Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic Diseases

Abstract

Purine derivatives like adenosine 5′-triphosphate (ATP) is the powerhouse of the cell and is essential to maintain the cellular homeostasis and activity. Besides this they also act as a chemical messenger when released into the extracellular milieu because of stress and cellular insult. The extracellular ATP (eATP) as well as its metabolite adenosine triggers purinergic signaling affecting various cellular processes such as cytokine and chemokine production, immune cell function, differentiation, and maturation, and mediates inflammatory activity. Aberrant purinergic signaling had been implicated in several diseased conditions. This chapter will focus on the dynamics of purinergic signaling and immune signaling in driving under various diseased conditions like autoimmunity and infectious disease.

1. Introduction

Adenosine 5′-triphosphate (ATP) is abundantly generated in the cytosol through respiration and glycolysis. Primarily, these are the “energy currency” of the cell as ATP hydrolysis release energy and is essential to maintain the cellular homeostasis and activity [1]. Extracellular activity of ATP was first described by Drury and Szent-Györgyi in 1929 [2]. Later, in 1970s, ATP was shown to be involved in non-adrenergic, non-cholinergic nerve-mediated responses, and further its function as a neurotransmitter was established that led to introduction of the term “purinergic signaling” [3]. Burnstock has described about the purinergic signaling and purinergic systems in very detail, which consists of (a) purine or pyrimidine derivatives that serve as an “extracellular messenger,” (b) “membrane transporter” that are responsible for the extracellular release of these nucleotides or nucleosides, (c) “metabolizing enzymes” present on the cell surface that hydrolyze the ATP to adenosine diphosphate (ADP) then to adenosine monophosphate (AMP) and adenosine and, (d) “purinergic receptors” that sense the extracellular purine or pyrimidine derivatives [3, 4, 5, 6, 7].

In the beginning, purinergic signaling was determined to have a role in neuronal signaling but now, their role in immune responses, inflammation, pain, exocrine and endocrine secretion, platelet aggregation, and endothelial-mediated vasodilatation had been explored and established [3, 4, 6, 8]. Additionally, cross talk of purinergic signaling with other signaling network also associates with the impact on cell proliferation, differentiation, and death that occur during the development and regeneration processes. Under normal condition, purinergic signaling operate in a very well-regulated manner to maintain the physiological function of different organ systems. Dysregulation in any component of the purinergic signaling network depending on the expression or activation of purinergic receptors, ectonucleotidases or release of agonist from damaged cell resulting from stress, inflammation serves as a potent modulator of inflammation and key promoters of host defenses, immune cells activation, pathogen clearance, and tissue repair that contributes to the disease pathogenesis [9]. Thus, their knowledge is of great importance for a full understanding of the pathophysiology of acute and chronic inflammatory diseases and will give an insight on novel therapeutic approaches to overcome inflammation. This chapter describes the component of purinergic system, its cross talk with immune signaling. Major focus of this chapter is to present the dynamics of purinergic signaling under normal physiological condition and its role in modulating the immune and inflammatory response under various diseased conditions like autoimmunity, and microbial infection.

2. Purinergic system and its component on immune cells: an immunomodulator

2.1 Mechanism of release of nucleotides

Extracellular ATP (eATP) has been well established as a ligand for autocrine and paracrine signaling that has a pathophysiological role. In addition, to eATP other nucleotides and nucleosides such as the adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), uridine diphosphate (UDP), uridine triphosphate (UTP), and nicotinamide adenine dinucleotide (NAD+) also serve as a potent purinergic signaling modulator. The release of nucleotides into the extracellular space occurs via regulated and unregulated mechanisms. Regulated mode of release of nucleotide is mediated through classical exocytosis [10] or conductive ATP release through ATP-permeable channels [11]. Currently, five groups of ATP-release channels are known such as: connexin hemichannels, Pannexin (PANX), calcium homeostasis modulator 1 (CALHM1), volume-regulated anion channels (VRACs), and maxi-anion channels (MACs) [12].

The ATP release by exocytosis is an active release mechanism that involves vesicular nucleotide transporter (VNUT). It is responsible for the accumulation and exocytosis of ATP from exocytotic vesicles that occurs in a proton-dependent electrochemical gradient manner generated by a vacuolar-ATPase (v-ATPase). Further, intracellular Ca⁺² level and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) drives the fusion of the exocytotic vesicles with the plasma membrane ultimately resulting in the release of nucleotides into the extracellular space [13, 14]. Hemichannels are the ATP permeable channels that support the release of ATP under specific pathological condition. Primarily, these channels contribute to various cellular and physiological functions by forming gap junctions or hemichannels, to allow intercellular communication. They are categorized into two based on their functions, [1] connexins that has both gap junction and form hemichannel function whereas [2] PANX only form hemichannel [15]. These channels are in closed state under normal condition to avoid the loss of vital ionic, energetic, and metabolic gradients. However, chemical and biochemical stimuli resulting from pathological conditions trigger their opening and lead to release of ATP. Till date, 21 isoforms of connexins are reported in human of which connexin-43, -37, -26, and -36 have been shown to support ATP release [16]. Connexin-43 is widely expressed and very well studied. It is activated by increase in the intracellular Ca⁺² concentration, plasma membrane depolarization, reactive oxygen species (ROS) or nitric oxide (NO) [17, 18]. The PANX (PANX) family is comprised of three members, PANX-1, -2, and -3 of which PANX-1 and -3 are widely expressed in different tissues, while PANX-2 is exclusively found in the brain [19]. In resting state, PANX channels are closed, mainly due to the blockage of the pore by C-terminal tail from the intracellular side [18]. However, in response to apoptosis or pyroptosis, C-terminal tail gets cleaved by caspase-3, -7, or -11 leading to opening of PANX-1 and allows nucleotides to cross the plasma membrane [20, 21]. Additionally, other stimuli such as intracellular calcium increase, redox potential changes, mechanical stress, and activation of the P2X7R can trigger PANX-1 channel opening [22].

