The Purinergic System as a Pharmacological Target for the Treatment of Immune-Mediated Inflammatory Diseases

A. Multiple Sclerosis

MS is a complex, chronic, progressive immune-mediated demyelinating disease causing focal damage to the white matter attacking different regions of the central nervous system. The disease has a relapsing-remitting course and a range of clinical symptoms (e.g., autonomic, visual, motor, and sensory problems), depending on where the demyelination and axonal loss have occurred (Trapp and Nave, 2008). The inflammatory process is characterized by marked infiltration of monocytes, DCs, T and B cells, as well as by activation of resident microglia and macrophages, induction of oxidative stress pathways, and alterations of the blood-brain barrier permeability (Dargahi et al., 2017). Of note, experimental autoimmune encephalomyelitis (EAE) in rodents is the most commonly used model for MS, mimicking several of the key pathophysiological features of the human disease, such as demyelination, axonal loss, inflammation, and gliosis (Constantinescu et al., 2011).

Brain sections from MS patients were immunopositive for P2X₁, P2X₂, P2X₃, P2X₄, and P2X₇ receptors (Amadio et al., 2010). By contrast, the P2X₆ receptor was undetectable (Amadio et al., 2010). P2X₇ receptor expression is increased on astrocytes in active brain lesions (Narcisse et al., 2005; Amadio et al., 2017) and on microglia of the optic nerve (Matute et al., 2007) of MS patients. P2X₇ receptor expression is increased on oligodendrocytes also in normal-appearing axon tracts in patients with MS; this may indicate an early role for P2X₇ receptors in disease progression (Matute et al., 2007). In addition to the brain, increased P2X₇ receptor immunoreactivity was also observed in microglia/macrophages in the spinal cord of MS patients (Yiangou et al., 2006).

The analysis of blood monocytes obtained from MS patients did not reveal any differences in P2X₇ receptor expression in comparison with healthy controls (Caragnano et al., 2012). However, a reduction of P2X₇ receptor expression was observed in monocytes from patients undergoing treatment with glatiramer acetate (Copolymer 1, Cop-1, or Copaxone), an immunomodulatory drug used to reduce the frequency of relapses in MS (Caragnano et al., 2012).

In the perfused rat optic nerve, oligodendrocytes are vulnerable to sustained activation of P2X₇ receptors, and this P2X7 receptor-mediated oligodendrocyte death is associated with microgliosis, demyelination, and axonal damage (Matute et al., 2007). Pharmacological blockade of P2X₇ receptors of mice with EAE attenuated tissue damage and neurologic symptoms (Matute et al., 2007). Of interest, the incidence of myelin oligodendrocyte glycoprotein (MOG)-induced EAE in mice deficient in P2X₇ receptors was decreased compared with wild-type mice (Sharp et al., 2008). However, once EAE was established in P2X₇ receptor-deficient mice, its severity and course were not different from that of wild-type mice. This indicates that P2X₇ receptors are necessary for the efficient initiation of EAE, but that EAE can occur, albeit at a decreased level, in the absence of P2X₇. In another MOG-induced EAE study, P2X₇ receptor-deficient mice developed more severe clinical and pathologic signs of EAE than wild-type mice and antigen-induced proliferation of spleen and lymph node cells from P2X₇ receptor-deficient mice was increased compared with cells from wild-type mice (Chen and Brosnan, 2006). Further studies will be necessary to explain the conflicting results between these two studies.

An early study showed that P2X₄ receptors were expressed on macrophages infiltrating the brain and spinal cord of rats with EAE (Guo and Schluesener, 2005). P2X₄ receptors are also upregulated on microglia during EAE (Vazquez-Villoldo et al., 2014). Both pharmacological blockade and genetic deficiency exacerbated EAE severity, whereas the P2X₄ allosteric activator Ivermectin (Stromectol), originally an antiparasitic medication, was beneficial, indicating that P2X₄ receptors are protective (Zabala et al., 2018). Mechanistically, P2X₄ receptors favored a switch in microglia to an anti-inflammatory phenotype and promoted remyelination.

It is not surprising that of the P2X receptors, the role of P2X₇ and P2X₄ have been studied in detail, as they are expressed at high levels on immune cells (Suurvali et al., 2017; Csóka et al., 2018). The role of other P2X receptors has not been addressed and should be the subject of future studies.

Histologic analysis revealed an increase in the expression of P2Y₁₁, P2Y₁₂, and P2Y₁₄ receptors in the frontal cortex of MS patients (Amadio et al., 2010). The cellular localization of P2Y₁₂ receptors was studied in detail, and they were found mostly on myelin and interlaminar astrocytes (Amadio et al., 2010). Although the significance of the increase in P2Y₁₁ and P2Y₁₄ receptors is unclear, P2Y₁₂ receptor expression was inversely correlated with myelin lesion formation in patients with MS (Amadio et al., 2010).

In one study, P2Y₁₂ receptor-deficient mice displayed an exacerbated EAE phenotype (Zhang et al., 2017b). In this model, bone marrow-derived DCs from P2Y₁₂ knockout mice undergoing EAE released increased amounts of IL-23, an essential factor for the differentiation of CD4⁺ T cells toward pathogenetic Th17 cells (Zhang et al., 2017b). In another study, EAE was ameliorated in P2Y₁₂ receptor-deficient mice with decreased brain leukocyte infiltration, less extensive demyelination, and decreased IL-17 expression (Qin et al., 2017). In addition, the anticoagulant drugs clopidogrel (P2Y₁₂ inverse agonist, Plavix) and ticagrelor (P2Y₁₂ neutral antagonist, Brilinta) alleviated the severity of EAE and inhibited Th₁₇ differentiation (Qin et al., 2017). It is unclear why the two studies had opposing results, as they appeared to use the same mice and model. Therefore, this discrepancy as well as the role of other P2Y receptors awaits further clarification.

Early studies reported that A₁ receptor mRNA and protein levels were reduced in blood and brain monocytes, macrophages, and microglia from patients with MS (Johnston et al., 2001) and microglia in mice during EAE (Tsutsui et al., 2004). This deficit in A₁ receptors appears to contribute to the course of EAE, as A₁ receptor-deficient mice showed exacerbated disease and had increased myelin and axonal loss (Tsutsui et al., 2004). Macrophages from A₁ receptor-deficient mice had increased expression of proinflammatory cytokines and metalloproteinase-12.

Vincenzi et al. (2013) demonstrated increased expression A_2A receptors on lymphocytes from MS patients compared with healthy individuals (Vincenzi et al., 2013). The authors speculated that this A_2A receptor overexpression is a compensatory mechanism aimed at curbing the inflammatory process, as in vitro stimulation of A_2A receptors of lymphocytes from multiple sclerosis patients suppressed the release of proinflammatory cytokines (TNF, IL-1β, IL-6, IL-17, and IFN-γ) and decreased cell proliferation, NF-κB activation, and the expression of the adhesion molecule VLA-4 (Vincenzi et al., 2013). Positron emission tomography demonstrated increased A_2A receptor expression in the brains of MS patients (Rissanen et al., 2013). At this point it is unclear which cell types were responsible for this increase.

Increased A_2A receptor expression was also observed on lymphocytes from EAE mice. Pharmacological stimulation of A_2A receptors with the selective agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 starting at the time of immunization with myelin oligodendrocyte glycoprotein (MOG) caused a significant amelioration of EAE clinical severity (Liu et al., 2016). In in vitro cultured lymphocytes from immunized mice, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 caused a marked decrease in lymphocyte proliferation and Th₁, Th₂, and Th₁₇ lymphocyte count and an increase in T_reg numbers (Liu et al., 2016). Using both pharmacological and genetic manipulation of A_2A receptors in mice with EAE, Ingwersen at al. (2016) demonstrated that while early activation of A_2A receptors ameliorated the course of EAE, A_2A receptor activation after disease onset aggravated the disease process (Ingwersen et al., 2016). In addition, bone marrow transfer studies demonstrated that A_2A receptor expression on nonimmune cells such as neurons in the brain, choroid plexus, meninges, hippocampus, and cerebellum contributed to EAE development, while A_2A receptor expression on immune cells (most likely lymphocytes) was essential for limiting the severity of the inflammatory response and disease progression (Mills et al., 2012b). A further complicating factor is that A_2A receptors appear to be important for the maintenance of the integrity of blood-brain barrier, and thereby modulating immune cell influx into the brain (Carman et al., 2011; Kim and Bynoe, 2015; Liu et al., 2018). Thus, the picture is complex and further studies will be required to unravel the precise role of A_2A receptors in EAE.

