Critically ill patients are routinely exposed to high concentrations of supplemental oxygen for prolonged periods of time, which can be life-saving in the short term, but such exposure also causes severe lung injury and increases mortality. To address this therapeutic dilemma, we studied the mechanisms of the tissue-damaging effects of oxygen in mice. We show that pulmonary invariant natural killer T (iNKT) cells are unexpectedly crucial in the development of acute oxygen-induced lung injury. iNKT cells express high concentrations of the ectonucleotidase CD39, which regulates their state of activation. Both iNKT cell–deficient (Jα18−/−) and CD39-null mice tolerate hyperoxia, compared with wild-type control mice that exhibit severe lung injury. An adoptive transfer of wild-type iNKT cells into Jα18−/− mice results in hyperoxic lung injury, whereas the transfer of CD39-null iNKT cells does not. Pulmonary iNKT cell activation and proliferation are modulated by ATP-dependent purinergic signaling responses. Hyperoxic lung injury can be induced by selective P2X7-receptor blockade in CD39-null mice. Our data indicate that iNKT cells are involved in the pathogenesis of hyperoxic lung injury, and that tissue protection can be mediated through ATP-induced P2X7 receptor signaling, resulting in iNKT cell death. In conclusion, our data suggest that iNKT cells and purinergic signaling should be evaluated as potential novel therapeutic targets to prevent hyperoxic lung injury.
lung injury; immunology; ATP; P2X7; oxygen
Hypoxia-adenosinergic suppression and re-direction of the immune response has been implicated in the regulation of anti-pathogen and anti-tumor immunity, with Hypoxia-inducible factor 1α (HIF-1α) playing a major role. In this study, we investigated the role of isoform I.1, a quantitatively minor alternative isoform of HIF-1α, in anti-bacterial immunity and sepsis survival. By using the cecal ligation and puncture model of bacterial peritonitis we studied the function of I.1 isoform in T cells using mice with total I.1-isoform deficiency and mice with T cell-targeted I.1 knockdown. We found that genetic deletion of the I.1 isoform resulted in enhanced resistance to septic lethality, significantly reduced bacterial load in peripheral blood, increased M1 macrophage polarization, augmented levels of pro-inflammatory cytokines in serum, and significantly decreased levels of the anti-inflammatory cytokine IL-10. Our data suggest an immunosuppressive role of the I.1 isoform in T cells during bacterial sepsis that was previously unrecognized. We interpret these data as indicative that activation-inducible isoform I.1 hinders the contribution of T cells to the anti-bacterial response by affecting M1/M2 macrophage polarization and microbicidal function.
Animal models; Hypoxia-inducible Factor; Sepsis; T lymphocytes
The recent approval by the FDA of cancer vaccines and drugs that blockade immunological negative regulators has further enhanced interest in promising approaches of the immunotherapy of cancer. However, the disappointingly short life extension has also underscored the need to better understand the mechanisms that prevent tumor rejection and survival even after the blockade of immunological negative regulators. Here, we describe the implications of the “metabolism-based” immunosuppressive mechanism, where the local tissue hypoxia-driven accumulation of extracellular adenosine triggers suppression via A2 adenosine receptors on the surface of activated immune cells. This molecular pathway is of critical importance in mechanisms of immunosuppression in inflamed and cancerous tissue microenvironments. The protection of tumors by tumor-generated extracellular adenosine and A2 adenosine receptors could be the misguided application of the normal tissue-protecting mechanism that limits excessive collateral damage to vital organs during the anti-pathogen immune response. The overview of the current state of the art regarding the immunosuppressive effects of extracellular adenosine is followed by an historical perspective of studies focused on the elucidation of the physiological negative regulators that protect tissues of vital organs from excessive collateral damage, but, as a trade-off, may also weaken the anti-pathogen effector functions and negate the attempts of anti-tumor immune cells to destroy cancerous cells.
adenosine; A2A adenosine receptor; cyclic AMP; hypoxia; inflammation; tumor; cancer immunotherapy; adoptive immunotherapy; tumor microenvironment; immunosuppression; T lymphocytes; regulatory T cells; cytokines; cytotoxicity
Extracellular adenosine-dependent suppression and redirection of pro-inflammatory activities are mediated by the signaling through adenosine receptors on the surface of most immune cells. The immunosuppression by endogenously-produced adenosine is pathophysiologically significant since inactivation of A2A/A2B adenosine receptor (A2AR/A2BR) and adenosine-producing ecto-enzymes CD39/CD73 results in the higher intensity of immune response and exaggeration of inflammatory damage. Regulatory T cells (Treg) can generate extracellular adenosine, which is implicated in the immunoregulatory activity of Tregs. Interestingly, adenosine has been shown to increase the numbers of Tregs and further promotes their immunoregulatory activity. A2AR-deficiency in Tregs reduces their immunosuppressive efficacy in vivo. Thus, adenosine is not only directly and instantly inhibiting to the immune response through interaction with A2AR/A2BR on the effector cells, but also adenosine signaling can recruit other immunoregulatory mechanisms, including Tregs. Such interaction between adenosine and Tregs suggests the presence of a positive feedback mechanism, which further promotes negative regulation of immune system through the establishment of immunosuppressive microenvironment.
adenosine; A2A-adenosine receptor; A2B-adenosine receptor; regulatory T cell; immunosuppression; tumor microenvironment
Liver ischemia(I)/reperfusion(R) injury(I) is a known risk factor for the postoperative outcome of patients undergoing liver surgery/transplantation. Attempts to protect from organ damage require multidisciplinary strategies and are of emerging interest in view of patients with higher age and ASA-status. Ischemic preconditioning has been successfully applied to prevent from IRI during liver resections/transplantation. Since even short periods of ischemia during preconditioning inevitably lead to hypoxia and formation of anti-inflammatory/ cytoprotective acting adenosine, we reasoned that short non-ischemic hypoxia also protects against hepatic IRI.
