Therapeutic Targeting of Hypoxia-A2-Adenosinergic Pathway in COVID-19 Patients.

The hypoxia-hypoxia-inducible factor (HIF)-1α-A2-adenosinergic pathway protects tissues from inflammatory damage during antipathogen immune responses. The elimination of this physiological tissue-protecting mechanism by supplemental oxygenation may contribute to the high mortality of oxygen-ventilated COVID-19 patients by exacerbating inflammatory lung damage. Restoration of this pathway with hypoxia-adenosinergic drugs may improve outcomes in these patients.

Introduction

Hypoxia-hypoxia-inducible factor (HIF)-1α and A2-adenosinergic signaling in immune cells is a physiological delayed negative-feedback mechanism that protects tissues from collateral damage during antipathogen immune responses (1–5). Recent attention has focused on the role of this immunosuppressive pathway in protecting cancerous tissues by inhibiting the antitumor immune response (6–11). Indeed, promising clinical data targeting the hypoxia-HIF-1α-A2-adenosinergic axis are beginning to emerge (7). However, here we return to the original discovery of this immunosuppressive pathway and its critical role in dampening the flames of inflammation to prevent tissue damage (1). The elimination of this physiological tissue-protecting mechanism may contribute to dire consequences in the context of COVID-19 patients. Alarmingly, the oxygenation of patients with acute lung inflammation, although necessary, may unintentionally exacerbate inflammatory lung damage because of the loss of the hypoxia-driven and adenosine-mediated immunosuppression and protection of still healthy lung tissue. Engagement of this hypoxia-triggered pathway by drugs such as adenosine ligands of A2A and A2B adenosine receptors may prevent the exacerbation of inflammatory lung damage and thereby improve outcomes in oxygen-ventilated COVID-19 patients.

Physiological Hypoxia-A2-Adenosinergic Pathway Inhibits Antipathogen and Antitumor Immunity

Adenosine mediates its effects through four subtypes of adenosine receptors: A1, A2A, A2B, and A3 (12, 13). The high-affinity A1 receptor (A1R) and low-affinity A3 receptor (A3R) are Gi protein coupled, whereas the high-affinity A2A receptor (A2AR) and low-affinity A2B receptor (A2BR) are cAMP-elevating Gs protein-coupled receptors. Initially, adenosine signaling was of clinical interest because of its heart rate-reducing properties in which intravenous administration of adenosine lessened atrioventricular nodal conduction (14). This was later attributed to signaling via A1R, as genetic deletion of this receptor subtype led to elimination of the adenosine-induced bradycardia (15). Thus, previous clinical use of adenosine has centered on the treatment of patients with supraventricular tachycardia because of its effects on heart rate and blood pressure via A1R (15). However, it was landmark studies by Sitkovsky that established the critical and nonredundant role of the A2AR-mediated pathway of immunosuppression in protecting tissues from collateral damage by downregulating the immune response (1). It was shown that extracellular adenosine signaling through A2AR increased levels of immunosuppressive cyclic AMP in immune cells and attenuated inflammation and tissue damage in vivo (1). Observations of this conserved immunosuppressive pathway were consistent among several different animal models of inflammation including viral fulminant hepatis and bacterial endotoxin-induced septic shock (1, 3, 16). These genetic and pharmacological studies established that A2AR signaling is an essential physiological negative-feedback mechanism that terminates tissue-specific and systemic inflammatory responses to protect host tissues.

Work by Ohta and Sitkovsky also demonstrated that A2AR protects cancerous tissues by inhibiting antitumor T cells. Once again, both genetic (A2AR gene deletion) and pharmacological (A2 antagonists and siRNA) approaches were employed to show that blockade of A2AR signaling promoted tumor rejection and prevented neovascularization in a T cell-autonomous manner (6). These studies were the first to propose and show that 1) the adenosine-rich tumor microenvironment (TME) inhibited antitumor T cells via the A2AR and that 2) targeting the hypoxia → adenosine → A2AR pathway is an important strategy to improve cancer immunotherapy (6).