Similar to A_2A receptors, A_2B receptors are upregulated on peripheral blood leukocytes of MS patients (Wei et al., 2013). A_2B receptor-deficient mice or mice treated with the selective A_2B receptor neutral antagonist CVT-6883 (3-ethyl-3,9-dihydro-1-propyl-8-[1-[[3-(trifluoromethyl)phenyl]methyl]-1H-pyrazol-4-yl]-1H-purine-2,6-dione; 3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-2,3,6,7-tetrahydro-1H-purine-2,6-dione);developed less severe EAE compared with wild-type or vehicle-treated mice, respectively (Wei et al., 2013). This decrease in EAE severity was associated with decreased Th17 and Th1 cell responses in vivo.

Given the central role of CD39 in switching from ATP-mediated proinflammatory responses to the overall anti-inflammatory nature of adenosine-mediated responses (Antonioli et al., 2013c), the role of CD39 in regulating MS is an important question. In an early study, Borsellino et al. (2007) found a strikingly reduced number of CD39⁺ T_regs in the blood. Fletcher et al. (2009) confirmed this finding, as they observed a deficit in the relative frequency and the suppressive function of CD4⁺CD25⁺CD127^lowFoxP3⁺CD39⁺ T_reg cells in multiple sclerosis patients. Despite these findings, the role of CD39 in modulating the development and course of MS is still elusive. CD39 on DCs was found to be important for limiting the onset and severity of EAE (Mascanfroni et al., 2013) and CD39-deficient CD4 T cells showed an enhanced capability to drive EAE progression (Wang et al., 2014b). Nucleoside, triphosphate diphosphohydrolase-2, which can also degrade ATP, was found to be downregulated in EAE, and this decrease was associated with the severity of disease symptoms (Jakovljevic et al., 2017).

CD73-deficient mice were resistant to EAE, as the entry of lymphocytes into the central nervous system was inhibited in the absence of CD73 (Mills et al., 2008, 2012a). This may be because adenosine generated by CD73 is a key factor for opening the blood-brain barrier and therefore allowing lymphocyte influx (Bynoe et al., 2015).

Despite the observation that CD38 is highly expressed in brain and lymphocytes obtained from MS patients (Penberthy and Tsunoda, 2009), the role of CD38 and the CD38-CD203 axis, is still completely unexplored. Thus, more research is needed to better appreciate the potential relevance of the CD38-CD203 axis in MS pathogenesis and progression.

An increase in adenosine deaminase (ADA) activity was observed in the serum (Polachini et al., 2014) and cerebrospinal fluid (Samuraki et al., 2017) of MS patients. By contrast, ADA enzymatic activity in lymphocytes and platelets was reduced (Vivekanandhan et al., 2005; Spanevello et al., 2010a,b). As no preclinical studies have been performed to study the role of ADA in regulating MS, the role of these changes in ADA expression and activity is unclear.

Animal models of MS as well as studies performed on samples obtained from MS patients highlighted a key role of purinergic receptors, such as P2X₇, P2Y₁₂, or A_2A and A_2B in the onset and progression of this disorder. Indeed, both in vitro and in vivo experiments showed that the pharmacological or genetic manipulation of these receptor subtypes altered disease features, indicating their putative relevance as molecular targets for the development of novel pharmacological entities useful to counteract MS.

B. Uveitis

Uveitis is an inflammatory condition directed against the uvea, the vascular, pigmented middle coat of the eye wall, composed of the iris anteriorly, the ciliary body intermediately, and the choroid posteriorly (Read, 2006). Uveitis, especially if untreated, can lead to significant visual deficit and blindness (Caspi, 2010). Based on the etiology, uveitis can be distinguished as 1) infectious uveitis caused by an innate inflammatory reaction triggered by environmental “danger” signals (microbial) and 2) noninfectious uveitis, which is believed to be autoimmune or immune mediated (Caspi, 2010; Chen and Sheu, 2017).

The role of P2 receptors in regulating uveitis is not well understood. Zhao et al. (2016) demonstrated that oxidized ATP (oxATP) almost completely abolished the onset of experimental autoimmune uveitis (EAU) elicited by immunization of mice with the human interphotoreceptor retinoid-binding protein peptide IRBP_1–20 (Zhao et al., 2016). OxATP-treated mice displayed fewer autoreactive T cells, especially Th₁₇ autoreactive T cells, indicating that P2X₇ receptors promote disease through inducing Th₁₇ cell expansion (Zhao et al., 2016).

The CD25⁺CD11c⁺ DC subset has a critical role in eliciting the Th₁₇ autoreactive T cell response in IRBP_1–20-elicited EAU, and activated γδ T cells promote Th₁₇ cell development (Liang et al., 2012, 2015). Adenosine receptor stimulation differentially affects the immune response depending on the disease stage (Liang et al., 2014). Treating mice with the nonselective adenosine receptor agonist NECA during the induction phase (0 days postimmunization) had a suppressive effect on disease development and Th₁₇ responses (Liang et al., 2014), whereas when administered once the autoimmune response had already been initiated (7 days postimmunization), NECA enhanced disease activity and Th₁₇ responses (Liang et al., 2014). The selective A_2A agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 recapitulated the biphasic effect of NECA, whereas BAY 60-6538 (2-[(3,4-dimethoxyphenyl)methyl]-7-[(1R)-1-hydroxyethyl]-4-phenylbutyl]-5-methyl-imidazo[5,1-f][1,2,4]triazin-4(1H)-one), an A_2B agonist, moderately augmented disease activity irrespective of the timing of the treatment (Liang et al., 2014). The stimulatory effect of BAY 60-6538 was confirmed in another study where four injections of this drug between days 0 and 10 postimmunization enhanced EAU development and Th17 cell numbers and IL-17 production (Chen et al., 2015). The enhancing effect of BAY 60-6583 on the Th₁₇ response was markedly reduced in mice lacking γδ T cells, suggesting that the proinflammatory effect of BAY 60-6583 required γδ T cells (Chen et al., 2015)). Since BAY 60-6583 was unable to directly stimulate γδ T cells but augmented the ability of DCs to activate γδ T cells, the authors suggested that the proinflammatory effects of BAY 60-6583 were primarily mediated by A_2B receptors on DCs (Chen et al., 2015). In another EAU model, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 administered at the peak of EAU accelerated the resolution of disease (Lee et al., 2016). Taken together, exogenous A_2A receptor stimulation can both increase and decrease disease activity in EAU depending on the timing of stimulation and the model used, whereas exogenous A_2B receptor stimulation is proinflammatory across time points and models. In contrast, A_2A receptor KO and wild-type mice had a comparable course of EAU, indicating that endogenous adenosine stimulating A_2A receptors has no effect on the disease (Lee and Taylor, 2013).

A study performed by Bar-Yehuda et al. (2011) demonstrated that the selective A₃ receptor agonist CF101 was protective in an experimental animal model of uveitis. CF101 orally administered improved uveitis clinical scores and ameliorated the pathologic manifestations of the disease (Bar-Yehuda et al., 2011). In addition, CF101 acted as an immunomodulatory agent and suppressed antigen-specific proliferation and cytokine production of autoreactive T cells (Bar-Yehuda et al., 2011).

