P2X4 receptors, immunity, and sepsis

Abstract

Sepsis is life-threatening systemic dysfunction caused by a deregulated host response to an infectious insult. Currently, the treatment of sepsis is limited to the use of antibiotics, fluids, and cardiovascular/respiratory support. Despite these interventions, septic mortality remains high, with reduced life quality in survivors. For this reason, the identification of novel drug targets is a pressing task of modern pharmacology. Based on recent research, it appears that P2 purinergic receptors, which can regulate the host’s response to infections, have been identified as potential targets for treatment of sepsis. Among P2 receptors, the P2X₄ receptor has recently captured the attention of the research community owing to its role in protecting against infections, inflammation, and organ injury. The present review provides an outline of the role played by P2X₄ receptors in the modulation of the host response to sepsis and the promise that targeting this receptor holds in the treatment of sepsis.’

Introduction

In 2016, the 3^rd International Consensus Conference for Sepsis and Septic Shock defined sepsis as a life-threatening multiorgan dysfunction arising from a dysregulated host response to an infection, which has a high risk of death [1]. According to early estimates in this millennium, there are about 751,000 cases of sepsis (3.0 per 1,000 people) each year, resulting in over 200,000 deaths in the United States of America [2]. More recent studies indicate that sepsis causes, or contributes to, from one-third to one-half of all deaths occurring in hospitals in the United States, with the majority of patients presenting to hospital with sepsis rather than acquiring sepsis in hospital [3]. The overall sepsis-related health care cost in the US has been estimated to be $16-25 billion annually [4]. Taking into account the rapid expansion of the elderly population, which is affected disproportionately by sepsis due to physiologic frailty and immune senescence, the prevalence of sepsis and the associated health care costs are expected to rise significantly over the next two decades [5]

Several risk factors contribute to the development of sepsis, including chronic diseases impairing the immune system (e.g., acquired immunodeficiency syndrome, chronic obstructive pulmonary disease, cancers) and treatment with immunosuppressive drugs [6]. In addition, age, sex, race and ethnic group are important determinants of sepsis prevalence, as it occurs more frequently in infants and elderly persons than in other age groups, at higher rates in males than in females, and at increased frequencies in African Americans as compared to Caucasians [7].

The clinical signs of sepsis are highly variable, depending on the initial site of infection, the pathogen, the pattern of acute organ dysfunction, as well as the underlying health status of the patient, and the interval before initiation of treatment [8]. Septic patients frequently display fever, shock, and respiratory failure [9]. Our understanding about the pathophysiological mechanisms underlying the onset and development of sepsis has evolved over the time. Initially, the development of sepsis was ascribed to a hyper-inflammatory condition characterized by an unrestrained immune response (systemic inflammatory response syndrome or SIRS) with a systemic cytokine storm, which is the result of an overproduction of several pro-inflammatory molecules including TNF-α, IL-1β, IL-2, IL-6, IL-8, and IFN-γ [9]. Subsequent hypotheses refined this original view, and proposed that sepsis develops through a sequential or parallel (SIRS)[10] and compensatory anti-inflammatory response syndrome (CARS), a condition with suppresses immunity and enhances the susceptibility to secondary infections[11].

Despite over 100 clinical trials conducted on sepsis, no FDA-approved treatment options exist that can improve sepsis survival [12]. For this reason, the identification of novel pharmacological approaches to manage sepsis is of extreme interest to the scientific community. Over the past two decades, increasing attention has been paid to the involvement of the purinergic system in the pathophysiology of sepsis [13-19]. The purine ATP is a well-recognized signaling molecule released at sites of infection, inflammation and cell injury. In this setting, extracellular ATP acts as a “danger” signal, which, through the activation of P2 receptors plays a critical role in immune cell migration, chemotaxis, and cytokine release [19-21].

In this brief review article, we have delineated the role of one of the P2 receptors, the P2X₄, in regulating immunity during infections, inflammation and sepsis.