Another mechanism involves the disruption of the cell membrane by apoptosis, necrosis, pyroptosis, or netosis, which leads to the unregulated leakage of ATP as well as other large cytosolic molecules including enzymes [11, 23, 24].

2.2 Metabolism of extracellular nucleotides and nucleosides

The life span of eATP is controlled by purinergic ectoenzymes that coordinate a sequential two-step process of hydrolyzing ATP into AMP and then into the potent anti-inflammatory adenosine. This make ectonucleotidases enzymes a crucial component of the purinergic system, which balance the level of eATP as well as other nucleotide derivatives UTP, NAD+, and their metabolites, thereby controlling the activation of purinergic receptors and biochemical composition of the inflammatory microenvironment. These enzymes are classified into four major families: (a) ectonucleoside triphosphate phosphohydrolases (NTPDases): This group of enzymes are further classified into 8 subfamilies—NTPDase 1 (CD39), 2, 3, and 8, which are expressed on the cell surface, whereas NTPDases 4–7 are present in the intracellular organelles. Of these 8 subfamilies, NTPDase1 (CD39) is the best characterized that hydrolyses ATP to ADP and further to AMP. CD39 is expressed on wide variety of immune cell, e.g., monocytes, dendritic cells (DCs), T regulatory (Treg) cells, and natural killer (NK) cells. (b) nicotinamide adenine dinucleotide glycohydrolase (NAD glycohydrolase/CD38): CD38 is a cell surface glycoprotein highly expressed in hematopoietic tissues such as the bone barrow and lymph nodes. Among immune cells, CD38 is highly expressed on monocytes, macrophages, DCs, neutrophils, innate lymphoid cells (ILC), NK cells, T and B cells. It hydrolyses NAD+ to cyclic-ADP ribose (cADPR) and then to AMP. (c) ecto-5′-nucleotidase (NT5E/CD73): CD73 degrades AMP generated by CD39 or CD38 to adenosine. It is expressed on stromal cells, follicular DCs, endothelial cells, neutrophils, macrophages, and subpopulations of T cells. and (d) ectonucleotide pyrophosphatase/phosphodiesterase (NPPs): NPPs include 7 members NPP 1–7. NPP1–3 degrade nucleoside triphosphates and diphosphates, NAD+, UDP-sugars, and di-nucleoside polyphosphates. NPP2 also known as autotaxin (ATX) has unique property of hydrolyzing nucleotide as well as phospholipids but acts more efficiently on later to generate the bioactive phospholipid mediator’s lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). NPP6 and 7 hydrolyzes phospholipids only, whereas catalytic properties of NPP4 and 5 are not known. Some NPPs are expressed on liver and intestinal epithelia, neuronal cells; NPP1 is also expressed on B and T cells [25, 26, 27, 28]. The ectonucleotidases are present on almost all types of immune cells, but their expression pattern changes in a function dependent manner and controls the pro-inflammatory and anti-inflammatory condition to avoid any pathological conditions like autoimmunity, cancer, and infectious disease.

Briefly, CD39 has an anti-inflammatory property that controls the extracellular level of ATP by converting it into adenosine in conjunction with CD73. CD39 and CD73 exhibit an immunosuppressive activity as shown by its expression on Tregs cells [29, 30, 31]; CD8 T cells [32] and B cells [33] and inhibits the pathogenic T cells. Breakdown of eATP by CD39 prevents the activation of P2X7R and attenuates the secretion of IL-1β and IL-18 [34]. The expression pattern of CD38 varies during the differentiation and maturation of B and T cells [35, 36]. The enzymatic activity of CD38 generates cADPR/ADPR and triggers Ca⁺² release from intracellular stores and Ca⁺² influx from the extracellular space that have role in transmigration and chemotaxis of neutrophils, monocytes and DCs, and cytokine release [37]. Elevated level of cADPR/ADPR and intracellular Ca⁺² regulates cellular chemotaxis [38], phagocytosis [39], and antigen presentation [40] in a CD38 dependent manner. Thus, dysregulation of CD38 has been implicated in several inflammatory pathologies such as autoimmunity and cancer [37, 41]. It is important to note that cADPR is generated by hydrolysis of NAD+, disruption in the metabolism of NAD+ has been associated with multiple pathological conditions [42]. Different types of NPPs have been implicated in a various of pathologic conditions such as tumor invasion and metastasis, inflammation, and angiogenesis (NPP2), tissue calcification and bone development (NPP1), and hemostasis and platelet aggregation (NPP4) [43]. However, NPP2 (ATX) is widely studied, ATX-LPA signaling axis induces inflammatory mediators such as IL-8, IL-6, TNF-α, and growth factors such as the vascular endothelial growth factor (VEGF) and the granulocyte colony-stimulating factor (G-CSF) thereby augmenting the cytokine production and lymphocyte infiltration that ultimately aggravates the inflammation in conditions such as asthma, pulmonary fibrosis, and rheumatoid arthritis [44, 45].

2.3 Purinergic receptors

Purinergic receptors are divided into two subtypes based on their binding tendency to different purine derivatives—P1 receptor (P1R) has affinity to bind adenosine only, whereas P2 can bind ATP, ADP, UDP-glucose, UDP and UTP [46]. Adenosine receptors (AR) belong to rhodopsin-like family of G protein receptors and consist of four subtypes such as A1, A2A, A2B, and A3. Adenosine generated by the hydrolysis of extracellular ATP, ADP, or AMP are either metabolized by adenosine deaminase (ADA) or shuttled back to the cells via two types of transporters, the equilibrate nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs) to stimulate various intracellular pathways like AMP-activated protein kinase, adenosine kinase and S-adenosyl homocysteine hydrolase [47]. Although it may depend on the concentration of adenosine and the given P1 receptor subtype engaged, but adenosine primarily, have anti-inflammatory and immune suppressive functions. The immunosuppressant activity of adenosine relies on the inhibition of virtually all immune cell populations such as T and B lymphocytes, NK cells, DCs, granulocytes, monocytes, and macrophages.