The role of purine metabolic enzymes in regulating uveitis is incompletely defined. CD73 expression was downregulated on γδ T cells during the preclinical or immunization phase (before day 7) of uveitis, whereas it was restored later during the clinical phase (Liang et al., 2016a). CD73 on αβ T cells remained unchanged throughout the course of EAU. As mice lacking γδ T cells adoptively transferred with CD73 KO cells had increased clinical score of EAU compared with mice transferred with CD73 sufficient γδ T cells, it was concluded that CD73 on γδ T cells protect against EAU (Liang et al., 2016a).

In a study aimed at evaluating the role of ADA in EAU, it was observed that ADA suppressed the course of EAU when given 8–14 days postimmunization and increased it when given either before or after this period (Liang et al., 2016b). Mice that received ADA at 8–14 days postimmunization had milder disease and recovered earlier than the untreated animals. In addition, ADA treatment at this time decreased serum IL-6 and IL-17 levels but induced a slight increase in serum IFN-γ and IL-10 concentrations (Liang et al., 2016b). The protective role of increased ADA activity at day 8 was confirmed by treating mice with the ADA inhibitor EHNA, which augmented both the course of EAU and IL-17 plasma levels (Liang et al., 2016b). The differential effect of ADA at different time point may reflect the differential role of adenosine receptors described above.

At present, the evidence about the role of the purinergic system in the pathophysiology of uveitis is few and fragmentary and further investigations are needed. In particular, there is a significant knowledge gap of the role of purinergic enzyme machinery in regulating the onset and development of uveitis. Nowadays, the available data indicate encouraging anti-inflammatory effects of adenosine and their agonists {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and CF101, acting via A_2A and A₃ adenosine receptors, respectively, thus prompting their use for the treatment inflammatory ophthalmic conditions. However, future studies are needed to evaluate better the relative contribution of adenosine receptors in shaping the activity of specific immune cells during uveitis.

C. Myasthenia Gravis

Myasthenia gravis (MG) is a chronic autoimmune disease caused by antibody-mediated blockade of neuromuscular transmission that results in muscle weakness and fatigue (Conti-Fine et al., 2006). The autoantibodies are generated by B cells in a T cell-dependent fashion and are reactive against nicotinic acetylcholine receptors (Vrolix et al., 2010; Meriggioli and Sanders, 2012).

Li et al. (2012) observed decreased A_2A receptor expression on both CD4⁺ T cells and B cells residing in spleen and lymph nodes of animals subjected to experimental autoimmune myasthenia gravis (EAMG) (Li et al., 2012). The administration of {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 29 days post EAMG induction (therapeutic treatment) ameliorated disease severity and decreased the number of Th₁ and Th₂ cells while increasing the number of T_reg cells, thus indicating that targeting A_2A receptors may represent putative therapeutic applications for MG (Li et al., 2012).

A recent paper by Oliveira et al. (2015) proposed that insufficient adenosine levels may contribute to deregulated immune cell function and neuromuscular transmission in myasthenic animals (Fig. 2). In EAMG animals, the expression of CD73 on T_reg cells was found to be decreased (Oliveira et al., 2015), which may result in impaired suppression of effector T cells contributing to the disease process (Li et al., 2012). In addition, the increased ADA activity observed in EAMG rats may also potentially aggravate adenosine deficiency and therefore autoimmunity (Oliveira et al., 2015).

Open in a separate window

Fig. 2.

Schematic representation depicting the role of the purinergic system in neuroimmunological alterations in an experimental model of experimental autoimmune myasthenia gravis. In healthy conditions, endogenous adenosine generated from the activity of CD73 counteracts the proinflammatory activity of T_eff cells, by acting on A_2A receptor expressed on them and by increasing the activity of T_reg cells. In addition, in the motor endplate, adenosine, arising from ATP degradation, facilitates acetylcholine release via the stimulation of A_2A receptors expressed at the prejunctional level. In myasthenic animals, there is reduced production of endogenous adenosine. In addition, adenosine degradation is increased, which is related with an increase in plasma adenosine deaminase activity. This results in increased production of autoAbs against nAChR and thus a loss of peripheral tolerance to nAChR. ACh, acetylcholine; ADA, adenosine deaminase; nAChr, nicotinic receptor.

In line with what was observed in this EAMG rat model (Oliveira et al., 2015), myasthenic patients showed an increase of total ADA activity (Chiba et al., 1990, 1995). Of note, the level of ADA2 was positively correlated with clinical MG grade (Chiba et al., 1995). Clearly, further studies aimed at unraveling the role of purinergic signaling in MG are needed.

D. Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a common autoimmune disease, which causes both joint inflammation and systemic complications, progressive disability, early death, and high socioeconomic costs (McInnes and Schett, 2011). Macrophages, DCs, T cells, and B cells accumulate in the joints, where they produce autoantibodies, proinflammatory cytokines, and bone destructive mediators (Weyand et al., 2009). Interactions between chronically stimulated immune cells and stromal cells, such as endothelial cells, vascular smooth muscle cells, and synovial fibroblasts are also critical to the onset and progression of this disorder (Weyand et al., 2009).

Extracellular ATP levels are increased in the synovial fluid of RA patients (Dowd et al., 1998). Human synoviocytes were shown to express P2X₁, P2X₂, P2X₄, P2X₅, P2X₆, and P2X₇ but not P2X₃ receptors at the messenger RNA level (Caporali et al., 2008). Pharmacological studies have shown that P2X₇ stimulation on these cells increases the production of IL-6 (Caporali et al., 2008), a major driver of the pathophysiology of RA. In a rat streptococcal cell wall arthritis model, P2X₇ receptor expression was detected in inflamed synovial tissue after the onset of disease, and P2X₇ receptor blockade with the selective antagonist AZD9056 suppressed articular inflammation and erosive progression (McInnes et al., 2014). Blockade of P2X₇ receptors also ameliorated pathologic changes in a collagen-induced arthritis model in mice, which was associated with decreased differentiation of pathogenic Th17 cells (Fan et al., 2016). Unfortunately, AZD9056 failed to improve clinical outcomes in a phase 2 clinical trial in RA (Keystone et al., 2012). Similarly, CE-224,535 (2-chloro-N-[(1-hydroxycycloheptyl)methyl]-5-[4-[(2R)-2-hydroxy-3-methoxypropyl]-3,5-dioxo-1,2,4-triazin-2-yl]benzamide), another P2X₇ receptor antagonist was also not efficacious, compared with placebo, for the treatment of RA in patients with an inadequate response to methotrexate (an allosteric inhibitor of dihydrofolate reductase), a commonly used disease modifying immunosuppressive drug (Stock et al., 2012).

Klein et al. (2012) reported that P2X₄ receptor stimulation increased the release of brain-derived neurotrophic factor, a neuromodulator involved in nociceptive hypersensitivity in the central nervous system, by synoviocytes from RA patients (Klein et al., 2012). The question of whether P2X₄ receptors contribute to pain in RA remains to be investigated. P2X₄ receptors were recently shown to promote joint inflammation and destruction in collagen-induced arthritis in mice (Li et al., 2014). Mechanistic studies showed that in both synovial cells obtained from human patients with RA and arthritic mice, targeting P2X₄ by antisense RNA suppressed the production of the pro-inflammatory cytokines IL-1β, TNF, and IL-6 (Li et al., 2014). In addition, P2X₄ receptor silencing suppressed NLRP1 inflammasome activation (Li et al., 2014).

mRNA for all four adenosine receptors was detected on human synoviocytes (Boyle et al., 1996; Hasko et al., 2008, 2018). Varani et al. (2009) demonstrated increased expression of A_2A and A₃ receptors on lymphocytes and neutrophils isolated from RA patients in comparison with healthy subjects. No changes in A₁ or A_2B receptors were observed (Varani et al., 2009, 2011; Ravani et al., 2017). The incubation of lymphocytes from RA patients with the selective A_2A and A₃ receptor agonists CGS 21860 and 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA) respectively, reduced nuclear factor-κB activation and diminished the release of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and matrix metalloproteinases MMP-1 and MMP-3 (Varani et al., 2011; Ravani et al., 2017). A₃ receptors were found to be highly expressed in inflammatory tissues isolated from rats with adjuvant-induced arthritis, especially synovia, peripheral blood mononuclear cells and draining lymph nodes (Fishman et al., 2006; Rath-Wolfson et al., 2006). A₃ receptors were also overexpressed in peripheral blood mononuclear cells of RA patients compared with healthy subjects (Madi et al., 2007).

