The antihypoxia–adenosinergic pathogenesis as a result of collateral damage by overactive immune cells

Short abstract

The consumption of natural A2A adenosine receptor antagonists, such as caffeine, as well as the use of supplemental oxygen during acute inflammation episodes may have undesirable effects.

Abstract

Here, we attract attention to the possibility of iatrogenic exacerbation of immune–mediated tissue damage as a result of the unintended weakening of the tissue–protecting, hypoxia–adenosinergic pathway. These immunosuppressive, anti–inflammatory pathways play a critical and nonredundant role in the protection of normal tissues from collateral damage during an inflammatory response. We believe that it is the tissue hypoxia associated with inflammatory damage that leads to local inhibition of overactive immune cells by activating A2AR and A2BR and stabilizing HIF–1α. We show in an animal model of acute lung injury that oxygenation (i.e., inspiring supplemental oxygen) reverses tissue hypoxia and exacerbates ongoing inflammatory lung tissue damage. However, little has been done to carefully investigate and prevent this in a clinical setting. Similarly, the consumption of caffeine antagonizes A2ARs, resulting in exacerbation of ongoing acute inflammation. It is suggested that although the elimination of hypoxia–adenosinergic immunosuppression is desirable to improve vaccines, it is important to take into account the unintentional effects of supplemental oxygen and caffeine, which may increase collateral, inflammatory tissue damage.

Abbreviations

A2AR/A2BR

A2A and A2B adenosine receptors,

AMP

adenosine monophosphate,

ARDS

acute respiratory distress syndrome,

HIF–1α

hypoxia–inducible factor–1α,

PMN

polymorphonuclear neutrophil

Introduction

The etiology of autoimmune diseases is often unclear, but it is understood that the balance of proinflammatory and anti–inflammatory mechanisms may play an important role in rheumatoid arthritis, multiple sclerosis, and other diseases, where pathogenesis could be traced to dysregulated proinflammatory cytokines. It is important to better understand the regulation of inflammatory processes and the mechanisms that down–regulate inflammation, which are a result of the inter–related and cooperative effects of immunological and physiological, negative regulators of the immune response.

The development of an immune response against pathogens and tumor cells is controlled by several important “checkpoints”, where there is additional stimulation or down–regulation of the immune response by negative regulators such as CTLA–4 and T regulatory cells [ ¹ ]. Here, we will review the clinical implications of our studies of the immunosuppressive effects of the hypoxia–adenosinergic pathway, as an example of a potent physiological, negative regulator of the immune response [ ² , ³ ]. We will emphasize the under–appreciated dangers of unintentionally eliminating important physiological, negative regulators of the immune response.

THE HYPOXIA–ADENOSINERGIC IMMUNOSUPPRESSIVE MECHANISM THAT PROTECTS TISSUES OF VITAL ORGANS FROM EXCESSIVE COLLATERAL DAMAGE

The hypoxia–adenosinergic immunosuppressive pathways are likely to have been developed to protect tissues from excessive collateral damage by overactive immune cells during the antipathogen immune response. Indeed, the inflammatory response to pathogens is often accompanied by significant damage to innocent bystander cells. Of interest, as part of the normal host response to infection, it has been proposed that in addition to danger signal 1, which triggers an antipathogen immune response, there exists a danger signal 2 to indicate the need to down–regulate or to stop the immune response and minimize collateral damage to normal tissues [ ⁴ ].

This suggested the existence of molecular sensors that trigger immunosuppression, thereby enabling the fine balance between proinflammatory and anti–inflammatory processes and to ensure that the majority of humans survives infections without permanent loss of vital organ function [ ⁴ ]. We believe it is the collateral damage to blood vessels that is the initial event that triggers the hypoxia–adenosinergic immunosuppression as a result of the diminished capacity to deliver oxygen in local inflamed tissue microenvironments. The ensuing low–oxygen tension (i.e., hypoxia) in local tissues leads to the accumulation of extracellular adenosine. In turn, extracellular adenosine stimulates A2AR/A2BR, elevating the immunosuppressive, intracellular second messenger cAMP, which inhibits overactive immune cells [ ³ , ⁵ ]. Hypoxia also leads to the stabilization of HIF–1α, which we have shown to cooperate with A2AR in the inhibition of activated T cells [ ³ , ⁶ , ⁷ , ⁸ ]. Specifically, we proposed that overactive T cells are down–regulated by the combined action of immunosuppressive A2AR [ ⁹ , ¹⁰ , ¹¹ ] and HIF–1α [ ⁹ ], which is considered to function as the “master regulator of oxygen homeostasis” [ ⁹ , ¹² , ¹³ , ¹⁴ ]. A2AR/A2BR and HIF–1α in T cells may act in concert [ ³ ]. Recent studies have provided more evidence that HIF–1α and adenosine receptors cooperate in inhibiting activated T cells.