Purinergic signalling: receptors, enzymes, transporters

Purinergic signaling is initiated by the release of nucleotides into the extracellular space through volume regulated anion channels, maxi-anion channels, transporters, connexins and pannexins [22], as well as exocytotic pathways and membrane damage (Fig. 1) [23]. Once released into the extracellular space, the nucleotides, which include ATP, ADP, UDP, and UDP-glucose, can trigger a series of cellular responses through the engagement of P2 receptors. These are classified into ionotropic P2X (P2X_1–7) and metabotropic P2Y (P2Y_{1,2,4,6,11–14}) receptors (Fig. 1) [23]. P2X receptors display a trimeric structure, where each monomer has two transmembrane domains. They are ion channels and gate primarily Na⁺, K⁺ and Ca²⁺, and, in some cases, Cl⁻ [24]. The stimulation of G_q/11-coupled P2Y_1,2,4,6 and P2Y₁₁ receptors causes the activation of phospholipase C, leading to the downstream production of inositol-(1,4,5)-trisphosphate and diacylglycerol (DAG) [25]. Inositol-(1,4,5)-trisphosphate increases intracellular Ca²⁺ levels were DAG stimulates protein kinase C (PKC) [25]. Moreover, P2Y₁₁ receptor activation can stimulate whereas P2Y_12,13 receptor engagement can inhibit, adenylate cyclase [25]. As asserted above, in addition to ATP, UTP and its degradation product, UDP, can play important regulatory roles also in modulating the immune cell activities (i.e. neutrophil and monocyte chemotaxis, activation of granulocytes, dendritic cells, monocytes, neutrophils and lymphocytes)[26]. Indeed, UTP, UDP and UDP-glucose act as agonists on four P2Y receptor subtypes (P2Y₂, P2Y₄, P2Y₆ and P2Y₁₄), whereas no affinity has been observed for P2X receptors [26]. In general, the engagement of P2 receptors has been shown to facilitate immune cell function.

Figure 1.

Once released into the extracellular environment, through channels or other extrusion systems, ATP, ADP, AMP, and adenosine, are metabolized by a set of nucleotidases [nucleoside triphosphate diphosphohydrolases, ecto-ATPase, NTPD-1 (CD39), ecto-5’nucleotidase (CD73), purine nucleoside phosphorylase PNP), xanthine oxidase], leading to their sequential dephosphorylation and subsequent generation of other bioactive metabolites, which selectively interact with purinergic P1 (A₁, A_2A, A_2B and A₃) or P2 (P2X and P2Y) receptors. Several cell types are endowed with nucleoside transporters (NT), which operate the uptake of extracellular adenosine, thus actively participating to the careful regulation and termination of adenosine signaling.

Following their release into the extracellular space, ATP and/or ADP are quickly converted by CD39 (ecto-nucleoside triphosphate diphosphohydrolase 1, E-NTPDase1) into AMP, and then, via CD73 (ecto-5'-nucleotidase) AMP is dephosphorylated into adenosine [27]. The latter is a purine nucleoside, which exerts its cellular actions through the engagement of specific G protein-coupled receptors classified as A₁, A_2A, A_2B and A₃. In particular, A₁ and A₃ receptors are coupled to G_i, G_q and G_o proteins, while the A_2A and A_2B receptors activate adenylyl cyclase via G_s or G_olf [28] (Fig. 1). The stimulation of A_2B receptors can trigger also phospholipase C via G_q [28,29]. In general adenosine exerts immunosuppressive effects [28,29]. Of note, since the CD39/CD73 enzyme axis is critical in the degradation of ATP, ADP, and AMP into adenosine, it can be considered as a an “immunological switch” which is able to shift an ATP-driven pro-inflammatory immune cell response toward an anti-inflammatory environment steered by adenosine [23] [27,30,31] (Fig. 1).