P2 receptors further categorized into two families based on molecular structure and second messenger systems, namely P2X ionotropic ligand-gated ion channel receptors that only binds to ATP and P2Y metabotropic G protein-coupled receptors (GPCR) can bind to ADP, UDP- glucose, UDP, and UTP [46]. The family of P2X receptors comprises seven members (P2X1–7), which perform tissue-specific functions by forming homo- or hetero-trimeric complexes. At least three P2X subunits assemble to form hetero- (e.g., P2X2/3 and P2X1/5) or homo-trimeric (P2X7) channels. This kind of assembly confers to P2X receptors a large repertoire of physiological functions in different tissues. Among P2XRs, the P2X7R has a special place in inflammation since its stimulation promotes NLRP3 inflammasome assembly and the associated IL-1β secretion. There are eight subtypes of P2Y receptors, which is further characterized into two subfamilies P2Y1 and P2Y12 based on their coupling to G_q and G_i, respectively. P2Y1 subfamily includes P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors. The second subfamily is P2Y12, which contains P2Y12, P2Y13, and P2Y14 receptors. Each P2Y receptors has different affinity towards different nucleotides and has a tissue-specific function. For instance, P2YR11 has affinity for ATP; P2YR1, P2YR12, and P2YR13 for ADP; P2YR2 and P2YR4 for UTP; P2YR6 for UDP; and P2YR14 for UDP-glucose and UDP-galactose [5].

The purinoceptors are expressed on almost all kinds of peripheral tissues and are involved in short-term as well as long-term regulation of variety of functions, ranging from neuromuscular and synaptic transmission to secretion in gut, kidney, liver, and reproductive systems. Their contribution in immune signaling is enormous, as these receptors are expressed on almost all types of immune cells. The purine nucleotides orchestrate the onset, magnitude duration, and resolution of the inflammatory response through the activation of purinergic receptors, which is also governed by the activity of ectonucleotidases (Figures 1 and 2). Any alterations in the purinergic machinery could contribute to the pathophysiological processes underlying the onset and development of immunological diseases, neurodegeneration, cancer, diabetes, and hypertension [7, 46, 48].

Figure 1.

Cartoon depicting the components of purinergic system and their functions. Mechanism of nucleotide release from the intact cells via exocytosis or transport channels as well as leakage of ATP from apoptotic and netosis (bottom of the image). The nucleotide triggers the activation of immune cells via specific purinergic receptor (top of the image). Activation of purinergic receptors ATP or their hydrolyzed metabolites ADP/AMP and adenosine by ectonucleotidases (middle of the image).

Figure 2.

Pictorial representation of cross talk of purinergic and immune signaling during normal physiological condition and inflammatory condition.

3. Interplay of purinergic signaling and immune signaling on inflammatory response

Beyond the physical and chemical barrier of skin and mucous lining, our body is guarded from the pathogens as well as self-attacking/cancerous cells by two different kinds of immune responses that acts in a coordinated manner. This includes (a) innate immune response comprising myeloid lineage derived cells (monocytes, macrophages, neutrophils, and DCs) and NK cells derived from lymphoid progenitors, and (b) adaptive immune response consists of B and T cells. Innate immune response provides the first line of defense against pathogens. It is an antigen-independent defense mechanism that is elicited when immune cells encounter pathogens. This response has no memory and remains similar during the lifetime. On the other hand, adaptive immune response is an antigen dependent, antigen specific, and has the tendency to form memory cells to elicit rapid response based on the previous encounter with the similar kind of antigen or pathogenic exposure. Innate and adaptive immune responses are not mutually exclusive defense mechanisms. They work in a very organized fashion and complement the functions, as activation of T cells requires antigen presentation by professional antigen presenting cells (dendritic cells, B-cells, or macrophages), together with the major histocompatibility complex (MHC) type I or II [49]. Defects in any of the component increases the vulnerability towards infection and disease.

ATP and adenosine are the key modulators of the immune response, ATP being an immunostimulant, whereas adenosine has an immunosuppressive effect thus balance between the two is crucial for the proper functioning of immune system. Extracellular signals by ATP and adenosine are detected and transduced by P2 and P1 receptors (Figure 2), respectively which is present on all kinds of immune cells, thus purinergic signaling affects all aspects of immunity and inflammation [50], which is described in detail in further section.

3.1 Effect on innate immune signaling

3.1.1 Monocytes/macrophages

Macrophages are the subset of myeloid cells that have immune surveillance function and sense even a minute changes in the tissue microenvironment. They express a variety of pattern recognition receptors (PRRs) that are present either on the surface, cytosol or in the endosome such as toll like receptors (TLRs), NOD like receptor (NLRs), retinoic acid inducible gene I like receptors (RLR), transmembrane C-type lectin receptors, and absent in melanoma (AIM)2-like receptors (ALRs) that recognize either pathogen associated or damage associated molecular patterns (PAMP and DAMP, respectively). These cells are highly plastic that could undergo profound metabolic modifications after sensing the pathogens or damage signal via PRRs to elicit the immune response. In addition, macrophages are endowed with purinergic P1, P2X, and P2Y receptors that also respond to damage associated molecules, extracellular nucleotides, and their derivatives, and undergo reprogramming from pro-inflammatory profile M1-like phenotype to an anti-inflammatory M2-like phenotype. As indicated by Elliott et al., that monocytes or macrophages sense extracellular nucleotide as a danger signal for “find me” or “eat me” to engulf and phagocytose the dying cells [24]. These cells not only sense the distant signal but also amplify the signaling for chemotaxis by releasing ATP by “autocrine purinergic loop” via P2Y2 and A3 receptors [51, 52]. Macrophages control their activation state in an autoregulatory mechanism by inducing the production of ATP and extracellular degradation to adenosine. Deficiency of CD39 promotes a sustained inflammatory activation state and inhibits the switch to an immunosuppressive phenotype [53]. Presence of extracellular adenosine stimuli in macrophages drives the polarization towards M2 phenotype with diminished expression of inflammatory genes TNF-α and IL-6 and increased expression of anti-inflammatory cytokines such as IL-10 and VEGF via A2A and A2B receptors [54]. Furthermore, macrophages exhibit a unique repertoire of P2X receptors such as expression of P2X1, P2X4, as well as P2X7 [55]. Among P2Y receptors, P2Y1 and P2Y4 receptors play minor roles, whereas the functions of P2Y2, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 are more established in the macrophage biology as described elsewhere [56]. A recent study demonstrated that bone marrow derived macrophages display unique expression pattern of purinergic receptors that correlates with a M1or M2 inflammatory phenotype. M1 phenotype exhibit a unique and more pronounced P2X7 negative macrophage population, which associates with decreased inflammasome formation. P1 receptors A2A and A2B are upregulated in M1 and M2. P2Y1 and P2Y6 exclusively upregulated in M2, whereas P2Y13 and P2Y14 are overexpressed in M1 [57]. This unique feature demonstrates capability of purinergic receptors on macrophages to adapt to pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.

