the ability of the microorganism to inhibit phagolysosomal fusion and subsequently survive and proliferate within a membrane-bound compartment called the inclusion [11]. Toxoplasma parasites infect virtually all mammalian cells, including macrophages, by active invasion [12]. The vacuole that surrounds Toxoplasma lacks the membrane proteins that normally deliver the endosomes to the cell fusion machinery. Thus, the parasitophorous vacuole remains at a neutral pH, allowing the parasite to survive [13]. Similarly, intracellular survival of Burkholderia cenocepacia in macrophages is associated with the pathogen’s ability to delay maturation of vacuoles that harbor these bacteria [14].

But the host immune system has also evolved to counteract the evasion strategies of these pathogens; and binding of extracellular nucleotides to purinergic receptors, especially the P2X₇ receptor, can block development of pathogens that survive in an intracellular vacuole. Thus, treatment of infected macrophages with ATP kills Mycobacterium tuberculosis or Mycobacterium bovis, through a process requiring phospholipase D (PLD) activation, fusion between lysosomes and mycobacterial vacuoles, and acidification of the vacuoles [15, 16]. Likewise, P2X₇-dependent PLD activation and fusion between vacuoles and lysosomes is involved in inhibition of Chlamydia trachomatis growth in macrophages [17]. These results have been extended by recent studies, which show that P2X₇ activation also inhibits chlamydial infection in a cervical epithelial cell line and in vaginally infected mice [18]. Activation of PLD may be a general mechanism of elimination of parasites that normally reside within intracellular vacuoles that avoid fusion with lysosomes [19] (Fig. 1). Consistent with this view, we have observed that extracellular ATP decreases the parasite load in Toxoplasma gondii-infected macrophages and that this effect is mediated by P2X₇-mediated acidification of the parasitophorous vacuole (manuscript in preparation).

Fig. 1.

Pathogen clearance by infected cells. Ligation of P2X₇ by extracellular ATP can promote elimination of intracellular pathogens (left side). P2X₇ signaling can lead to PLD activation and/or ROS production, both of which can lead to killing of the pathogens. PLD has its effect primarily through stimulation of fusion between parasitophorous vacuoles and lysosomes, and subsequent killing of pathogens in acidic phagolysosomes (right side)

Avoidance of the toxic effects of reactive oxygen species

Production of ROS such as H₂O₂ was considered for many years as an unfortunate, deleterious consequence of aerobic metabolism. However, it has now become clear that ROS can also participate as physiological mediators in cell signaling pathways [20, 21]. Moreover, ROS can contribute to pathogen killing, and the role of ROS in nucleotide-mediated cell signaling is attracting growing attention. Hence, nucleotide receptors have been implicated in modulating the production of superoxide, H₂O₂, and other ROS by several cell types [22–26]. Ferrari et al. [27] had previously described that nuclear factor (NF)-κB activation by P2X₇ ligation is sensitive to antioxidants, suggesting that ROS might contribute to this outcome. In addition, nucleotide receptor signaling in murine macrophages is linked to ROS generation [26], and ATP treatment can activate an ROS-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages [28] (Fig. 1). Interestingly, one of the mechanisms by which ATP triggers apoptosis in macrophages may also involve ROS, probably via NOX2 [29].

Leishmania parasites enter into macrophages by phagocytosis. But unlike the pathogens cited above, Leishmania does not seek to inhibit fusion between entry vacuoles and lysosomes. Instead, Leishmania amastigotes display the interesting ability to survive and replicate within the hostile, low-pH environment of phagolysosomes [1]. Leishmania promastigotes interfere with reactive oxygen and nitrogen species responses in phagocytes [1].

We have observed that Leishmania infection of macrophages modulates P2X₇ activity and that extracellular ATP treatment reduces the parasite load via P2X₇ activation (submitted). In addition, we observed an increase in ROS levels in infected macrophages after treatment with ATP and increased parasite survival in ATP-treated macrophages treated with antioxidants (unpublished data). These findings suggest that ROS production by the immune system may contribute to clearance of parasites such as Leishmania that survive within phagolysosomes.

Prevention of host cell apoptosis

Intracellular pathogens obtain many of their nutrients from the host cell and also require that their host cells survive long enough for the pathogen to complete its infectious cycle (reviewed in [30, 31–34]). Apoptosis is a widespread mechanism that is central to the maintenance of cellular homeostasis in all tissues, including the immune system [35]. Apoptosis, or the lack of apoptosis, contributes to the pathogenesis of a number of diseases, including acquired immunodeficiency syndrome, autoimmune disease, and, in particular, cancer [36, 37]. One may argue that the natural tendency of infected cells would be to die, mainly in response to the stress represented by the infection, and that therefore any successful intracellular pathogen should delay host cell apoptosis as long as possible.