It is interesting to note that P1 and P2 purinergic receptors have distinct and divergent effects on immunity and inflammation regulation [32]. Whereas adenosine-mediated P1 purinergic receptor activation dampens inflammation and attenuate immune-mediated tissue injury overall [14,15,33], P2 purinergic receptor activation by ATP drives inflammasome activation and stimulates bacterial killing [34,35]. These divergent effects of P1 and P2 purinergic receptors on immunity and inflammation is clinically significant as sepsis is a disease characterized by initial hyper-inflammatory phase followed by hypo-immune state where the subject succumbs from immune paralysis [36]. Depending on the immune state of the septic subject, one can tailor therapy with either P1 (to dampen inflammation) or P2 (to promote immunity) purinergic receptors.

P2X₄ receptor: structure and molecular biology

P2X receptors are a family of non-selective trimeric ligand-gated channels, permitting Na⁺, K⁺ and Ca²⁺ ion fluxes upon binding extracellular ATP [37]. Seven subtypes of P2X receptors have been cloned and classified as P2X₁ to P2X₇, with functional channels assembled as homo- or heterotrimers [37]. Each monomer comprises two transmembrane domains linked by a large extracellular loop and intracellularly located N- and C-termini [38]. The activity of P2X receptors is modified by several factors including the extracellular concentration of ions (Zn²⁺, Cu²⁺, Hg²⁺, Ni²⁺, Cd²⁺), protons, lipids, steroids, and ethanol [39].

The P2X₄ receptor is one of the most sensitive purinergic receptors, as it is activated by nanomolar concentrations of extracellular ATP [39]. Of note, the Ca²⁺ permeability of P2X₄ is the highest among the P2X family [40].

Immunoprecipitation studies have revealed that the P2X₄ subtype can heteromerically assemble with other P2X members, including P2X₁, P2X₂ and P2X₆ [41,42]. In addition, structural interactions have been reported between P2X₄ and P2X₇ receptors [41,43]. In this regard, Schneider et al. [44] recently demonstrated that P2X₄ and P2X₇ subunits can form heterotrimeric P2X₄/P2X₇ receptors. Of note, the P2X₄ and P2X₇ subunit isoforms are widely co-expressed particularly on immune/inflammatory cells [45].

P2X₄ receptors are distributed throughout the body [46]. Indeed, they are widely expressed in central and peripheral neurons, microglia, and various glandular tissues such as pancreatic acinar cells and salivary glands as well as endothelial cells [47]. They are expressed also throughout the gastrointestinal tract, liver, lung, kidney and reproductive system [23][24]. At the cellular level, the P2X₄ receptor is located on plasma membrane, but also in intracellular compartments, such as lysosomes, vesicles, vacuoles and lamellar bodies[48].

Acute kidney injury and inflammation is a major complication of sepsis and has extremely high mortality (>70% in severe sepsis) [49]. Although several renal cells express P2X₄ purinergic receptor, the role for P2X₄ receptor in acute kidney injury and inflammation is unclear unlike the better-characterized pro-inflammatory role of P2X₇ receptor [50]. It appears that P2X₄ dampens renal fibrosis response during recovery from kidney injury as mice deficient in P2X₄ receptors have exacerbated renal fibrosis in a mouse model of chronic interstitial inflammation [51]. Furthermore, renal tubular P2X₄ receptor activation may promote kidney injury in early sepsis by promoting NOD-like receptor 3 inflammasome activation and facilitating IL-1β and IL-18 maturation [52].

P2X₄ receptor and immune system

All immune cells, whether from the myeloid or lymphoid lineage, express at least one P2X or P2Y receptor subtype (see Table 1) [53]. Consistently with this knowledge, an increasing body of evidence supports the critical role played by both P2X and P2Y receptors in immune cell biology [54-57]. In accordance with its wide distribution, the P2X₄ receptor has been shown to regulate a variety of pathophysiological processes, such as neuropathic pain and autoimmune diseases [39,58,59]. Many of these modulating roles are mediated by cells of the immune system.

Table 1.