3.1.2 Dendritic cells

DCs are professional antigen-presenting cells (APCs), which has a crucial role in initiating and regulating the adaptive immune response by directing the activation and differentiation of naive T cells. Immature DCs (iDCs) sense the danger signals in the similar fashion as monocytes and macrophages do, however upon exposure, DCs lose their phagocytotic capacity, migrate to secondary lymphoid organs and transition to a mature DC (mDC) by acquiring MHC and costimulatory molecules, such as CD54, CD80, CD83, and CD86. Migration of DCs to the inflamed tissue is mediated by A1 and A3 ARs [58]. Adenosine upregulates the expression of co-stimulatory molecules on mDCs [59]. Both, A2A and A2B ARs suppress maturation of DCs as well as their capacity to initiate Th1 response, however, it increases pro-angiogenic VEGF, IL-10 and cytokines that contribute to Th17 cell polarization [59, 60]. Adenosine also mediates the attraction of DC and Treg cells, which is crucial for the immunosuppressive activity of Treg cells [61]. Similarly, ATP also acts as a chemoattractant for iDCs, and enhance the migration by autocrine signaling loop mechanism via P2X7. This signaling is further amplified by the release of ATP by PANX-1 channels [62]. Furthermore, eATP had been shown to activate P2X7R to promote the maturation of dendritic cells via NF-κB (p65) pathway [63]. On the other hand, P2Y6 has inhibitory role in the maturation and activation of DCs via NF-κB by inhibiting the production of IL-12 and IL-23 and the polarization of Th1 and Th17. Loss of P2Y6 enhances the DC mediates differentiation of Th1 and Th17 subsets [64]. The ATP-P2X7 signaling axis of DCs also promotes interleukin (IL)-1β and IL-18 secretion by activating NLRP3 inflammasome and induces Th2/Th17 differentiation [65]. P2X4 acts in conjunction with P2X7 to regulate IL-1β production by DCs [66]. As described previously, the balance of proinflammatory-ATP and anti-inflammatory adenosine is regulated by CD39 and CD73 present on the immune cells. In context of DCs, their expression fine tunes the DCs function either as tolerance (higher expression) or as immunity (lower expression) ensues [67, 68].

3.1.3 Neutrophils

Neutrophils belongs to the granulocyte family, which has a major role during the early stages of the inflammatory response. They are the first cell to arrive at the inflammation site, which employ an extracellular ATP-dependent mechanism to generate a chemotactic gradient and orientate its migration. Remarkably, the purinergic system regulates many effector functions of neutrophils such as phagocytosis, oxidative burst, degranulation, and neutrophil extracellular traps (NETs) formation via Netosis [69, 70]. Apoptotic neutrophils release ATP to stimulate mononuclear phagocytic cell influx and promote engulfment and clearance functions. Nucleotides released as a result of the apoptosis and netosis serve as danger or find me signal to initiate immune cell chemotaxis via P2Y2 receptor towards inflamed tissue and fine-tuned control local inflammation and promote phagocytosis and clearance [24, 71]. Similar to other phagocytic cells such as monocytic and dendritic cells, neutrophils in the immune microenvironment also release ATP via PANX-1 to induce chemotaxis by autocrine stimulation of P2Y2 [51, 52, 62, 72]. On the other hand, P1 receptor, A2A (activated by adenosine) blocks the chemoattractant signaling, whereas alternative binding of adenosine to A3 receptors, stimulate immune migration. Thus, P2Y2 and A3 receptors are responsible for the amplification of the chemotaxis signal via feedback loop mechanism [52]. P2Y2 receptors play crucial role in neutrophil activation by regulating the release of IL-8, a major chemokine for neutrophils chemotaxis [73]. IL-8 secretion is in turn controlled by CD39 [74]. Thus, the local microenvironment composed by ATP and the consequent degradation to adenosine by CD39 and CD73 ectoenzymes influence reprogramming of the innate immune cells and their response towards pathogens and other diseased condition.