Supplemental Oxygen Disrupts the Hypoxia-A2-Adenosinergic Anti-Inflammatory Pathway

Insights from the studies above enabled Sitkovsky and Thiel to predict the role of the hypoxia-adenosinergic pathway in protecting tissues from inflammatory damage during acute respiratory distress syndrome (ARDS) (17). It was hypothesized that administration of oxygen to ARDS patients with pulmonary inflammation could cause severe side effects and may contribute to patient mortality. ARDS often leads to supportive therapy by intubation and mechanical ventilation with supplemental high oxygen concentrations. Although oxygen therapy is a life-saving measure, these studies demonstrated that oxygenation eliminated the physiological tissue-protecting hypoxia-A2-adenosinergic pathway and exacerbated acute inflammatory lung injury (17). Preclinical studies showed that oxygenation (60% or 100% O2) increased alveolocapillary permeability and caused severe lung gas exchange impairment in murine models of polymicrobial-induced acute lung injury (17). Counterintuitively, oxygenation decreased the arterial oxygen partial pressure in the periphery to near-death levels because of inflammatory lung tissue damage and was accompanied by a decrease in extracellular adenosine (17).

Of note, five times more mice with inflamed lungs died after oxygenation compared with those breathing ambient 21% O2. These studies also provided evidence of the life-saving intervention by administration of an A2AR agonist to restore the adenosine-A2AR axis during oxygenation. Treatment with the A2AR agonist CGS21680 rescued the majority of mice from oxygenation-induced death in a severe model of bacteria-induced ARDS (17). Additional mechanistic evidence of the protective effects of A2AR agonist demonstrated that treatment with CGS21680 resulted in 1) decreased polymorphonuclear leukocyte (PMN) infiltration into the lung, 2) decreased levels of reactive oxygen metabolites, 3) decreased lung vascular permeability, 4) significantly improved lung gas exchange, and 5) prevention of oxygenation-induced inflammatory lung damage (17). These studies were the first to suggest that the hypoxia-adenosinergic mechanism may be an iatrogenic complication of oxygen therapy during acute lung inflammation and discuss the need of clinical studies to address this issue. Here, it was suggested that oxygen therapy in patients with ARDS should be combined with inhalation of A2AR agonists to induce the lung tissue-protecting hypoxia-A2-adenosinergic pathway.

Importantly, signaling through the A2BR has also been shown to dampen acute lung injury and exhibit a protective role during ARDS (18, 19). Through sophisticated genetic and pharmacological approaches, Eltzschig’s group revealed that A2BR signaling in the alveolar epithelium contributes to lung protection, suggesting that A2BR agonists may be effective in the treatment of acute lung injury (20). The expression of A2BR on vasculature and its role in barrier protection may be especially important in diseases that lead to vascular disruption, including COVID-19. A2BR may also be important in the regulation of immune cell migration. It was recently shown that inhibitors of CXCR4 and CXCR7 attenuate acute pulmonary inflammation via A2BR on blood cells (21). Parallel studies demonstrated an induction of A2BR following ventilator-induced lung injury that was shown to be dependent on HIF-1α (22). These and other studies suggest that hypoxia/HIF-1α signaling may not only contribute to levels of extracellular adenosine by increasing the adenosine-generating enzyme CD73 but also increase the expression of adenosine receptors themselves, promoting a feedback loop that restrains inflammation (22–25). Other key studies by Blackburn’s group have demonstrated that hyperoxic lung injury (95% O2) alone can also increase A2BR expression (26). It seems possible that the pulmonary inflammation, damage to tissue and endothelium, and edema associated with hyperoxic lung injury may also promote the HIF-1-A2BR axis. However, it is noteworthy to mention the complexities associated with HIF signaling due to its critical role in cell metabolism. Recent work has also shown that SARS-CoV-2 infection stimulates reactive oxygen species (ROS) production that stabilizes HIF-1α (27). The subsequent metabolic changes in monocytes were shown to inhibit T cells as well as the survival of epithelial cells (27). Ongoing preclinical and clinical studies may clarify the complex role of HIFs in the pathogenesis of COVID-19.

Other studies by Choukèr and colleagues showed that hyperoxia could also worsen hepatic injury. In models of hepatic ischemia-reperfusion injury (a morbidity risk factor after liver surgery), postoperative hyperoxic treatment increased tissue damage that was dependent on PMNs (28). Supplemental oxygenation enhanced immune cell-mediated oxidative burst and production of reactive oxygen species (28). These studies also highlight the need for clinical evaluation of the effects of high-oxygen therapy in multiple organs and strategies to avoid the associated inflammatory tissue damage.