Expression of P2 receptors on immune cells

Immune cell population	Purinergic receptors	References
Monocyte/macrophage	P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14 P2X1, P2X4, P2X7	[55,96]
Dendritic cells	P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y14, P2X1, P2X4, P2X7	[76,97]
Neutrophils	P2Y1, P2Y4, P2Y6, P2Y11 P2X1, P2X4, P2X7	[56]
Eosinophils	P2Y1, P2Y4, P2Y6, P2Y11, P2Y14, P2X1, P2X4, P2X7	[98,99]
Mast cells	P2Y1, P2Y12, P2Y13, P2Y14 P2X1, P2X3, P2X4, P2X7,	[100,101]
T lymphocytes	P2Y1, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14, P2X1, P2X4, P2X7	[102]
B lymphocytes	P2Y1, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14 P2X1, P2X4, P2X7	[57]
Natural killer cells	P2Y11, P2X1, P2X4, P2X7	[103]

Monocytes, and macrophages.

Monocytes and macrophages play a pivotal role in the immune response against micro-organisms and the pathogenesis of sepsis [60] [61]. These cells phagocyte and kill micro-organisms, release cytokines, and present pathogens to T cells, thus triggering both cellular and humoral immune responses [60]. All monocyte and macrophage cell types have been found to express P2X and P2Y receptors. In monocytes, the most abundant P2X receptor transcripts are P2X₄, followed by transcripts for P2X₇ and P2X₁ [62], thus suggesting an important role for these receptor subtypes in monocyte functions.

Murine peritoneal macrophages express functionally active P2X₄ and P2X₇ receptors [63,64]. In particular, the exposure of these cells to low concentrations of ATP evoked a small P2X₄-driven ion current, while higher ATP concentrations evoked a large P2X₇-driven ion current [64]. However, the physiological role of the P2X₄ receptor-driven current was not explored in this study [64]. P2X₄ expression has been detected also on both human and rat alveolar macrophages [65], and the authors described a dynamic regulation of P2X₄ receptors during distinct phases of macrophage activation [65]. In resting macrophages, functional P2X₄ receptors as measured using ATP-evoked currents were maintained at very low levels, whereas they underwent a rapid up-regulation in response to phagocytosis [65]. By contrast, classical activation of macrophages elicited by their incubation with IFN- γ and TNF-α or IFN- γ and LPS, reduced the surface expression of P2X₄ receptors and decreased ATP-evoked currents without altering total P2X₄ receptor protein levels [65]. Of note, the alternative activation of these cells with IL- 4 or IL- 13 did not alter the total surface or the functional expression of these receptors [65]. Based on these observations, the authors hypothesized a scenario in which an initial bacterial or inflammatory stimulus elicits rapid trafficking of P2X₄ receptors to the macrophage cell surface causing increased Ca²⁺ influx, thus promoting their activity [65]. After the termination of macrophage activation, a feedback mechanism develops to curb P2X₄ receptor trafficking to the cell membrane and function, probably aimed at facilitating the resolution inflammation [65].

Recent observations highlighted molecular and functional interactions between P2X₄ and P2X₇ receptors on macrophages [66-68]. In particular, a physical protein–to protein interaction takes place between P2X₄ and P2X₇ receptors, where the two receptors are bridged by the C-terminus of P2X₇ receptor [66]. This physical interaction was facilitated by the presence of extracellular ATP [66]. Kawano et al. provided evidence about a role of P2X₄ receptor in modulating P2X₇ receptor-dependent inflammatory functions [67]. Indeed, by means of in vitro assay, it was observed that treatment of RAW264.7 cells with ATP elicited a P2X₇-dependent release of HMGB1 and IL-1β [67]. Of note, this event was blunted by the genetic ablation of P2X₄ receptor via short hairpin RNA transfection as well as by removing extracellular Ca²⁺[67], thus confirming a functional interplay between P2X₄ and P2X₇ receptors, driven by a modulation of intracellular Ca²⁺ concentrations.