2-(Cyclohexylethylthio) adenosine 5′-monophosphate, a prodrug that is degraded to a selective A_2A receptor agonist by CD73, suppressed joint inflammation in mice with collagen-induced arthritis. The cellular targets of the drug appeared to be monocytes and neutrophils. Interestingly, synovial cytokines were not affected (Flogel et al., 2012).

An early study showed that A₃ receptor stimulation with N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA also called CF101) reduced the severity of joint inflammation and inhibited the production of MIP-1α, IL-12, and reactive nitrogen species in the paws and suppressed neutrophil infiltration (Szabo et al., 1998). Subsequent studies confirmed the salutary effects of IB-MECA in a rat RA model as well (Bar-Yehuda et al., 2009). Mechanistic studies demonstrated that the anti-inflammatory effects of IB-MECA were associated with reduced expression and activation of phosphoinositide 3-kinase, protein kinase B/Akt, and NF-κB (Fishman et al., 2006; Ochaion et al., 2008). Despite the promising results with IB-MECA in animal models, it failed to improve the course of RA in human patients (Silverman et al., 2008).

Several studies reported increased CD39 expression on CD4⁺ T cells of RA patients (Potocnik et al., 1990; Berner et al., 2000; Dos Santos Jaques et al., 2013). FOXP3⁺ CD39⁺ T_reg cells are enriched in the joints of patients suffering from RA, and these cells are potent suppressors of many effector T-cell functions, including production of IFN-γ, TNF-α, and IL-17F (Herrath et al., 2014). Although no studies have directly tested the role of CD39 in regulating the course of RA, there is evidence that CD39 mediates the anti-arthritic effects of various treatment modalities. For example, in a murine model of arthritis, CD39 blockade reversed the anti-arthritic effects of methotrexate, a mainstay of RA therapy (Peres et al., 2015). Fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, also attenuates experimental murine arthritis through CD39 (Veras et al., 2015). Adoptive transfer of human gingiva-derived mesenchymal stem cells ameliorated murine collagen-induced arthritis in a CD39-dependent manner (Chen et al., 2013).

In contrast to CD39, CD73 was downregulated on CD4⁺ cells and Foxp3⁺ T_regs at the site of inflammation in patients with RA (Herrath et al., 2014). On the other hand, CD73 was increased on neutrophils and monocytes recovered from the synovial fluid of arthritic mice (Flogel et al., 2012). CD73-deficient mice were found to be more susceptible to collagen-induced arthritis compared with wild-type mice (Chrobak et al., 2015). They had increased production of proinflammatory cytokines in their joints and increased Th1 responses. Studies using bone marrow chimeric mice demonstrated that CD73 on non-hematopoietic cells was responsible for the CD73 protection against arthritis (Chrobak et al., 2015).

ADA levels are increased in plasma of patients with RA (Vinapamula et al., 2015; Valadbeigi et al., 2019 and reviewed in Antonioli et al., 2012). Synoviocytes obtained from RA patients had increased ADA mRNA expression (Nakamachi et al., 2003). It is conceivable that increased ADA is a pathogenic factor, as increased deamination of adenosine will result in its lowered bioavailability and decreased adenosine receptor-mediated suppression of inflammation.

Methotrexate has been in use for the treatment of RA since the 1980s, and it is still often the first line medication for RA patients (Friedman and Cronstein, 2018). There is a large body of evidence that methotrexate mediates its anti-inflammatory effect through increasing ATP release (Morabito et al., 1998; Montesinos et al., 2007), which is subsequently degraded to adenosine through ectonucleotidases (Montesinos et al., 2007), which in turn suppresses inflammation (Montesinos et al., 2000). Recently, it was observed that methotrexate nonresponsiveness in RA patients was associated with low expression of CD39 on T_regs (Peres et al., 2015; Gupta et al., 2018). This suggests that CD39 expression on T_regs could be a noninvasive biomarker for the early identification of patients who are unlikely to respond to methotrexate therapy.

Based on the above mentioned evidence a promising novel therapeutic approach for the treatment of RA may involve targeting adenosine receptors (mainly the A_2A and A₃ receptor subtypes). Alternatively, indirect targeting of adenosine receptors by enhancing endogenous adenosine concentration at inflamed sites by the pharmacological blockade of ADA or nucleoside transporters, may represent a novel therapeutic approach.

E. Scleroderma

Scleroderma, which is also called systemic sclerosis, is an autoimmune connective tissue disease characterized by fibrosis of the skin and internal organs as well as by vasculopathy (Denton and Khanna, 2017). Salient features of the tissue lesions in scleroderma are early microvascular damage, mononuclear-cell infiltrates, and slowly developing fibrosis (Gabrielli et al., 2009). In later stages of scleroderma, the main findings are very densely packed collagen in the dermis and other organs, loss of cells, and atrophy (Gabrielli et al., 2009; Denton and Khanna, 2017).

There is a growing body of evidence indicating that adenosine has an important role in tissue remodeling and dermal fibrosis (Chan et al., 2013; Hu et al., 2013; Perez-Aso et al., 2014; Zhang et al., 2017a; Karmouty-Quintana et al., 2018). A_2A receptor activation causes dermal wound closure and increased dermal matrix deposition in vitro (Montesinos et al., 2004; Cronstein, 2006a; Scheibner et al., 2009). In agreement with these in vitro profibrotic effects of A_2A receptor activation, both genetic deletion and pharmacological inhibition of A_2A receptors with ZM241385 (4-[2-(7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a] [1,3,5]triazin-5-yl-amino]ethyl phenol), a neutral antagonist, prevented dermal fibrosis in mice challenged with subcutaneous bleomycin, a model of human scleroderma (Chan et al., 2006). Another study utilizing a structurally different A_2A antagonist, KW6002 ((E)-8-(3,4-dimethoxystyryl)-1,3-diethyl-7-methylxanthine, 8-[(1E)-2-(3,4-dimethoxyphenyl)ethenyl]-1,3-diethyl-3,7-dihydro-7-methyl-1H-purine-2,6-dione), also confirmed reduced severity of bleomycin-induced dermal fibrosis (Zhang et al., 2017a).

Subcutaneous treatment of mice with bleomycin upregulates A_2B receptor transcript levels in the skin (Karmouty-Quintana et al., 2018). Pharmacological blockade of A_2B receptors by GS-6201 reduced the production of profibrotic mediators (fibronectin, MCP1, IL-6, and α-SMA) in the skin and attenuated dermal fibrosis of mice in bleomycin-induced as well as genetic [mutant tight-skin (TSK1/+) mice] models of human scleroderma (Karmouty-Quintana et al., 2018). While no differences in A_2B receptor expression were found between healthy and sclerotic human skin (Karmouty-Quintana et al., 2018), a reduction in density and function of A_2B receptors was noted in neutrophils of patients affected by scleroderma compared with healthy patients (Bazzichi et al., 2005). The significance of this decrease of A_2B receptor expression and function in scleroderma patients is unclear.

Consistent with the generally profibrotic effects of adenosine, CD39 knockout (KO) animals as well as CD39/CD73 KO exhibited reduced skin fibrosis upon bleomycin challenge (Fernandez et al., 2013).

Genetic deletion of ADA leads to elevated adenosine levels and spontaneous dermal fibrosis in mice (Fernandez et al., 2008). Although increased ADA activity has been reported in plasma of scleroderma patients (Sasaki and Nakajima, 1992; Meunier et al., 1995), it is unclear whether the increased ADA is a causative factor in scleroderma.

In summary, increased adenosine and A_2A and A_2B receptors contribute to scleroderma development in mice. The role of P2 receptors is unknown and will need to be defined in the future.