3.1.4 Natural killer cells

NK cells are considered as a component of innate immune system due to the lack antigen-specific cell surface receptors but morphologically they resemble lymphocytes as they originate from the common lymphoid progenitor cell in the bone marrow. NK cells exert sophisticated biological functions that attribute to both innate and adaptive immunity, thus the functional boundary between these two arms of the immune response is obscure [75]. These cells express a repertoire of activating (NKG2 C-H) and inhibitory receptors (NKG2 A and B) through which it interacts with pathogens by recognizing MHC-I molecule [76]. Activation of the NK cell leads to cytolytic killing of infected cells. Adenosine receptors A1, A3 and A2A, A2B have an antagonistic effect in controlling the intracellular cAMP levels. A1, A3 inhibits the adenylyl cyclase and decreases the intracellular cAMP level, which has a stimulatory effect on NK cell and promote the cytotoxic activity whereas, A2A and A2B has the immunosuppressive effects on NK cells [76, 77]. NAD+ and ADP-ribose inhibited human NK proliferation [78]. Nucleotide triphosphates (ATP, GTP) have high potency in inhibiting NK cell-mediated cytotoxicity, this tendency however decreases with reduced negative charge due to less phosphate group [(ADP, GDP) > (AMP, GMP)]. Ectonucleotidases do not have any significant role in modulating the cytolytic effect of NK cells by extracellular ATP/ADP/AMP [79]. NK cells express lower level of CD73 even with IL-15 and IL-12 priming [74]. Decreased expression of P2Y6 promotes the development of the NK precursor cells into immature NK and mature NK cells suggesting P2Y6 as a negative regulator of NK cell maturation and function [80]. Among other extracellular purine derivatives, NKT cells display higher sensitivity to NAD+ and induce cell death via P2X7 pathway [81, 82]. Furthermore, another phenotypically heterogeneous NKT cells subset includes invariant natural killer T (iNKT), which are CD4 and CD8 negative but express NK cell marker and produce IL-4 and IFNγ. iNKT recognizes lipid antigens combined with CD1d on the surface [83]. Activation of iNKTs in vitro induces the expression of purinergic signaling genes A2A, P2X7R, CD38, CD39, NPP1, CD73, PANX-1, and ENT1, which has an anti-inflammatory role [84]. iNKT cells interact with DCs and monocytes via P2X7 dependent and an independent manner, respectively [85, 86]. Overall, NK cells alter their functional responses to adenosine signaling via mechanisms that are sensitive to specific cytokine activation programs.

3.2 Effect on adaptive immune signaling

3.2.1 T cells

Activation of T cell immune response is the key in adaptive immune system functions, which elicits both cellular and humoral immunity. Naïve T cells are activated by APCs, but they require two subsequent signals, first one is the binding of TCR to peptide–MHC complex and the second one is the co-stimulatory interaction at the interface between APCs and T cells via B7/CD28, LFA-1/ICAM-1 and ICAM2, and CD2/LFA-3 ligand and receptor complex [87]. Di Virgilio et al. was the first to show T cell responsiveness to extracellular ATP (eATP), back to 1989 [88]. Once T cells are activated, they release ATP via PANX-1 channels, resulting in the activation of P2X1, P2X4, and P2X7 receptors that promotes downstream signal transduction pathways leading to IL-2 expression and T cell proliferation via Ca⁺² influx [89]. P2X7 receptor stands out among P2X family members as the most important regulator of T cell function [90]. The released ATP stimulates purinergic receptors that also contributes to the amplification of co-stimulatory TCR/CD28 signal at the immune synapse by autocrine stimulation of P2X7 [91] and P2Y1 receptors [92]. In addition, the T cell activation via P2X7R inhibits the immunosuppressive Tregs cells [93]. P2X7R is also crucial for the activation of CD8 T cells, and its expression increases as they differentiate to T_CM (central memory) and T_RM (tissue-resident memory) suggesting its key role in generating long-lived memory CD8 T cells [94]. However, another study demonstrated that eATP treatment can trigger cell death in the naive CD8 (CD44^loCD45RB^hi) subset, but it is unable to induce these cellular activities in the effector/memory CD8 (CD44^hiCD45RB^hi) subset. Even though both subsets express similarly low levels of P2X7R, but they demonstrate different sensitivity to ATP depending on the stage of differentiation instead of P2X7R expression levels [95]. Importantly, expression of CD39 and CD73, the ecto-5′-nucleotidase that degrades extracellular AMP into adenosine, by other immune and tissue-resident cells can dramatically condition the outcome of T cell responses [96]. On the other hand, A2A receptor signal inhibits Th1 cell generation and IFN-γ production, triggering the induction of FoxP3 + Treg cell subset and the production of TGF-β. ATP catabolism and generation of retaliatory metabolite adenosine is a typical suppression mechanism of regulatory cells involving Treg, type-1 regulatory (Tr1) T cells, and myeloid-derived suppressor cells (MDSCs) [97]. These regulatory cells express CD39 and CD73 to abrogate ATP-related effects and enable the inhibitory properties. P2X7R can imprint distinct outcomes to the T cell depending on the metabolic fitness and/or developmental stage via autocrine signaling or microenvironment’s clues. The peculiarity of P2X7R function as cationic channel and cytolytic pore could be responsible for some apparently contradictory findings on P2X7R dependent responses in particular T cell subsets in different experimental settings [94, 95, 96].

3.2.2 B cells

Another important arm of adaptive immune response is the humoral immunity, which is mediated by B cells. These cells are also necessary for the development of T-cell immunity because they serve as an APC, providing costimulatory signals and producing cytokines necessary for effector functions of T cells. B cells exhibit expression of the membrane B cell receptor (BCR), which can recognize antigens in their native forms, thus B cells do not need antigen presentation for activation. Antigen recognition, together with signals from activated Th2 cells, induces B cells to proliferate and generate effector plasma cells and memory B cells. B cells expresses ectonucleotidases—CD39 and CD73, P1 receptors—A1, A2A, and A3, and P2 receptors—P2X1, P2X2, P2X4, and P2X7 [33, 98, 99]. The function and activity of B cells are largely governed by the concentration of adenosine and ATP in the microenvironment. Adenosine imposes suppressive effect on B cells, whereas increased ATP release and production are associated with activated B cells thereby exerting pro-inflammatory effect on the target tissue and IgM release [100]. In vitro activated B cells exhibit downregulation of CD73, which mainly produces AMP, and inhibits T-cell proliferation and cytokine production, whereas overexpress A3 receptor in activated state [98]. Accumulation of pericellular ATP occurring in B cells activates the P2X7 receptor, which results in shedding of CD21, CD23, and CD62L from the cell surface [101, 102]. This process is involved in transendothelial migration of B cells. There is also evidence showing that P2X7 is directly involved in the release of IgM from B cells after T cell independent activation [103]. Moreover, CD73 is progressively upregulated on germinal center (GC) B cells following immunization, and is expressed at even higher levels among T follicular helper cells but is absent among plasma cells and plasmablasts. CD73-dependent adenosine signaling is prominent in the mature GC, maintenance of plasma cell compartment and necessary for immunoglobulin class switching [100, 104]. Thus, any disruption in the balance of ATP signaling that is dominant in activated B cells and adenosine signaling, which seems crucial in achieving immunocompetence by activated cells [100] could lead to severe immunological disorder.