Of interest is the recent finding that HIF–1α–dependent induction of netrin–1 prevented neutrophil transmigration in hypoxic epithelia, which was shown to be dependent on A2BRs [ ¹⁵ ]. Decreased neutrophil trafficking and the attenuation of inflammation by netrin–1 were not seen in A2BR–deficient mice, suggesting HIF–1α and adenosine receptors may work together to mitigate inflammatory effects during hypoxia [ ¹⁵ ].

HYPOXIA AND INTRACELLULAR SOURCES OF EXTRACELLULAR ADENOSINE

This mechanism may be triggered by local tissue hypoxia that follows excessive collateral immune damage to endothelial cells and microcirculation with ensuing interruption of normal blood and oxygen supply. Hypoxia is associated with a decrease in intracellular ATP; an increase in intracellular AMP; the inhibition of adenosine kinase [ ¹⁶ ]; the accumulation of intracellular adenosine [ ¹⁶ ]; and subsequent transport or diffusion of intracellular adenosine and the resulting accumulation of adenosine in the extracellular space. Recently, transcription factor–binding assays and HIF–1α loss– and gain–of–function studies have suggested that HIF–1α plays a role in the transcriptional repression of adenosine kinase, a critical enzyme in adenosine modulation [ ¹⁷ , ¹⁸ ].

HYPOXIA AND EXTRACELLULAR SOURCES OF EXTRACELLULAR ADENOSINE

The hypoxia–triggered ecto–enzymes CD39 (ATPase/ADPase) and CD73 (5′–nucleotidase) represent another important pathway in the generation of extracellular adenosine [ ¹⁹ , ²⁰ , ²¹ ]. It is likely that nucleotides released into the extracellular space are phosphohydrolyzed by CD39 (which converts ATP/ADP to AMP) in tandem with CD73 (which converts AMP to adenosine). Studies using CD39 and CD73 null mice concluded that these enzymes serve as critical control points for endogenous adenosine generation. These studies also implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation. Furthermore, the hypoxia–induced ecto–nucleotidase cascade has been shown in studies of CD39 and CD73 gene–altered mice to be critical in adenosine accumulation [ ¹⁹ , ²⁰ , ²¹ ].

FOUR DIFFERENT ADENOSINE RECEPTORS (A1, A2A, A2B, AND A3)

There are four subtypes of adenosine receptors. The high–affinity A1R and low–affinity A3R are inhibitory G protein–coupled, and the A2Rs are subdivided into the high–affinity A2AR and low–affinity A2BR and are stimulatory G protein–coupled [ ²² , ²³ , ²⁴ , ²⁵ ]. It has been well established that adenosine receptors are immunosuppressive when activated pharmacologically [ ²⁴ , ²⁶ , ²⁷ ]. CD8+ and CD4+ T cells express A2AR/A2BR predominantly [ ²⁷ ]. Signaling through A2AR or A2BR in T cells results in the intracellular accumulation of cAMP and the subsequent inhibition of the TCR–triggered activation of T cells [ ²⁸ , ²⁹ , ³⁰ , ³¹ ] and of many effector functions, including proliferation, expansion, and secretion of important proinflammatory cytokines such as IFN–γ [ ¹⁰ , ³¹ , ³² ] and TNF–α [ ³³ ].

HIF–1α

HIF–1α is a subject of great interest in cancer research [ ³⁴ , ³⁵ ] but of relevance to antibacterial immunity as well. In this regard, we have established that HIF–1α levels in T cells increase, not only after exposure to hypoxia but also through antigen receptor (TCR)–triggered activation of T cells [ ⁶ ]. This suggests that activated T cells may be inhibited by the accumulation of HIF–1α, even after exiting hypoxic tissue areas. Therefore, the stabilization of HIF–1α in activated and proliferating T cells not only ensures sufficient energy supply by switching to glycolysis, but it also inhibits the production of important proinflammatory cytokines such as IFN–γ [ ⁷ ].

HIF–1α IS A NEGATIVE REGULATOR OF T CELLS

Our interest in HIF–1α was prompted by observations of inhibition that T cells are inhibited by low oxygen tension [ ³⁶ ]. This led us to ask whether it was because of HIF–1α activities and if so, whether HIF–1α may suppress effector T cells during immune attack in hypoxic tissues. Our experiments using recombination activating gene 2/blastocyst complementation chimeras [ ⁹ ] were the first to indicate the immunosuppressive and tissue–protecting functions of HIF–1α. This conceptually novel view of HIF–1α has been confirmed further and extended in in vitro studies using tissue–specific deletion of the HIF–1α gene in T cells [ ⁷ ]. We show that T cells lacking HIF–1α (Lck–Cre/LoxP system) have increased TCR–triggered production of cytokines such as IFN–γ. The negative role of HIF–1α in the regulation of activated T cells was also supported by in vivo studies of T cells in hypoxic–inflamed areas [ ⁸ ]. Thus, by inhibiting HIF–1α, we expect to accomplish better antibacterial immunity, as we observed a stronger T cell response and antimicrobial immunity when HIF–1α was knocked down [ ⁸ ].