Recently, our team also demonstrated the proinflammatory effects of oxygenation in the context of the antitumor immune response (29–31). It was shown that through the reversal of hypoxia, oxygenation depleted many powerful biochemical and immunological negative regulators that are recruited by hypoxia and adenosine (29, 30). This, in turn, unleashed the cytotoxic capacities of activated immune cells to inflict inflammatory damage to surrounding tissues. Our novel motivation to use oxygen to eliminate hypoxia in the tumor microenvironment (TME) was to prevent hypoxia/HIF-1α-mediated immune suppression and inhibit the hypoxia-driven accumulation of immunosuppressive extracellular adenosine and A2AR signaling in antitumor immune cells. This was hypothesized to weaken the upstream stage of the hypoxia-A2-adenosinergic axis of immunosuppression, thereby enhancing antitumor immunity.

This was confirmed by proteomics analyses of the TME showing that respiratory hyperoxia (60% O2) 1) decreased tumor hypoxia, HIF-1α, and HIF-1α gene targets and 2) inhibited the adenosine pathway by reducing adenosine-generating enzymes CD39/CD73, levels of extracellular adenosine, and A2AR/A2BR expression in the TME (30). Systemic oxygenation also reprogrammed the TME toward immunopermission by promoting infiltration of tumor-reactive T cells and enhancing inflammatory cytokines and chemokines, while decreasing immunosuppressive factors [e.g., transforming growth factor (TGF)-β, cytotoxic T lymphocyte-associated protein (CTLA)-4, regulatory T cells (Tregs)] (29). This, in turn, promoted tumor regression and prolonged survival of lung tumor-bearing mice and was shown to be dependent on T cells and natural killer (NK) cells (29).

Oxygenation-Mediated Interruption of Natural Anti-Inflammatory Mechanisms May Contribute to High Lethality of ARDS

Even before COVID-19, the high mortality of oxygenated ARDS patients in the ICU was a major problem. An estimated 190,600 ARDS cases occur each year in the United States, with 74,500 of these cases associated with deaths (32). This nearly 50% mortality rate suggests a paucity of effective methods to treat ARDS patients in the ICU. This alarming—and growing—statistic was exposed by COVID-19. ARDS is life-threatening because of poor oxygenation, pulmonary infiltrates, and severe inflammation (33). Patients are diagnosed with ARDS when the ratio of oxygen in arterial blood () to the fraction of oxygen in inspired air () is <300 (/ < 300). This can occur when pathogenesis of infectious disease, including COVID-19, is exacerbated by overactive immune responses. Immune cells inflict collateral tissue damage via production of abnormally high levels of proinflammatory molecules (34). Indeed, pneumonia, either community acquired or nosocomial, is the third most frequent cause of death among all other lethal pulmonary diseases. Regardless of the origin, ARDS typically results in the destruction of the pulmonary architecture by uncontrolled inflammatory responses (34, 35) mediated by a complex interplay of cellular effectors, including myeloid cells, NK cells, natural killer T (NKT) cells, T cells, and Tregs (35). T cells, in particular, have been reported to be critical for early proinflammatory cytokine gene expression during murine Klebsiella pneumoniae infection (35). Cytokines (IL-1β, TNF-α) and chemokines contribute to the exacerbation of lung inflammation and damage (36). Thus, the unacceptably high lethality of ARDS may be due to oxygenation unintentionally disrupting natural anti-inflammatory mechanisms and exacerbating disease pathogenesis.

Engaging the Hypoxia-HIF-1α-Adenosine-A2R Tissue-Protecting Mechanism in COVID-19 ARDS Patients

Insights from the studies above may have important implications for decreasing the mortality of severely ill COVID-19 patients. SARS-CoV-2 infection triggers severe inflammation, pneumonia, and ARDS (37). COVID-19 patients with interstitial pneumonitis often develop severe lung inflammation that leads to invasive ventilation and hyperoxic breathing with often fatal outcomes (38). This high mortality rate is impacted by uncontrolled release of inflammatory cytokines and mediators as well as excessive tissue and microvascular damage (39–44), thereby increasing hypoxia. In severe cases, patients become hypoxemic and receive mechanical ventilation.