4. Implication of purinergic signaling in various pathological conditions

A healthy individual has a practically insignificant amount ATP in the extracellular microenvironment (at the nanomolar range), whereas, they have significantly higher concentration of ATP in the intracellular environment (reaching several millimolar), as ATPs are the powerhouse of the cell. Inflammatory stress due to the increased production of proinflammatory mediators associates with release of ATP and other nucleotides into the extracellular space (Figure 1). These extracellular nucleotides trigger a stimulation of purinergic receptors, which is a normal physiological phenomenon and beneficial for preventing tissue damage ensuring host survival, it may also be detrimental for clearance of pathogens or dying cells. However, failure in the fine tuning of the immune response alters inflammatory and regulatory microenvironments, leading to unbalanced stimulation and culminates a hyperinflammatory condition generating numerous pathologies such as autoimmunity, chronic infectious diseases, and cancer (Figure 2).

4.1 Purinergic signaling in autoimmune disease

Autoimmune diseases are characterized by diverse clinical manifestations including dysregulated innate and adaptive immune signaling, chronic inflammation, autoreactive immune cells, generation of autoantibodies to self-nuclear and cytoplasmic component. Based on the target organ and tissues, they are represented as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren’s syndrome (SS), systemic sclerosis (SSc), etc. As described previously that some of the purinergic receptors are coupled with inflammasome assembly, pro-inflammatory cascades, secretion of IL-1β, IL-18, and T and B cell activation, and maturation, all these events play a pivotal role in autoimmunity [105].

4.1.1 Systemic lupus erythematosus

SLE is an inflammatory autoimmune disease that affects many organs, including the skin, joints, the central nervous system, and the kidneys. A frequent and serious manifestation of SLE includes glomerulonephritis (GN), a condition that can cause proteinuria and progresses to kidney failure. These diverse clinical features include hematological and serological abnormalities, such as decreased levels of complement and increased levels of autoantibodies [106, 107]. SLE has multiple etiology like genetic, environmental, and hormonal factor but involvement of dysfunctional innate and the adaptive system is prominent [108]. Purinergic signaling is another key pathway that connects with the inflammatory signaling cascade and contributes to the immunopathogenesis of SLE.

Till date, more than 180 autoantibodies have been documented in SLE patients [107]. Source of the diverse pool of autoantigens are apoptosis [109], netosis [110], and pyroptosis [111]. Simultaneously, SLE patient also exhibit impairment of phagocytotic clearance and NET degradation [112, 113], which together represent a mechanism that trigger to breakdown of the self-tolerance against autoantigens and leading to initiation of SLE. Defects in the purinergic signaling and its role in SLE pathogenesis and disease severity had been described at several instances. Therefore, P2X7R activation by ATP or by extracellular complexes, such as NETs, might have a dual pathogenetic role in promoting inflammation in lupus: on one hand, it directly triggers inflammation by stimulating the NLRP3 inflammasome, and on the other it has an indirect pro-inflammatory effect by inducing pyroptotic cell death [114]. Presence of the NETs in the microenvironment induce NLRP3 inflammasome, in macrophages and results in the amplification of inflammation by releasing of IL-1β and IL-18, which is mediated via P2X7R [115]. Induction of inflammasome and IL-1β and IL-18 release have been shown to contribute to the cardiovascular, skin, and nephritis manifestations [116, 117, 118]. Evidence suggests the higher P2X7R in renal tissue of lupus nephritis patients [119]. In that context, a study demonstrated substantial up-regulation of P2X7R, NLRP3, and ASC, in the kidneys of MLR/lpr mice compared to control mice and inhibition of P2X7R ameliorates the disease phenotype mainly diminished both the severity of nephritis and levels of circulating anti-dsDNA antibodies [120, 121]. The presence of single nuclear polymorphism (SNP) 489C>T in P2X7 receptor had been associated with increased inflammasome activation in SLE patients and shows involvement in pericarditis [122, 123]. Th1, Th17, and Regulatory T (Treg) cells in SLE patients display higher expression of P2X7 receptor, which correlates with active SLE disease and increased levels of IFN-$\gamma$, IL-1$\beta$, IL-6, IL-17A, and IL-23 cytokines [124]. Monocytes and lymphocytes from SLE patients and RA patients show reduced expression of P2X7R gene. They show reduced tendency to induce apoptosis and cytokine release in vitro compared to cells from healthy individual [125].

Furthermore, P2X7R has an important function of restricting the expansion of T follicular helper (Tfh) cells by pyroptosis and controls the development of pathogenic ICOS+ IFN-γ–secreting cells and in turn prevents the overproduction of autoantibodies and activation of T cells that ultimately controls the production of autoantibodies conditions [126]. SLE patients exhibit deletion of P2X7R genes that have deleterious effect of autoantibody generation [126]. Another study had reported that deletion of P2X7R could amplify the defect in peripheral T cell homeostasis due to the FAS mutation and thus contribute to the autoimmune pathology [127]. In similar way, another purinergic receptor P2Y8R restricts the proliferation of self-tolerant B cells. Distinct variant of P2Y8R had been shown to be downregulated in SLE patients and these are associated with the loss of function, which leads to increased expansion of self-reactive B cells, resulting in the increased autoantibody production. P2Y8R correlated with lupus nephritis and increased age-associated B cells and plasma cells indicating a role of P2Y8R in immunological tolerance and lupus pathogenesis [128].