POSSIBLE CONSEQUENCES OF THE UNINTENDED ELIMINATION OF THE HYPOXIA–ADENOSINERGIC PATHWAY

It was not appreciated sufficiently that local tissue hypoxia caused by inflammatory damage may be beneficial in preventing a prolonged inflammatory assault on healthy tissues by triggering the hypoxia–adenosinergic pathway. For example, the elimination of hypoxia by inspiring high oxygen–containing gas mixtures and/or the weakening of A2AR signaling as a result of pharmacological antagonism by synthetic drugs or natural compounds such as 1,3,7 trimethylxanthine (caffeine) may relieve immunosuppression, resulting in enhanced immunity and exacerbated inflammatory damage. The most alarming feature is that this tissue–protecting mechanism can be weakened easily by the inhalation of oxygen gas mixtures higher than 21%, as is the case in hospitals, where patients receive supplemental oxygen. The consumption of caffeine–containing drugs and/or recreational beverages during episodes of acute inflammation can also manipulate this tissue–protecting mechanism, exacerbating the inflammatory damage.

The potentially dangerous effects of inspiring high oxygen–containing gas mixtures have been demonstrated in animal studies that model ARDS. Patients with ARDS usually require symptomatic, supportive therapy by inspiring supplemental high–oxygen concentrations. It was demonstrated that oxygenation not only does, as intended, eliminate the tissue hypoxia, but it also may weaken the local tissue hypoxia–driven and A2AR–mediated anti–inflammatory mechanism. This was shown to exacerbate inflammatory lung injury further. Comparison of wild–type and A2AR–deficient mice confirmed that the elimination of lung tissue hypoxia by oxygenation was accompanied by the loss of hypoxia–driven extracellular adenosine accumulation and the weakening of immunosuppressive A2AR signaling in lung neutrophils. Treatment with selective A2A antagonists was also sufficient to increase PMN infiltration and protein leakage in a model of LPS–induced lung injury. This, in turn, resulted in excessive damage to lung tissues, impaired lung function, and accelerated death [ ³⁷ ]. Conversely, hypoxia was shown to down–regulate neutrophil activity and accumulation, thereby protecting lung tissue from inflammatory damage [ ³⁷ ]. As the molecular mechanism of this potential iatrogenic complication is known, the authors offered simple preventative measures. In patients that must receive supplemental oxygen, it may be possible to compensate for the oxygen–associated loss of extracellular adenosine in inflamed lung tissues by inhalation of a selective A2AR agonist. This drug prevented the exacerbation of inflammation by oxygen in mice completely [ ³⁷ ]. Such studies suggest that antihypoxia–adenosinergic exacerbation of inflammatory lung damage may be important in attracting attention to the possibility of a “doctor–cure–made” pathogenesis. Additionally, there is a possibility of antihypoxia–adenosinergic disease exacerbation as a result of food/drink consumption or “over–the–counter” drugs.

The behavioral drug caffeine is so widely consumed, as its psychoactive effects are mediated largely by its antagonistic action on A2AR in the brain [ ³⁸ , ³⁹ ]. Experiments to test whether the caffeine consumed during an acute inflammation episode would lead to the exacerbation of immune–mediated tissue damage established that caffeine at lower doses (10 and 20 mg/kg) strongly increased acute liver damage and increased levels of proinflammatory cytokines in wild–type mice but not in A2AR–deficient mice [ ⁴⁰ ]. This confirmed that the exacerbation of liver inflammation was a result of caffeine–mediated antagonism of the A2AR–mediated tissue–protecting mechanism. Interestingly, caffeine increased liver damage even when consumed chronically, but caffeine exacerbated liver damage more strongly in “naïve” mice, which had never consumed caffeine.

CONCLUSION

The pharmacological recruitment of the life–saving, hypoxia–adenosinergic immunosuppressive pathway could be beneficial when the goal is to prevent inflammatory tissue damage during pathogenesis of autoimmune diseases or endotoxemia [ ⁵ , ⁴¹ ]. Conversely, this very same mechanism should be eliminated when it protects cancerous tissues [ ⁴² ] or prevents the development of an antipathogen immune response [ ⁸ , ⁴³ ]. Importantly, the use of drugs in different clinical applications should be re–evaluated if the drug under consideration may interfere with the hypoxia–adenosinergic mechanism. Indeed, we described here the exacerbation of inflammation after the weakening or elimination of the hypoxia–adenosinergic mechanism as a result of the delivery of oxygen to tissues [ ³⁷ ] or when food or drinks may contain A2AR antagonists [ ⁴⁰ ].