The P2X₄/P2X₇ linkage has been shown to hold a critical role also in eliciting macrophage death[68]. In particular, the recruitment of P2X₇ receptors in RAW264.7 cells with high concentrations of ATP determined an increase in Ca²⁺ influx, pore formation, and activation of ERK1/2 and p38MAPK, thus leading to cell death [68]. In this context, despite the activation of P2X₄ receptor alone did not induce cell death, the P2X₄ receptor-dependent acute-phase Ca²⁺ influx, elicited by the high levels of ATP, contributed to P2X₇ receptor-dependent cell death in activated macrophages [68].

Recently, our experiments revealed a role of ATP in regulating bacterial killing in macrophages via P2X₄ receptors [18]. We observed that the selective P2X₄ receptor antagonist 5-BDBD and the shRNA-mediated receptor silencing, prevented the stimulant effect of ATP on E. coli killing by macrophages [18]. In addition, in macrophages isolated from P2X₄ receptor knockout mice, ATP failed to stimulate bacterial killing [18]. Thus, we concluded that P2X₄ receptors augment bacterial killing by macrophages. Mechanistically, we found that P2X₄ receptor activation specifically increased mitochondrial, but not cellular, ROS production, which was a likely mediator of the antibacterial effect of P2X₄ receptor activation [18].

In parallel, using the murine cecal ligation and puncture model, considered by several investigators as the “gold standard” mouse model of sepsis [69], we demonstrated also that macrophage P2X₄ receptors control bacterial spread and inflammation in sepsis [18]. That is, P2X₄ receptor knockout mice showed an increased bacterial load and a decreased survival in comparison with wild type animals [18], indicating that endogenous ATP released during sepsis, exerts a protective effect through P2X₄ receptor engagement [18]. Of note, we also provided evidence that protective effect of P2X4 receptor activation was mediated though P2X₄ receptor signaling on macrophages, as both adoptive transfer of P2X₄^−/− receptor macrophages or myeloid-specific P2X₄^−/− receptor mice emulated the deleterious phenotype of P2X₄ deficient animals.

Dendritic cells.

Dendritic cells (DCs) are the primary antigen-presenting cells in the body and therefore they are pivotal in linking innate and adaptive immunity [70]. Dendritic cell function is compromised during sepsis [71,72] and sepsis is associated with widespread dendritic cell depletion [73,74]. DCs are endowed with both P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁ and P2Y₁₄) and P2X receptors (P2X₁, P2X₄, P2X₇) [75,76]. Our understanding of the role of P2X4 receptors in regulating dendritic cell function is in its infancy. Sakaki et al. demonstrated recently that P2X₄ receptor activation elicited a rapid and substantial initial Ca²⁺ influx in dendritic cells. This Ca²⁺ influx was required for optimal P2X₇-dependent IL-1β and IL-18 release from DCs [77]. Clearly, further studies are required to unravel the precise role of P2X₄ receptors in regulating DC function during immune responses and sepsis.

Neutrophils.

Neutrophils are professional phagocytes that play a central role in host defense against extracellular bacteria and fungi [78]. These cells are endowed with a unique capacity to very efficiently and rapidly engulf and thereby eliminate pathogens and cell debris [79]. Patients with sepsis display aberrant neutrophil activity, characterized by an impaired recruitment of these cells to sites of infection, abnormal longevity, and overproduction of toxic mediators leading to bystander organ injury [80].

A substantial body of evidence supports the notion that ATP regulates tightly several neutrophil functions, such as chemotaxis, rolling, adhesion, transmigration, phagocytosis, oxidative burst, extracellular trap formation, degranulation and apoptosis [56]. The role of P2X₄ receptors in neutrophils is incompletely understood. The only study dealing with this issue showed that P2X₄ knockout mice demonstrated reduced infiltration of neutrophils to the injury site in a murine spinal cord injury model [81]. It was however unclear from this study whether P2X₄ receptor signaling in neutrophils was required for optimal neutrophil infiltration or P2X₄ signaling indirectly affected neutrophil infiltration.

Considering our data, showing a stimulant role of P2X₄ receptors on macrophages in bacterial killing [18], it would be interesting to test whether P2X₄ receptors have a similar role in neutrophils.

T lymphocytes.