Supplemental oxygenation given in the ICU may enhance inflammation and contribute to the poor clinical outcomes of severely hypoxemic COVID-19 patients. Oxygenation, although necessary and unavoidable, eliminates the tissue hypoxia-HIF-1α-driven and adenosine-A2AR-mediated anti-inflammatory mechanism (FIGURE 1). This powerful immunosuppressive pathway also recruits many other biochemical and immunological negative regulators (PD-1, CTLA-4, LAG-3, TGF-β, IL-10, Tregs) (29, 45–48). When this tissue-protecting mechanism is eliminated by oxygenation, immune cells are no longer inhibited and are unleashed to destroy remaining healthy lung tissue. In addition, hyperoxia may also alter the pulmonary vasculature and alveolar barrier function and have long-term effects on the lung parenchyma of COVID-19 patients (49).

FIGURE 1.

A2A receptor (A2AR)/A2B receptor (A2BR) agonists prevent oxygen-mediated exacerbation of inflammatory damage and death during acute respiratory distress syndrome (ARDS) Oxygen eliminates lung tissue-protecting hypoxia and extracellular adenosine that signals through cAMP-elevating A2AR/A2BR to downregulate inflammation. Treatment with A2 agonists restarts hypoxia-adenosinergic immune suppression, allowing for safe oxygen therapy and ARDS patient survival. HIF, hypoxia-inducing factor; TLR, toll-like receptor.

The COVID-19 pandemic has again attracted attention to the 15-yr-old study by Sitkovsky and colleagues (17). In this study, it was predicted that the oxygenation-associated loss of hypoxia-driven protection of acutely inflamed lungs in preclinical ARDS models contributes to deadly outcomes. Thus, it is the otherwise life-saving hyperoxic breathing that leads to the loss of the tissue-protecting hypoxia and extracellular adenosine-A2AR signaling. This, in turn, exacerbates the acute inflammatory lung injury. This initial discovery was confirmed and extended by others demonstrating that oxygenation unleashes not only lung neutrophils but also macrophages and pulmonary NKT cells (50, 51).

This study also offered a therapeutic solution to the iatrogenic complication. It was shown that pharmacological compensation for the oxygenation-mediated loss of naturally generated adenosine in inflamed lungs could prevent the deadly outcomes. This approach is now of great interest to intensivists in the ICU to treat COVID-19 patients since it may solve this pathophysiological dilemma. Clinical use of drugs to induce the anti-inflammatory A2-adenosinergic pathway may allow for the life-saving benefits of oxygenation while protecting lungs from exacerbation of inflammatory injury by oxygen-induced hyperactive immune cells.

Therapeutic Countermeasures That Can Be Immediately Tested With COVID-19 Patients

We, and others, propose that oxygen ventilation of COVID-19 patients with ARDS should be combined with hypoxia-A2 adenosinergic drugs to prevent the exacerbation of acute inflammation and death. Since the pathogenesis of the exacerbation of inflammation by oxygenation may be due to the loss of hypoxia-A2-adenosinergic signaling, therapeutic pharmacological engagement of this immunosuppression can be accomplished by 1) intratracheal administration of adenosine or synthetic agonists of A2AR/A2BR (17, 52, 53), 2) recombinant CD39 and CD73, which generate extracellular adenosine, 3) drugs that target adenosine-modifying enzymes [e.g., inhibitors of adenosine deaminase (ADA), adenosine kinase (ADK), equilibrative nucleoside transporters (ENTs)], or 4) drugs that stabilize hypoxia-inducible factors. In favor of the feasibility of these interventions, proof of principle was provided by the demonstration that the exacerbation of inflammation by supplemental oxygen was prevented by intratracheal inhalation of a selective A2AR agonist (17). Indeed, the most straightforward approach may be to combine oxygen ventilation of COVID-19 ARDS patients with inhalation of adenosine or A2R agonists.