The role of CD39 in the maintenance of immune tolerance is associated with its capacity of degrading ATP and consequently inhibiting the production of IL-17, which stimulates B cells to produce autoantibodies. Ectonucleotide provides protection in by converting eATP to adenosine. Deletion of ectonucleotides mainly, CD39 and CD73 lead to higher levels of anti-RNP antibodies in response to pristane, with CD73 deletion in particular promoting expansion of splenic B cell and T cell populations that likely contribute to autoantibody production [129]. B cells show the highest CD73 surface expression among human circulating immune cells. In SLE patients, the activity of CD73 and CD38 was found to be selectively silenced in B cells. Since CD73 is the bottleneck of extracellular nucleotide degradation to anti-inflammatory adenosine, this pathway is likely to be a crucial step in the pathophysiology of SLE involving B cell immune cell interactions [130].

4.1.2 Rheumatoid arthritis

RA is a chronic inflammatory disease of joints characterized by damage of bone and cartilage, which leads to joint destruction and disability. Primarily, it is driven by proliferation of synovial fibroblasts, inflammatory response of innate and adaptive immune response, differentiation of macrophage into osteoclasts, and impaired differentiation of mesenchymal stem cells into osteoblasts. The incidence is about 5 per 1000 people and can lead to severe joint damage and disability [131]. Studies have shown a critical role for P2 receptors in osteoblastogenesis and mineralization, synoviocytes proliferation, inflammation of immune cells, and differentiation of macrophages into osteoclasts [132]. Specifically, P2X7, P2Y14, P2Y12, P2Y6, P2Y1, P2Y2, and P2X4 receptors are involved in modulating bone and joint biology [133]. Pain is the major symptom of RA, which associates with the involvement of P2X4R had been reported in chronic arthritis [134]. Knockout of this gene in mice model alleviates the pain [135]. P2X4R control the production of Th17 cells, as shown by the inhibition of P2X4 receptor which reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis [136]. Inhibition of P2X4R associated with the attenuation of synovial inflammation and joint destruction as well as decreased the levels of serum IL-1β, TNF-α, IL-6, and IL-17 via NLRP1 [137]. Similar to SLE, SNP in P2X7 is associated with increased inflammatory response and susceptibility to RA [123, 125]. P2X7 receptor-mediates the release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases and is important for osteoclastogenesis [138]. It also regulates the differentiation of Th17 cells and type II collagen-induced arthritis in mice [139]. P2Y receptor also contribute to the development of RA such as P2Y11 receptor induce inflammation in primary fibroblast-like synoviocytes [140], P2Y12 and P2Y14 receptors induce bone lysis by activating osteoclasts [141, 142, 143]. RA patients demonstrate differential expression of adenosine receptors on synovium with preferential expression of A3 and its variant. However, in a separate RA cohort treated with methotrexate shows overexpression A2A and A2B indicating the anti-inflammatory property via these adenosine receptors [144]. Under hypoxia condition, bone resorption is increased in RA patients via A2B receptors. Inhibition of A2B receptors potentially prevent the hypoxia-mediated pathological osteolysis in RA [145].

The expression of CD39 in Tregs is limited by single nucleotide polymorphisms (SNP). It has been shown that AA genotype of the rs10748643 SNP, a low-expressing CD39 variant, is involved in the regulation of the immune system in autoimmunity [146]. A reduced response to methotrexate (MTX) in patients with rheumatoid arthritis was also shown to be related to an SNP that decreases the frequencies of CD39-expressing Tregs, the rs7071836 SNP [147]. Lower expression of CD73 in lymphocytes at the sites of inflammation has been associated with disease severity in juvenile idiopathic arthritis [148].

4.1.3 Multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, characterized by the presence of focal lesions in white and gray matter, which is associated with pathological and progression neurological dysfunction. Presence of peripheral immune cells infiltration is a main diagnostic hallmark of the disease. Purinergic receptors control immune cell function as well as neuronal and oligodendroglia survival, and the activation of astrocytes and microglia, the endogenous brain immune cells. Genetic variation in P2X4 and P2X7 receptors show susceptibility to MS. Functionally, the variants impair the expression of P2X7 on the surface resulting in the inhibition of ATP-induced pore function and phagocytic activity [149]. Cortical microglia from MS patient exhibit loss of P2Y12 receptor, which associates with the pro-inflammatory and neuronal damaging profile in MS [150]. On the other hand, P2Y12 is the markers of platelet and megakaryocyte activation. Its increased expression in MS patients associates with cardiovascular disease [151]. A study in mice model show that the loss of P2Y6 develop more severe experimental autoimmune encephalomyelitis compared with wild-type mice as it has pivotal role in DCs regulation [64]. Lymphocytes from MS patients also exhibit upregulation of A2A receptor, which modulates the release of proinflammatory cytokine TNF-α, IFN-γ, IL-6, IL-1β, IL-17 via NF-κB. A2A receptor upregulation was observed in lymphocytes from MS patients in comparison with healthy subjects. The stimulation of these receptors mediated a significant inhibition of TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and cell proliferation as well as very late antigen (VLA)-4 expression and NF-κB activation [152].

CD39 expressing Treg cells controls the neuroinflammation in MS by suppressing the pathogenic Th17 cells and IL-17 production [31]. Its activity and the frequency were elevated in relapsing MS patients [153]. Furthermore, a study on animal model demonstrated that overexpression of CD39 on reactive microglia/macrophages that associates with either pro-inflammatory (M1-subtype) or neuroprotective (M2-subtype) at different stages of the disease. At the peak of EAE, CD39 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype [154]. Thus, modulation of purinergic signaling using an agonist or antagonist provides a new avenue for treatment of disease [155, 156].