F. Psoriasis

Psoriasis vulgaris, commonly known as plaque psoriasis, is a chronic inflammatory skin disease characterized by skin plaques and systemic symptoms. The immunopathogenesis of psoriasis involves both the innate and adaptive immune systems (Lowes et al., 2014). The immune circuits that normally participate in the regulation of skin homeostasis, become abnormally activated and amplified in psoriatic patients, leading to an excessive and rapid growth of the epidermal layer of the skin (Lowes et al., 2014). Activated myeloid DCs release IL-23 and IL-12, which stimulate Th₁₇, Th₂₂, and Th₁ cells to release copious amounts of psoriatic cytokines such as IL-17, IL-22, TNF, and IFN-γ, which promote keratinocyte hyperproliferation (Lowes et al., 2014).

Keratinocyte express all P2X and P2Y receptor subtypes so far cloned, except for P2Y₁₄ (Dixon et al., 1999; Burrell et al., 2003; Inoue et al., 2005; Pastore et al., 2007; Ishimaru et al., 2013). IFN-γ upregulated the expression of P2X₇ and P2Y₁ receptors on human keratinocytes, suggesting a possible involvement of these receptor subtypes in the pathophysiology of psoriasis (Pastore et al., 2007). In line with this view, P2X₇ and P2Y₁ receptors were found to be upregulated in lesional skin of patients with psoriasis (Pastore et al., 2007). An increase in P2X₇ receptor expression was also reported in nonlesional skin of psoriatic patients in comparison with healthy skin tissue, leading the authors to hypothesize that P2X₇ receptor dysregulation in psoriasis precedes the onset of inflammatory lesions (Killeen et al., 2013). In healthy human skin explants, the pharmacological stimulation of P2X₇ with BzATP induced a significant increase in vascular endothelial growth factor, IL-23, and IL-6 expression, indicating that P2X₇ receptor activation may be an initiating factor in psoriasis development (Killeen et al., 2013).

Although both P2Y₆ (Uratsuji et al., 2012) and P2Y₁₁ receptors (Ishimaru et al., 2013) can contribute to proinflammatory responses of keratinocytes in vitro, the relevance of these findings for psoriasis is still incompletely understood.

Normal human keratinocytes and normal human skin express mainly A_2B receptors and detectable levels of A_2A receptors, whereas the levels of A₁ and A₃ receptor mRNA are negligible (Andres et al., 2017). Psoriasis is associated with an upregulation of A_2A and downregulation of A_2B receptors in the psoriatic skin (Andres et al., 2017). Since A_2A receptors augment keratinocyte proliferation and A_2B receptors arrest it (Fig. 3) (Andres et al., 2017), it is conceivable that the increase in A_2A and decrease in A_2B receptor expression observed in psoriatic patients contribute the hyperkeratosis process (Fig. 3) (Lowes et al., 2014).

Open in a separate window

Fig. 3.

Role of adenosine in modulating keratinocyte proliferation. Psoriatic patients are characterized by an abnormal hyperproliferation and differentiation of keratinocytes. In this context, endogenous adenosine participates in the hyperkeratosis process by increasing the proliferation of keratinocytes via the engagement of A_2A receptors, which are overexpressed in psoriatic patients.

In contrast to keratinocytes, A_2A receptors are downregulated on effector CD4⁺ T cells from patients with psoriasis compared with healthy subjects (Han et al., 2018), while A₃ receptors are overexpressed in peripheral blood mononuclear cells of psoriatic patients (Ochaion et al., 2009). A randomized, double-blind, placebo-controlled trial demonstrated that IB-MECA improved the clinical symptoms of psoriasis (David et al., 2016). Although this study did not investigate the mechanisms underlying the beneficial effects of IB-MECA, a subsequent study found that A₃ receptor activation suppressed keratinocyte proliferation and IL-17 and IL-23 production by keratinocytes (Cohen et al., 2018).

In conclusion, novel therapies could be derived for hyperproliferative skin diseases, such as psoriasis, based on the intriguing dual roles played by A_2A and A_2B adenosine receptors in modulating keratinocyte proliferation. Future studies should inform us on whether A_2A agonists could be used to reduce inflammation. In addition, it should be of interest to evaluate the influence of existing therapeutic approaches for psoriasis in regulating adenosine receptor expression to determine whether adenosine receptor expression may serve as a biomarker in the trajectory of psoriatic pathology.

G. Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic, relapsing/remitting, and multisystemic autoimmune disease with heterogeneous clinical manifestations. The disease can have dermatological, musculoskeletal, renal, respiratory, cardiovascular, hematologic, and neurologic consequences (Moulton et al., 2017). Major pathogenetic factors of SLE comprise immune responses against endogenous nuclear antigens, increased autoantibody production, increased apoptosis, and deficient clearance of apoptotic cells (Moulton et al., 2017). As a result of these processes, immune cells secrete aberrant amounts of cytokines and other soluble proinflammatory mediators, which cause inflammation and the destruction of end-organs (Moulton et al., 2017). The majority of SLE patients display elevated production of type I interferons and increased expression of type I IFN-regulated genes (Ronnblom and Pascual, 2008).

There is an increasing appreciation of the notion that alterations of the purinergic machinery can contribute to the pathogenesis of SLE (Forchap et al., 2008; Portales-Cervantes et al., 2010; Loza et al., 2011; Sipka, 2011; Saghiri et al., 2012; Bortoluzzi et al., 2016). Portales-Cervantes et al. (2010) and Forchap et al. (2008) both studied the role of the most frequent, loss‐of‐function 1513 A→C single nucleotide polymorphism of P2X₇ receptors in human SLE. Both studies failed to find differences in allele frequencies of this polymorphism when comparing sporadic cases of SLE and healthy controls. Although ATP-induced IL-1β release was significantly decreased in SLE patients with the 1513 A→C genotype (Portales-Cervantes et al., 2010), the significance of this finding is unclear. In murine studies, pharmacological P2X7 receptor blockade attenuated lupus nephritis, the mechanism of which appeared to be related to decreased NLRP3 inflammasome activation and IL-1β release (Zhao et al., 2013). Le Gall et al. (2012) reported that B220⁺ CD4^–CD8^– lymphocytes, which accumulate in autoimmune MRL/lpr mice (a murine model characterized by a similar autoantibody profile to SLE patients, with serum antibodies directed against many nuclear antigens, such as DNA and histones) (Mandik-Nayak et al., 1999), became more resistant with age to P2X₇ receptor-induced shedding of CD62L, pore formation, phosphatidylserine exposure, and cell death compared with non-B220⁺ CD4^–CD8^– lymphocytes (Le Gall et al., 2012). The authors proposed that the decreased P2X₇ receptor-mediated cell death of the pathogenetic B220⁺CD4^–CD8^– population may contribute to disease progression in MRL/lpr mice. Apart from P2X₇ receptors, not much is known about the role of P2 receptors in the pathophysiology of SLE, which should be explored in further studies.

An early report described that adenosine was less efficacious in suppressing T lymphocyte function in patients with SLE compared with healthy subjects (Mandler et al., 1982). Since A_2A receptors mediate most of the immunosuppressive effects of adenosine on T cells (Csóka et al., 2008; Himer et al., 2010), this would suggest that A_2A receptor expression or coupling is altered in SLE T cells. Bortoluzzi et al. (2016), however, found higher expression of A_2A mRNA and protein in lymphocytes from patients with SLE compared with control subjects (Bortoluzzi et al., 2016). A_2A receptor density was inversely correlated with SLE disease activity index (Bortoluzzi et al., 2016). In addition, the A_2A receptor agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 was more efficacious in suppressing T lymphocyte proinflammatory cytokine production in SLE T lymphocytes than control T lymphocytes. The authors proposed that the upregulation of A_2A receptors on lymphocytes in SLE patients was a compensatory mechanism to counteract the proinflammatory milieu of SLE (Bortoluzzi et al., 2016). Murine studies confirmed the protective function of A_2A receptors in SLE, as {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment of MRL/lpr mice suppressed T cell activation, autoantibody production, and renal injury (Zhang et al., 2011). The role of A₁, A_2B, and A₃ receptors in SLE is unknown.