In fact, Heussler et al. [38, 39] showed that the intracellular apicomplexan parasite Theileria parva protects infected T cells from apoptosis through activation of the transcription factor NF-κB. Another apicomplexan parasite, Toxoplasma, also modulates activity of NF-κB as a way of protecting infected cells against apoptosis [40]. Similar strategies are also used by several bacteria, and NF-κB activation has been shown to insure host cell survival during Rickettsia rickettsii infection [41]. However, although Chlamydia infection renders host cells resistant to apoptosis, the evidence linking NF-κB activation with Chlamydia infection has been more controversial [33, 34]. Other parasites that protect the host cells against apoptosis include T. cruzi, Leishmania, and Plasmodium [42–44].

Since P2X₇ ligation can lead to apoptosis or necrosis of uninfected macrophages and epithelial cells [45, 46], it should come as no surprise that some intracellular pathogens also inhibit P2X₇-mediated cell death. In fact, inhibition of P2X₇ signaling appears to be critical for propagation of some infections, since P2X₇-mediated host cell death has a larger impact on development of intracellular pathogens than host cell death induced through other surface receptors. Thus, treatment of M. tuberculosis-infected macrophages with ATP results in killing of the intracellular mycobacteria [47, 48]. Treatment of monocytes infected with bacille Calmette-Guérin (BCG) with H₂O₂ or ATP kills the monocytes, but only ATP treatment of the infected monocytes kills the mycobacteria [48]. In a comparison with other conditions that can induce lysis of macrophages, such as complement-mediated cytolysis, Fas ligation, and CD69 activation, only ATP treatment results in death of both host cells and intracellular mycobacteria [47].

Infection by several pathogens has now been shown to inhibit ATP-induced apoptosis of the host cell, including mycobacteria, chlamydiae, Porphyromonas gingivalis, and Leishmania [45, 49–51]. However, the mechanism by which these pathogens protect the host cell has been characterized mainly for mycobacteria and P. gingivalis [49, 50], both of which secrete an enzyme, nucleoside diphosphate kinase (NDK), that consumes extracellular ATP, thus depriving P2X₇ of its physiological ligand.

NDK is ubiquitously expressed in most species with similar amino acid sequence; however, the quaternary structure of NDK varies among species. It exists as a hexamer (humans, rats, pigs, bovine, Drosophila) in most species and as a tetramer in some bacteria (Myxococcus xanthus, Escherichia coli) [52]. In 1997, Shankar et al. [53] purified the 18-kDa NDK from Mycobacterium smegmatis. Approximately 70% homology was reported between the N terminus of M. smegmatis NDK and those of P. aeruginosa, E. coli, and Myxococcus xanthus. Mycobacterium bovis BCG is capable of secreting NDK upon stimulation with eukaryotic proteins (bovine serum albumin or ovalbumin) [50]. Later, recombinant M. tuberculosis NDK was crystallized by Chen et al., who showed that it has 45% sequence identity to human NDK with similar secondary and tertiary structures and exists as a hexamer, unlike most other bacteria [54]. Another group purified NDK from M. tuberculosis and demonstrated similar structural results to the recombinant protein [55].

Other ectonucleotidases produced by parasites

Examples of membrane-bound ecto-ATPases have been described in several parasites. In 1981, a Ca^2 + -dependent nucleotidase was localized and characterized in Entamoeba histolytica, whose ability to hydrolyze extracellular ATP increases in the presence of MgCl₂ [56, 57] (Fig. 2). In 1987, a nucleoside triphosphate hydrolase was described in T. gondii [58]. This hydrolase is present in the tachyzoite form of T. gondii and was detected as a circulating antigen in the sera of infected mice. Moreover, avirulent T. gondii strains express only nucleoside triphosphatase (NTPase) II, whereas virulent strains express both NTPase I and NTPase II [59]. Another study showed the membrane localization of a nucleotide triphosphate hydrolase in Toxoplasma [60], and its isoform activity was linked with T. gondii virulence [61]. Neospora caninum, also an apicomplexan parasite, expresses a type I nucleoside triphosphate hydrolase [62]. Recently, ecto-ATPases have been identified in diverse trypanosomatids, including T. cruzi [63], T. rangeli [64], and T. brucei brucei [65]. While the physiological significance of some of these ecto-ATPases remains to be shown, virulent Leishmania amazonensis promastigotes can hydrolyze ATP by a Mg-dependent ecto-ATPase more efficiently than avirulent promastigotes [66]. Mg-dependent ecto-ATPase activity was also described in Leishmania tropica [67]. In general, high levels of surface expression of ecto-ATPases correlate with virulence of the pathogens [19].

Fig. 2.

Mechanisms of pathogen evasion. Parasites have several methods to avoid antimicrobial activity of infected cells, including interference of intracellular signaling pathways (in response to ROS) and membrane trafficking (via PLD). Successful pathogens (green) can thus replicate, complete their infection cycle, and escape to the extracellular environment (right side). In addition, some protozoan parasites express ecto-ATPases (green with orange hat), which can contribute to cleavage of extracellular ATP, preventing P2X₇R activation (left side). Some intracellular bacteria also protect the infected cell by secreting enzymes that consume extracellular ATP (not shown)

P2X₇ receptor polymorphisms and their association with disease susceptibility

Single nucleotide polymorphisms (SNPs) are variations in a DNA sequence that occur when a single nucleotide in the sequence is different from the norm in at least 1% of the population. When SNPs occur inside a gene, they create different variants, or alleles, of that gene. Genetic factors may confer protection or increase susceptibility to infectious disease [68, 69].