4.2 Purinergic signaling during bacterial and viral infection

Infectious diseases are caused by the invasion of pathogenic microorganisms. After infection, host immune system elicits the anti-microbial immune response and at the same time microorganisms develop strategies to evade host defense mechanism. This involves generation of a variety of inflammatory and suppressive responses along with regulatory feedback systems to eliminate the pathogens but also to restore the homeostatic condition following infection or injury [157]. The purinergic system has the dual function of regulating the immune response and triggering effector antimicrobial response against bacterial and viral infections. During the infections, the ATP release initiates a cascade that activates purinergic receptors. This receptor activation enhances the secretion of pro-inflammatory cytokines and performs the chemotaxis of macrophages and neutrophils, generating an association between the immune and the purinergic systems. Immunomodulation by purinergic signaling has been widely discussed elsewhere [26, 158]. Some instances of involvement of purinergic signaling in bacterial infection include, reduced CD73 expression was associated with macrophage phagocytosis and an efficient clearance of Salmonella infection [159]. Likewise, depletion of CD39 on CD4, CD8, and Treg cells augments the T cells response to Listeria and Mycobacterium infections [160, 161]. On the other hand, transgenic mice with overexpression of CD39 in lung epithelia shows increased recruitment of neutrophils and macrophages in lungs upon Pseudomonas aeruginosa infection. The CD39 activity associates with efficient clearance of infection [162]. CD39, due to ATP-scavenging property it limits P2X7 receptor mediated pro-inflammatory responses. Thus, deletion of CD39 exacerbates sepsis-induced liver injury [163]. P2X7R signaling has a detrimental role in severe tuberculosis infection. ATP release and activation of P2X7R cause macrophage necrosis resulting in the spread of bacterial particles, leukocyte infiltration, and tissue damage [164]. Deletion of P2X7 receptor or blockage of P2X7R, or scavenging of eATP may attenuated inflammation, largely preventing increased cytokine secretion and tissue damage [163, 165].

Immunomodulation of purinergic signaling had been implicated in wide variety of viral infections such as human immunodeficiency virus (HIV)-1, hepatitis virus, dengue virus, and SARS-CoV2 [166, 167, 168, 169]. HIV-1 primarily infects CD4 T cells, but also affects myeloid dendritic cells and monocyte, macrophages populations that express CD4 receptor. Infected patients exhibit decreased CD4 T cell counts and a reversed CD4/CD8 T cells ratio. Adenosine has an immunosuppressive effect, patients with HIV infection show upregulated CD39 on Treg cells which is inversely related with the CD4 T cell count [167, 170]. In contrast to CD39, CD73 expression was diminished on CD4 T cells, which represent a phenotypically and functionally different subpopulation of CD73+ CD4 T cells. This T cell subsets are preferentially reduced in HIV patients, which suggests the effect of an adenosine diminished microenvironment that cannot prevent persistent immune activation. CD73+ CD4+ T cell counts were inversely associated with T cell activation, as well as plasma C reactive protein levels [171]. Besides, CD73 is involved in the expansion of HIV-specific CD8 T cells, whereas CD73 expression is higher in memory CD8+ T cell subset. The frequency of CD73+ CD8+ T cells is inversely associated with cell activation and plasma viral load [172]. PANX-1 hemichannel opening, activation of P2Y2R, P2X1R are involved in the mediating the effective viral entry and replication in CD4 or target cells [173, 174, 175]. Blocking the P2X1 and P2X7 receptors inhibits the viral entry and fusion [176]. Similarly, the P2X1R, P2X4R and P2X7R expression increased in during hepatitis C virus infection and Dengue virus infection [168, 177]. Blocking P2X receptor with antagonist improves the anti-viral response and T cell function [168, 178].

Given the pathophysiological role of purinergic signaling in highly prevalent viral infections has developed a potential interest in investigating the effects of purinergic system in severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2). SARS-CoV-2 infection had impacted more than millions of people worldwide since its emergence in December 2019, in Wuhan, China. The clinical manifestations of SARS-CoV-2 include pneumonia, acute respiratory distress syndrome (ARDS), and hyperinflammation. SARS-CoV-2 primarily invade the alveolar epithelia of respiratory tract and lungs where they replicate, triggers the activation of the immune system resulting in the release of cytokines as a defense mechanism, but the response become exaggerated and prompt the so-called “cytokine storm.” This is a state of hyperinflammatory response, which develops acute respiratory syndrome (SARS). This is characterized by fever, cough, and difficulty breathing, which can progress to pneumonia, failure of different organs, and death. Patients with SARS-CoV-2 infection exhibit increased purinergic signaling, which has been suggested to have a role in hyperinflammatory state [179]. The mechanisms have been described in very detail in review articles [169, 180]. The increased inflammations resulting from activated purinergic signaling in SARS-Cov-2 infections are also associated with different pathological conditions such as neuropathy [181], thrombopathy [182, 183]. As observed in other viral infections patients with SARS-CoV-2 shows reduced expression of CD73 on circulating CD8, NK, and NKT cells. However, cells lacking CD73 exhibit increased cytotoxic effector capacity compared to their counterpart CD73+ [184]. P2X7R-NLRP3 signaling axis are the key driver of inflammation in SARS-CoV-2 [185]. Therefore, P2X7R could serve as a potential therapeutic target to control the inflammation [186]. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function [169]. Understanding this biology is very crucial as anti-inflammatory drugs are not effective and sometimes accompanied by serious adverse effects.

5. Conclusion

This chapter has highlighted the importance of purinergic signaling in modulating the immune system in various therapeutic areas. Purinergic system is capable of fine tuning the levels of nucleotides and their derivative in the extracellular space thereby controlling the chemotaxis, proliferation, differentiation of various immune cell presents locally or far from the infectious site. Dysregulation of purinergic signaling because of genetic factor or escape mechanism employed by the microbes or regulatory cell leads to overt inflammation that contributes to the disease. Special attention has been paid to the mechanisms through which alterations in the various compartments of the purinergic system could contribute to the patho-pathophysiology of autoimmune disease and microbial infection. This chapter could help in gaining insight on the possibility of counteracting such dysfunctions by means of pharmacological interventions on purinergic molecular targets.