CD39 expression was found to be defective on freshly isolated T_reg cells of lupus subjects with minimally active disease compared with patients with active disease or healthy controls (Loza et al., 2011). In addition, nonregulatory T cells were deficient in their capacity to upregulate expression of CD39 upon CD3 stimulation specifically in patients with minimally active disease. Although the reason why patients with minimally active disease had defective CD39 remained unclear, the authors proposed that defective CD39 expression might be a useful biomarker for early detection of the disease, prior to the onset of symptoms (Loza et al., 2011). A recent study employing CD39-deficient mice undergoing pristine-induced lupus demonstrated that CD39 controlled autoimmunity and disease symptoms, shedding some light on the role of CD39 in lupus (Knight et al., 2018).

A defective activity was reported also for 5′-nucleotidase in lymphocytes isolated from lupus patients (Stolk et al., 1999). Although this study did not provide more specific evidence, it is likely that the 5′-nucleotidase activity was due to CD73, as this enzyme is the major cell-associated 5′-nucleotidase (Yegutkin, 2008). Similar to CD39, CD73 protected against pristane-induced lupus (Knight et al., 2018), suggesting that the CD39-CD73-adenosine axis may be important for curbing inflammation in SLE.

H. Glomerulonephritis

Glomerulonephritis is a condition of glomerular inflammation, which manifests as hematuria and proteinuria (Anders, 2013; Liu and Chun, 2018). It encompasses a spectrum of kidney diseases that collectively are the third leading cause of end-stage renal disease. The incidence of primary glomerulonephritis varies between 0.2 and 2.5 per 100,000 per year (Liu and Chun, 2018). The pathogenesis of glomerulonephritis is complex. Several factors can trigger and contribute to the progression of glomerular injury. These include, but are not limited to, genetic predisposition, autoimmunity, malignancy, infections, diabetes, hypertension, and exposure to drugs (Liu and Chun, 2018). Major pathophysiological factors include glomerular infiltration of macrophages and neutrophils and mesangial cell activation (Scindia et al., 2010; Kitching and Hutton, 2016).

Several P2 receptors are expressed in the healthy kidney (Arulkumaran et al., 2013). Turner et al. (2007) reported that P2X₇ receptors were upregulated in the kidney of both patients and mice with glomerulonephritis (Turner et al., 2007). In animal models of antibody-mediated glomerulonephritis, P2X₇ receptor deficiency or pharmacological antagonism prevented macrophage infiltration and protected against kidney injury (Taylor et al., 2009). Pharmacological blockade or siRNA-mediated silencing also prevented kidney injury in MRL/lpr mice, a model of lupus-induced glomerulonephritis (Zhao et al., 2013). The protective effect of P2X₇ blockade was associated with decreased NLRP3 inflammasome activation and IL-1β release (Zhao et al., 2013). In a recent murine study, systemic injection of a nanobody against the P2X₇ receptors blocked the receptor on T cells and macrophages and ameliorated antibody-induced glomerulonephritis (Danquah et al., 2016). Other than the P2X₇ receptor, the only other receptor whose role has been tested in glomerulonephritis is the P2Y₁ receptor, the deficiency of which was protective in mice with antibody-mediated glomerulonephritis (Hohenstein et al., 2007).

Of all the adenosine receptors, the A_2A receptor is the only one whose role has been investigated in regulating glomerulonephritis. In the first study on this topic, increased expression of A_2A receptors on macrophages was found in the glomeruli of rats with antibody injection-induced glomerulonephritis (Garcia et al., 2008). Pharmacological activation of A_2A receptors with {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 prevented the infiltration of leukocytes into the kidney, counteracted glomerular inflammation, and protected the kidney from inflammatory injury in this model (Garcia et al., 2008). The protective effect of A_2A receptor stimulation was subsequently confirmed in MRL/lpr mice, as CGS-21680 treatment caused reduced proteinuria, and blood urea and creatinine levels, as well as an improvement in renal histology (Zhang et al., 2011). Renal tissue had reduced macrophage and T-cell infiltration, as well as attenuated MCP-1, IFN-γ, and MHC-II expression (Zhang et al., 2011). {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment also reduced serum anti-dsDNA levels and renal immune complex deposition (Zhang et al., 2011). A recent study using A_2A receptor KO mice showed that endogenous adenosine protected mice from glomerulonephritis through A_2A receptors, as the deficient mice were more prone to kidney injury and had increased renal inflammatory cytokines and glomerular hyalinosis compared with wild-type animals (Truong et al., 2016). Using macrophage depletion and reconstitution with wild-type or A_2A receptor-deficient macrophages during the established phase of glomerulonephritis, the authors demonstrated that macrophage A_2A receptors were central to the A_2A receptor-mediated protection against glomerulonephritis (Truong et al., 2016).

I. Chronic Obstructive Pulmonary Disease

COPD is a progressive inflammatory condition characterized by a progressive and irreversible deterioration of lung function due to airflow obstruction, destruction of parenchyma, and emphysema (MacNee and Tuder, 2009; Rovina et al., 2013). Tobacco smoking is the most common cause of COPD in the developed world, whereas indoor air pollution due to poorly ventilated cooking fires is a major cause in developing countries (Rovina et al., 2013). The major pathophysiological factor leading to COPD is airway inflammation caused by the inhaled irritants. The primary target cells of the irritants are epithelial cells and resident (alveolar) macrophages, which become activated and release chemotactic mediators leading to the recruitment of further inflammatory cells (CD8⁺ T cells, neutrophils, monocytes, and lymphocytes) into the lung. The resulting multicellular infiltrate is central to the maintenance of the chronic inflammatory process, which persists even after the exposure to irritants ceases (Rovina et al., 2013).

There has been a steadily increasing interest in the involvement of purinergic signaling in the pathophysiology of COPD (Polosa and Blackburn, 2009; Mortaz et al., 2010; Pelleg et al., 2016). Lommatzsch et al. (2010) measured ATP concentrations in alveolar bronchoalveolar lavage (BAL) fluid and found that COPD patients had elevated ATP levels compared with controls (Lommatzsch et al., 2010). In patients with COPD, BAL fluid ATP concentrations correlated inversely with lung function and positively with BAL fluid neutrophil counts. BAL fluid macrophages isolated from patients with COPD upregulated their P2X₇ receptor expression compared with control subjects (Lommatzsch et al., 2010). In line with the higher P2X7 receptor expression on macrophages from COPD patients, P2X₇ stimulation triggered higher production of proinflammatory cytokines and matrix metalloproteinase 9 by macrophages of patients with COPD compared with macrophages from control subjects (Lommatzsch et al., 2010). In contrast, P2X₇ receptor stimulation on macrophages induced a more pronounced suppression of tissue inhibitor of matrix metalloproteinase-1 release in patients with COPD (Lommatzsch et al., 2010). Since proinflammatory cytokines and matrix metalloproteinase-9 contribute to lung tissue destruction while tissue inhibitor of matrix metalloproteinase-1 prevents it (Daheshia, 2005; Demedts et al., 2005; Churg et al., 2012), the P2X₇ receptor modulation of proinflammatory cytokines and extracellular matrix regulating enzymes appear to be harmful in COPD. Blood neutrophils from COPD patients had upregulated P2Y₂ receptor expression as well as a marked increase in P2Y₂-mediated migration and elastase release compared with neutrophils from healthy subjects (Lommatzsch et al., 2010). Since elastase is important in extracellular matrix degradation (Churg et al., 2012; Bidan et al., 2015), this indicates that P2Y₂ receptors on neutrophils may contribute to lung tissue breakdown.