P2X₇ receptor polymorphisms have been described in several diseases associated with loss- or gain-of-functions of this receptor. Two P2X₇ alleles have been associated with human diseases. Recently the Thr²⁸³ was found to be a key determinant in P2X₇ receptor function [70]. Gu et al. [71] reported that the A1513C polymorphism is associated with normal P2X₇ protein expression levels and subcellular localization, but defective pore formation. Functional analysis in transfected HEK293 cells expressing P2X₇ confirmed increased ATP-dependent activation of the P2X₇ 489T mutant, compared to the wild-type receptor. These data identify 489C>T as a gain-of-function polymorphism of P2X₇ [71]. The A1513C allele has been correlated with resistance to ATP-induced apoptosis and an increased incidence of familial chronic lymphocytic leukemia [72–74]. In addition, a His-155 to Tyr polymorphism confers gain-of-function to the human P2X₇ receptor of human leukemic lymphocytes [75].

P2X₇ polymorphisms are also involved in murine and human lupus susceptibility [76] and Crohn’s disease [77]. Several lines of evidence link P2X₇ polymorphisms with various diseases such as Alzheimer’s disease [78], bipolar affective disorders [79], multiple sclerosis [80], and diabetes [81].

Skarrat et al. [82] identified a splice site mutation that is an inherited polymorphism in a Caucasian population and gives rise to a P2X₇ null allele in 1–2% of the population. Similarly, an Arg³⁰⁷ to Gln change within the ATP-binding site of human P2X₇ causes a loss of function of the receptor [83]. This work raises the possibility that low or absent P2X₇ receptor function due to inherited polymorphisms may be a genetic susceptibility factor in a range of infections as diverse as tuberculosis, toxoplasmosis, or Chlamydia. Individuals who carry two loss-of-function polymorphisms (compound heterozygotes) in P2X₇ may have the highest susceptibility to infections [83].

In the context of infections by intracellular pathogens, heterogeneity of ATP-induced killing of BCG was shown in a small number of patients, suggesting possible genetic differences in P2X₇ [47]. A Gambian study showed a weakly protective effect against pulmonary tuberculosis for a polymorphism in the putative promoter [84]. These findings suggest that P2X₇ polymorphisms may contribute to host immunity to M. tuberculosis infection in humans [84]. P2X₇ is now known to stimulate secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, following inflammasome and caspase-1 activation in macrophages [85, 86]. The Glu-496 to Ala polymorphism was shown to impair ATP-mediated immune responses such as the killing of mycobacteria by human macrophages and the release of IL-1β and IL-18 from human monocytes [87–89]. Three single loss-of-function mutations in human P2X₇ cause reduced ATP-induced macrophage apoptosis and killing of mycobacteria [90]. Shemon et al. [91] reported that another single polymorphism decreases the response against mycobacterial infection by the host: a Thr³⁵⁷ to Ser change in homozygous and compound heterozygous subjects causes absent or reduced P2X₇ function and impairs ATP-induced mycobacterial killing by macrophages. P2X₇ gene polymorphisms in Mexican mestizo patients with pulmonary tuberculosis was associated with increased susceptibility for M. tuberculosis infection [92]. In conclusion, various polymorphisms in P2X₇ abrogate ATP-induced killing of mycobacteria by human macrophages and, thus, may contribute to variability in susceptibility to mycobacterial infections [90].

Infection by T. gondii has also have been studied with respect to P2X₇ polymorphisms. Wiley et al. [93] have observed severe illness in two compound heterozygotes whose P2X₇ function was totally absent. Moreover Fuller et al. [94] identified three immunocompetent subjects with absent P2X₇ function due to single nucleotide polymorphisms and severe disease due to T. gondii infection.

Taken together, the results from studies on patients with tuberculosis or toxoplasmosis suggest an important role for P2X₇ polymorphisms in the ability of the immune response to control infection.

Concluding remarks

For many years, P2X₇ on macrophages was studied mainly with regards to its pharmacological properties and role in macrophage lysis. A physiological function for P2X₇ remained elusive, until more recent studies have shown a role for P2X₇ in promoting proinflammatory responses and controlling intracellular infection in vitro. Growing evidence also links P2X₇ polymorphisms in humans with susceptibility to infection, further confirming an antimicrobial role for P2X₇ in vivo. Several pathogens are now known to protect infected cells against P2X₇-dependent apoptosis by producing ATPases that consume extracellular ATP, and it is likely that more intracellular pathogens will be shown to similarly confer resistance against host cell death.

Abbreviations

NDK

nucleoside diphosphate kinase

NTPase

nucleoside triphosphatase

PLD

phospholipase D

ROS

reactive oxygen species

SNP

single nucleotide polymorphisms