Sepsis results in a deluge of pro- and anti-inflammatory cytokines and immunosuppressive mediators, leading to CD4⁺ and CD8⁺ T cell dysfunction and death [82]. Although the precise mechanisms, underlying this condition of T cell suppression, are not well defined, several lines of evidence indicate that purinergic signaling may control T cells during sepsis in many different ways [20,83]. Indeed, the release of ATP via pannexin-1 pores during lymphocyte activation, enhances IL-2 release, in an autocrine manner, through the engagement of P2X₁, P2X₄ and P2X₇ receptors [84,85]. Mechanistically, ATP elicits Ca²⁺ influx through these receptors and stimulates the nuclear factor of activated T cells and MAPK signaling resulting in increased IL-2 production [84,86,87]. The accumulation of several purinergic components, including pannexin 1 channels, P2X₁ and P2X₄ receptors, at the immune synapse facilitates T cell–accessory cell communication, antigen recognition and T cell activation [87]. Mitochondria also translocate to the immune synapse where by directly providing the ATP to be released, fuel the autocrine purinergic signaling in the synaptic cleft [87]. P2X₄ receptors colocalize with mitochondria in clusters at the front of cells, facilitating the Ca²⁺ influx to maintain local mitochondrial ATP production at the levels needed for cell migration [88].

In patients with sepsis, purinergic signaling appears defective [89]. Indeed, resting cells from septic patients lack the autocrine purinergic feedback loops that maintain basal cytosolic Ca²⁺ levels and mitochondrial function thus, leading to a condition of cellular paralysis that impairs vigilance and precludes proper functional responses to T cell stimulation [89]. However, the suppression of purinergic signaling is not due to a reduction of P2X receptor expression, since the mRNA levels of these receptors appear unchanged (P2X₄ and P2X₇) or elevated (P2X₁) in patients with sepsis [89]. Further studies will be needed to unravel the precise role of purinergic signaling in T cells in sepsis.

Conclusions

Although antibiotic therapy still represents an irreplaceable part of sepsis management, this therapeutic approach is facing hard challenges due to the development of multi-resistant bacterial strains [90,91]. In addition, antibiotic treatment does not prevent the various immune abnormalities occurring during sepsis. For these reasons, it is necessary to foster an integrated approach in the treatment of sepsis, which might take advantage of our increasing understanding of the role of purinergic signaling is sepsis [92].

So far, in the arena of purinergic research much attention has been focused on P2X₇ receptors, as they have been shown to be involved in a plethora of inflammatory disorders (i.e. osteoarthritis, rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn’s disease), and thus they appear to be one of the most appealing potential drug target [93]. However, recently there has been also growing interest on the P2X₄ receptors. Indeed, this receptor subtype, which is widely investigated in the context of pain transmission [94,95], also has a role in mediating the effects of ATP in shaping immune/inflammatory responses. For this reason, it is striking that the P2X₄ receptor, which is being widely expressed on cells of both the innate and adaptive arms of the immune system (see Table 1) remains poorly characterized in sepsis. Our pioneering preclinical studies demonstrated that P2X₄ receptor engagement is critical in limiting the dissemination of bacteria by enhancing killing of these pathogens[18]. This indicates that P2X₄ receptors are a potential target for antibacterial drug discovery. Unfortunately, the study of P2X₄ receptors has been seriously hindered by the lack of selective pharmacological tools. Indeed, for a long time, researchers had to rely on non-selective P2X receptor antagonists such as paroxetine, TNP-ATP, and BBG to characterize P2X₄ receptors (Fig. 2). We are hopeful that in the coming years medicinal chemists will generate selective and potent ligands for P2X₄ receptors

Figure 2.

Schematic structure of the P2X₄ receptor and the main commercially available ligands acting on this receptor subtype with indicated the IC₅₀ and EC₅₀ values.

Highlights.

• Sepsis is an organ dysfunction caused by a deregulated immune response to an infection

• At present, no approved treatment options exist that can improve sepsis survival

• Increasing attention has been paid to the role of the purinergic system in sepsis pathophysiology