Importantly, pharmacological interventions targeting any of the stages of the hypoxia-HIF-1α-adenosine-A2R pathway may restart this anti-inflammatory tissue-protecting mechanism. This notion was recently highlighted in a review by Geiger et al. (54) suggesting that adenosine-modifying enzymes (ADA, ADK, and ENT) may play a critical role in the pathogenesis of COVID-19 in at least three distinct mechanisms: 1) ADK and ADA increase extracellular adenosine levels preventing excessive inflammation via A2AR/A2BR; 2) ADA may compete with SARS-CoV-2 for binding to the ADA receptor (CD26) that can be used for cell entry; and 3) blocking the adenosine transporter ENT prevents intracellular transport of adenosine and may promote extracellular adenosine-mediated inhibition of platelet activation and thrombosis. This concept has been demonstrated preclinically with the ENT inhibitor dipyridamole, which was shown to promote adenosine-mediated lung protection (55). Interestingly, the attenuation of lung inflammation caused by dipyridamole was abrogated in mice with alveolar epithelial A2BR gene deletion (55). Additional support may be derived from the recent observation that individuals with cystic fibrosis, which is associated with excess levels of ATP and adenosine, have improved COVID-19 survival (56).

Finally, stabilization of HIF-1α signaling or enhancing activity of adenosine-generating enzymes such as CD73/CD39 may also be a therapeutic maneuver to improve the outcomes of COVID-19 patients. HIF-1α lies upstream of the hypoxia-adenosinergic pathway by increasing levels of enzymes that generate extracellular adenosine, which signal through immunosuppressive A2AR/A2BR. In addition, HIF-mediated regulation of alveolar-epithelial glucose metabolism has also been shown to suppress lung inflammation (25). Recently, a phase II clinical study (NCT04478071) led by Dr. Eltzschig and Dr. Bobrow is underway to evaluate the efficacy of vadadustat, a HIF-1α activator, in the prevention and treatment of COVID-19 patients. Recent work by the same group has also shown that activation of HIF-2α may also attenuate myocardial ischemia-reperfusion injury (57). Thus, HIF-2α may represent yet another pathway to dampen myocardial infarction and other coronary artery disease issues associated with COVID-19.

Clinical Evidence Supporting the Use of Anti-Hypoxia-Adenosinergic Drugs With COVID-19 ARDS Patients

Preclinical data (17) and their confirmations (50, 51), coupled with limited methods to treat COVID-19 patients, provided the justification for clinical testing of this approach (58, 59). Recently, a team of clinicians in Italy led by Dr. Correale treated 14 patients with COVID-19-related interstitial pneumonitis with off-label inhaled adenosine to restart A2AR/A2BR-mediated lung protection and prevent inflammatory lung tissue damage. Remarkably, 13 patients had accelerated clinical benefit from adenosine treatment with decrease in symptoms and improved -to- ratio and respiratory symptoms (58). Eight patients did not require further oxygen administration and could be released from the hospital within 1 wk from the beginning of adenosine treatment (58). In addition, a radiological response was demonstrated in 7 patients who received adenosine, and SARS-CoV-2 load rapidly decreased in 13 patients within 7 days (58). Such positive clinical changes were not observed in historical control subjects. Although the number of patients in this study was limited, the safety and therapeutic benefit of inhaled adenosine in severe COVID-19 patients warrants additional investigation in well-controlled clinical trials. Indeed, inhaled adenosine, or adenosine receptor agonists, may be the first treatment for COVID-19 aimed to prevent oxygen-mediated inflammatory damage to 1) rapidly improve clinical benefit and reduce mortality and 2) promote antiviral activity in severely ill patients (59). Currently these studies are informing the design of a phase II clinical trial led by Dr. Spiess at the University of Florida to treat ARDS COVID-19 patients with aerosolized inhaled adenosine (NCT04588441).

Concluding Remarks

The advance in understanding of the mechanisms of inflammatory damage of oxygenated/ventilated ARDS patients and the novel approach described here may be important to improve outcomes of not only COVID-19 patients but also patients with ARDS of different etiologies. This is emphasized by reports that half of COVID-19 nonsurvivors also experience a secondary infection, including signs of pneumonia or bacteremia, and a positive culture of a new pathogen (60). Thus, bacterial pneumonia-induced (or enhanced) ARDS may also be a major contributor to COVID-19 patient mortality, which could be prevented by engaging the hypoxia-A2-adenosinergic lung tissue-protecting mechanism. Although vaccination is expected to reduce COVID-19 cases, SARS-CoV-2 is not likely to disappear. In addition, new strains, or the next viral pandemic, may exhibit similar pathogenesis and ARDS-related symptoms. Thus, the treatment of ARDS and oxygenation/ventilation protocols require a better understanding of underlying molecular mechanisms that regulate inflammation and disease pathogenesis.