Murine studies have corroborated the proinflammatory and destructive role of ATP and P2 receptors in COPD. Mice exposed to cigarette smoke had increased P2X₇ receptors primarily in airway macrophages and neutrophils and in lung tissue (Lucattelli et al., 2011). Both pharmacological blockade of P2X₇ with KN62 and experiments performed using P2X₇ receptor KO mice revealed a prominent role of this receptor subtype in mediating the pro-inflammatory effects of ATP on cigarette smoke-induced lung inflammation and injury (Lucattelli et al., 2011). Similar to observations in human patients (Lommatzsch et al., 2010), P2Y₂ receptors contributed to neutrophil migration in mice (Cicko et al., 2010). In addition, as P2Y₂ receptor-deficient mice had reduced pulmonary inflammation following cigarette smoke exposure, the authors concluded that P2Y₂ receptors may be involved in the pathogenesis of cigarette smoke-induced COPD (Cicko et al., 2010).

Elevated levels of adenosine were detected in the airway lining fluid of patients with COPD compared with normal controls (Polosa, 2002). Using mass spectrometric analysis of exhaled breath condensate, Esther et al. (2011) evaluated AMP and adenosine concentrations on airway surfaces in COPD patients in comparison with healthy smokers and nonsmokers. The results demonstrated elevated airway AMP and adenosine levels in subjects with COPD, which were correlated with several markers of disease severity (Esther et al., 2011).

Adenosine receptors were also found to be altered in COPD patients. That is, in an early study, COPD subjects had decreased affinity but increased density and mRNA expression of A_2A and A₃ receptors in peripheral lung tissue (Varani et al., 2006). To explain these alterations, the authors speculated that increased adenosine in COPD patients might desensitize A_2A and A₃ receptors in the lung, which might result in the compensatory upregulation of these receptors. A₁ receptor affinity decreased and density increased, but no changes in mRNA expression levels were noted. The affinity of A_2B receptors was not altered, but A_2B receptor density and mRNA expression decreased in peripheral lung tissue of patients with COPD compared with the control group. These inconsistencies with regard to A₁ and A_2B receptor affinity, density, and mRNA expression in whole lung can potentially be ascribed to the differential expression of A₁ and A_2B receptor expression on the various cell types in the lung (Varani et al., 2006). For example, while A_2B receptors were found to be downregulated on macrophages (Varani et al., 2010), A_2B receptor expression was heightened on pulmonary artery smooth muscle cells of COPD patients (Karmouty-Quintana et al., 2013). The clinical significance of these changes in adenosine receptor subtype expression and function in patients with COPD is unclear at this point. Varani et al. (2006) detected a significant correlation between the density and affinity of A_2A, A_2B, and A₃ and the forced expiratory volume in one second/forced vital capacity ratio, an established index of airflow obstruction. In a mouse model, A_2A receptor stimulation with {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 was unable to suppress cigarette smoke-induced inflammation (Bonneau et al., 2006; Mantell et al., 2008). Although these findings hint that adenosine receptors may modulate the course of COPD, further studies will be necessary to precisely delineate the role of the various adenosine receptors in regulating the course of COPD in patients.

Aliagas et al. (2018) found decreased CD39 gene and protein expression as well as activity in the lungs of COPD patients in comparison with controls. This decrease in CD39 correlated with higher systemic inflammation and intimal thickening of muscular pulmonary arteries in the COPD group (Aliagas et al., 2018). Immunohistochemical analysis showed that CD39 was downregulated mainly in lung parenchyma, in epithelial bronchial cells, and in the endothelial cells of pulmonary muscular arteries (Aliagas et al., 2018). In contrast, another study demonstrated that CD39 expression and activity were higher in sputa and BAL cells of COPD patients compared with controls (Lazar et al., 2016). Experiments performed on mice chronically exposed to cigarette smoke confirmed increased CD39 in lung tissue (Lazar et al., 2016). In addition, the same study showed that CD39-deficient mice displayed a worsening of lung inflammation induced by both acute and chronic cigarette smoke exposure, which was partially rescued by the administration of apyrase, a CD39 analog. This indicates that CD39 is protective in COPD (Lazar et al., 2016). Another recent study reported increased CD39 expression on peripheral T cells in patients with acute exacerbations of COPD compared with both COPD patients without exacerbations and healthy controls.

CD73 expression was found to be upregulated and ADA downregulated in lung tissue of patients with COPD (Zhou et al., 2010), indicating that the lung environment in COPD may favor the accumulation of adenosine.

Of note, it will be important to address the role of the purinergic machinery in regulating COPD in more detail. In particular, it would be of interest to investigate how and to what extent the purinergic pathway is involved in the extensive immune dysfunction observed in COPD patients, with particular regard for the role of purines in shaping CD4⁺PD-1⁺ exhausted effector T cells, and myeloid-derived suppressor cells, which are involved in COPD pathophysiology.

J. Asthma

Asthma is a chronic inflammatory disorder of the airways (Colucci et al., 2007). Clinically, asthma is characterized by recurrent episodes of wheezing, breathlessness, chest tightness, and cough. Reversible airway obstruction, mucus overproduction, and bronchial hyperresponsiveness triggered by specific and nonspecific stimuli, such as allergens, chemical irritants, cold air, and exercise underlie the symptoms of asthma (Colucci et al., 2007). Mast cells, eosinophils, Th2 lymphocytes, group 2 innate lymphoid cell types, IgE-producing B lymphocytes, DCs, macrophages, and eosinophils are the key players of the type 2 immune response driving inflammation in asthma (Barnes, 2018). The type 2 immune response is driven primarily by the “classical” type 2 cytokines IL-4, IL-5, and IL-13, as well as by the damage-associated cytokines thymic stromal lymphopoietin, IL-25, and IL-33.

ATP accumulates in BAL fluid isolated from patients with asthma (Idzko et al., 2007). P2X₁ receptor-mediated currents and CD11b expression are reduced in in eosinophils from asthma patients compared with healthy controls (Wright et al., 2016). P2X₇ receptors are expressed at higher levels on BAL fluid cells of patients with asthma compared with healthy control subjects (Muller et al., 2011). Eosinophils isolated from asthmatic individuals express higher levels of P2Y₂ receptor compared with healthy controls. As a result, asthmatic eosinophils have increased chemotactic responses and reactive oxygen metabolite production in response to ATP compared with healthy individuals (Muller et al., 2010). A recent genome-wide association study identified P2Y13 and P2Y14 as genes associated with asthma risk (Ferreira et al., 2017). Bronchial provocation test with nebulized AMP is an objective test for airway hyperresponsiveness that is clinically useful to aid in the diagnosis of asthma. A study by Basoglu et al. (2005) compared the effect of ATP with that of AMP on airway hyperresponsiveness in patients with asthma. The study demonstrated that ATP was a more potent and efficacious inducer of airway hyperresponsiveness in asthmatic patients than AMP. As ATP but not AMP activates P2 receptors, this finding indicates that P2 receptors may contribute to the symptoms of asthma in humans (Basoglu et al., 2005).

Preclinical studies also point to a pro-inflammatory role of extracellular ATP and P2 receptors in asthma. Neutralization of ATP using apyrase or pharmacological P2 receptor antagonism with pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) or oxATP decreased inflammation in an ovalbumin (OVA) model of asthma (Idzko et al., 2007). In contrast, exogenously added ATP promoted sensitization to inhaled OVA in mice (Idzko et al., 2007). Zech et al. (2016) demonstrated that the P2X₄ receptor may be one of the mediators of the proinflammatory effects of ATP in asthma. They showed that both the P2X₄ receptor antagonist 5-BDBD (5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one) and P2X4 deficiency alleviated BAL fluid eosinophilia, peribronchial inflammation, Th2 cytokine production, and bronchial hyperresponsiveness in a murine OVA asthma model. P2X₄ receptors on DCs were implicated as having a central role in promoting asthma, as adoptive transfer of P2X₄ receptor-deficient DCs attenuated the Th2 response and inflammation in OVA-sensitized mice (Zech et al., 2016). P2X₇ receptors also promote asthma in mice. Both mice treated with the AZ9056, a specific P2X₇ receptor-antagonist and P2X₇-deficient mice had reduced features of lung inflammation, such as airway eosinophilia, goblet cell hyperplasia, and bronchial hyperresponsiveness to methacholine in both the OVA model and a house dust mite model of asthma (Muller et al., 2011). Adoptive transfer studies incriminated P2X₇ receptor signaling on DCs as a major proinflammatory factor in asthma (Muller et al., 2011).

P2Y₁ receptors were recently shown to be involved in regulating allergic inflammation. Treatment of OVA-sensitized mice with the selective and competitive P2Y₁ antagonist 2′-deoxy-N⁶-methyladenosine 3′,5′-bisphosphate (MRS2179) or (1R*,2S*)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester (MRS2500) inhibited leukocyte recruitment to the lung (Amison et al., 2015). Since platelet depletion followed by reinfusion of platelets preincubated with MRS2500 versus vehicle-preincubated platelets resulted in decreased inflammation, the authors concluded that P2Y₁ receptors on platelets are important for mediating the proinflammatory effects of P2Y₁ receptors. P2Y₂ receptors are also proinflammatory in asthma. In one study, P2Y₂ receptor-deficient mice showed decreased inflammation, decreased IgE levels, decreased VCAM expression on endothelial cells, and defective eosinophil infiltration in OVA-induced asthma (Vanderstocken et al., 2010). In another study, P2Y₂ receptor-deficient mice exhibited reduced allergic inflammation, which was explained by defective inflammatory cell migration into the lung and a reduced Th2 response in lymph nodes (Muller et al., 2010). One study showed that P2Y₁₂ receptors may also contribute to asthma by serving as receptors for LTE4, a major proinflammatory mediator of asthma (Paruchuri et al., 2009). However, a subsequent study questioned the P2Y₁₂ agonistic role of LTE4 (Foster et al., 2013).

Adenosine receptors have long been implicated in asthma (Polosa and Blackburn, 2009). Theophylline, a competitive nonselective phosphodiesterase inhibitor and also a nonselective adenosine receptor antagonist, has been used to treat asthma for a century (Barnes, 2013). Adenosine concentrations are increased in exhaled breath condensate (Huszar et al., 2002) or BAL fluid (Driver et al., 1993) of patients with asthma compared with healthy subjects. Inhaled adenosine is a potent bronchoconstrictor in human asthma patients but not in healthy subjects (Cushley et al., 1983). These results indicate that adenosine receptors contribute to asthma development and symptoms.

A₁ receptor expression is increased in bronchial biopsies from patients with asthma versus healthy subjects (Brown et al., 2008b). A_2A receptors are expressed at higher levels on peripheral blood mononuclear cells of patients with mild-to-moderate asthma than healthy patients or patients with severe asthma (Wang et al., 2018a). In sputum, asthma patients had a lower percentage of neutrophils expressing A_2B receptors than healthy subjects (Versluis et al., 2008). A₃ receptor transcript abundance was greater in lung tissue of asthmatic than in healthy patients (Walker et al., 1997).

Preclinical evidence supports the notion that adenosine signaling participates in the regulation of pulmonary inflammation and damage in asthma (Sun et al., 2006; Mohsenin et al., 2007). Mice lacking ADA and therefore having elevated extracellular adenosine levels develop pulmonary inflammation with features typically observed in patients suffering from asthma, such as an increase in alveolar macrophages, airway remodeling, increased mucin production, angiogenesis, and alveolar airway enlargement (Blackburn et al., 2000). Lung-specific IL-13 overexpression in mice produced eosinophil-, lymphocyte-, and macrophage-rich inflammation, alveolar enlargement, mucus metaplasia, and airway hyperresponsiveness on methacholine challenge (Zhu et al., 1999). Blackburn et al. (2003) demonstrated that extracellular adenosine was elevated in these mice and ADA therapy prevented the asthmatic phenotype, indicating that adenosine mediated the proinflammatory effects of IL-13. By crossbreeding adenosine receptor-deficient mice onto the ADA-deficient background, Blackburn and coworkers demonstrated that A₁, A_2A, and A_2B receptors protected against lung inflammation, whereas A₃ receptors contributed to it (Young et al., 2004; Sun et al., 2005; Mohsenin et al., 2007; Zhou et al., 2009). The protective role of A_2B receptors, however, was not confirmed using a pharmacological approach in ADA-deficient mice, as pharmacological blockade of A_2B receptors with CVT-6883 starting on postnatal day 24 reduced the number of immune cells in the BAL fluid, decreased the production of pro-inflammatory cytokines and chemokines, and attenuated pulmonary fibrosis (Sun et al., 2006). The reason for the discrepant results of the knockout and pharmacological studies is unclear at this point. One possible explanation is that A_2B receptors affect asthma in a stage dependent manner, where A_2B receptor inactivation from birth may be pro-inflammatory, whereas A_2B receptor blockade initiated after birth may be protective.

Allergen sensitization models have also been helpful in delineating the role of adenosine receptors in asthma. In a ragweed model, A₁ receptor-deficient mice exhibited decreased IL-5 and ICAM-1 expression and decreased airway hyperresponsiveness, indicating that A₁ receptors are proinflammatory in this model (Ponnoth et al., 2010). Pharmacological studies using a selective A₁ antagonist also pointed to a proinflammatory role of A₁ receptors in a house dust mite asthma model in rabbits (Obiefuna et al., 2005). A_2A receptor-deficient mice challenged with ragweed had increased inflammation, NF-κB activation, and airway reactivity to methacholine, indicating that A_2A receptors are protective in this model of asthma (Nadeem et al., 2007). Pharmacological stimulation of A_2A receptors is in general anti-inflammatory, although the protective effects are variable. After repeated ovalbumin challenges in mice, intranasally administered {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 inhibited BAL fluid inflammatory cell influx but had no effect on OVA-induced bronchoconstriction and airway hyperreactivity (Bonneau et al., 2006). In another murine OVA model, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 upregulated the T_reg transcription factor FoxP3 and the T_reg-derived cytokine TGF-β, decreased the Th-17-related transcription factor ROR-γT and IL-17, and improved lung function (Wang et al., 2018a). The anti-inflammatory effects of A_2A agonism in mice were not borne out in a human clinical trial, as the selective A_2A agonist GW328267X failed to affect the inflammatory response and airway hyperresponsiveness in patients with asthma (Luijk et al., 2008).

Genetic ablation of A_2B receptors in mice attenuated OVA-induced chronic pulmonary inflammation, IL-4 and TGF-β production, and pulmonary inflammation and injury (Zaynagetdinov et al., 2010), indicating that A_2B receptors contribute to the pathophysiology of asthma. Similarly, both global and myeloid A_2B receptor deficiency decreased pulmonary inflammation, Th2 cytokine production, and chemokine level in a cockroach-allergen murine model (Belikoff et al., 2012). Pharmacological studies with a selective A_2B receptor antagonist confirmed the proinflammatory role of A_2B receptors in murine asthma (Basu et al., 2017a,b). Using both A₃ receptor-deficient mice and treatment with IB-MECA, Young et al. (2006) demonstrated that A₃ receptors contribute to airway mucin secretion after OVA challenge of mice. However, pulmonary inflammation and function were not determined in this study.

The purinergic enzyme machinery also influences the course of asthma. Asthma patients have decreased proportions of CD39⁺ T_regs among all T_regs compared with healthy individuals (Wang et al., 2013). CD39 mRNA levels in both CD4⁺ T cells (Wang et al., 2013) and peripheral blood mononuclear cells are decreased in asthma patients versus healthy subjects (Wang et al., 2014a). This suggests that insufficient CD39-mediated immune suppression may contribute to the progression of asthma. Surprisingly, mice lacking CD39 displayed a milder asthma phenotype than wild-type mice when tested in both the OVA and house dust mite models (Idzko et al., 2013). This decrease in asthma severity in the CD39-deficient mice was due to aberrant migration of DCs with a consequent limitation of the capacity of these cells to prime Th₂ responses (Idzko et al., 2013).

Similar to CD39, the proportions of CD73⁺ T_regs among all T_regs, as well as CD73 mRNA expression in CD4⁺ T cells was lower in asthmatic patients than in healthy subjects (Wang et al., 2013). The role of CD73 in regulating asthma remains to be determined.