Mice underwent hypoxic preconditioning(HPC) by breathing 10%-oxygen for 10 minutes, followed by 10 minutes of 21%-oxygen prior to left-liver-lobe-ischemia(45 min) and reperfusion(4 hrs). The interactions of hypoxia->adenosine->adenosine-receptors were tested by pharmacologic antagonism at adenosine receptor(AR) sites in wild type mice and in mice with genetic deletions at the A1-;A2A-;A2B- and A3-ARs. Hepatocellular damage, inflammation and metabolic effects were quantified by enzyme activities, cytokines, liver-myeloperoxidase(MPO), blood adenosine and tissue-adenosinemonophosphate(AMP), respectively.
Hepatoprotection by HPC was significant in wild type and A1-, A2A-and A3 AR-knock-out mice as quantified by lower ALT serum activities, cytokine levels, histological damage-scores, tissue-myeloperoxidase-concentrations and as well as preserved AMP-concentrations. Protection by HPC was blunted in mice pretreated with the A2B-AR-antagonist MRS1754 or in A2B-AR“knock-outs”.
Because liver protective effects of HPC are negated when the A2B receptor is non-functional, the "hypoxia->adenosine->A2B receptor" pathway plays a critical role in the prevention of warm ischemia reperfusion injury in vivo. Hypoxic activation of this pathway warrants use of selective A2B-AR-agonists or even intermittent hypoxia (e.g. in deceased organ donors) to protect from liver ischemia/reperfusion injury.
hypoxia; murine liver ischemia; preconditioning; hepatoprotection
Asthma is a chronic condition with high morbidity and healthcare costs, and cockroach allergens are an established cause of urban pediatric asthma. A better understanding of cell types involved in promoting lung inflammation could provide new targets for the treatment of chronic pulmonary disease. Due to its role in regulating myeloid cell dependent inflammatory processes, we examined A2B R expression by myeloid cells in a cockroach allergen (CRA) model of murine asthma-like pulmonary inflammation. Both systemic and myeloid tissue-specific A2B R deletion significantly decreased pulmonary inflammatory cell recruitment, airway mucin production, and pro-inflammatory cytokine secretion after final allergen challenge in sensitized mice. A2B R deficiency resulted in a dramatic reduction on Th2 type airways responses with decreased pulmonary eosinophilia without augmenting neutrophilia, and decreased lung IL-4, IL-5, and IL-13 production. Chemokine analysis demonstrated that eotaxin 1 and 2 secretion in response to repeated allergen challenge is myeloid cell A2B R dependent. In contrast, there were no differences in the levels of the CXC chemokines KC and MIP-2 in the myeloid cell A2B R deficient mice strengthening A2B R involvement in the development of Th2 type airways inflammation. Pro-inflammatory TNF-α, IFN-γ and IL-17 secretion were also reduced in systemic and myeloid tissue-specific A2B R deletion mouse lines. Our results demonstrate Th2 type predominance for A2B R expression by myeloid cells as a mechanism of developing asthma-like pulmonary inflammation.
Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.
Strategies are needed to reverse the immune cell hyporesponsiveness and prevent bacterial overgrowth associated with high mortality rates in septic patients. Adenosine signaling may be mediating immunosuppressive signals within the inflammatory microenvironment that are safeguarding bacteria by rendering immune cells hyporesponsive. We examined A2A adenosine receptor (A2AR)–mediated immune responses in a chronic model of cecal ligation and puncture (CLP)–induced sepsis using both wild-type (WT) and A2AR knockout (KO) mice. In this model, chronic bacterial peritonitis was established that results in the first death on day 4. A2A adenosine receptors promoted bacterial overgrowth that was associated with a high 28-day sepsis mortality (WT 87% vs. A2AR KO 13%; P < 0.0001). Chronic bacteremia persisted in both WT and A2AR KO mice over the 28-day study period. Bacteremia was significantly decreased in A2AR KO mice 2 days after antibiotic therapy cessation (day 6 after CLP; P < 0.005). Local and disseminated bacteria levels were compared at the end of the 28-day study period or from moribund mice. A2A adenosine receptor deficiency dramatically decreased peritoneal (P < 0.05), splenic (P < 0.05), and blood (P < 0.01) bacterial levels. A2A adenosine receptor deficiency caused an early reduction in inflammatory mediators IL-6, macrophage inflammatory protein 2, TNF-srI, and TNF-srII (P < 0.05), but not in TNF-α, IL-1β, IL-10, or monocyte chemotactic protein 1 within 24 h after CLP. In response to an intravenous lipopolysaccharide (day 5 after CLP) challenge, A2AR KO mice showed enhanced secretion of TNF-α (2 h), IFN-γ, IL-6, monocyte chemotactic protein 1, IL-10, and macrophage inflammatory protein 2 (9 h) (P < 0.05), suggesting that A2ARs attenuate inflammatory responses to repeat infectious insults. These data demonstrate that A2AR blockade may be an effective immunotherapy treatment to prevent bacterial overgrowth and reduce mortality secondary to immunosuppression in septic patients.
Immune suppression; inflammation; bacterial overgrowth; compensatory anti-inflammatory response syndrome; systemic inflammatory response syndrome; cytokines
Strategy, Management and Health Policy
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The hexahistidine-tagged mouse P2X1 receptor (H-mP2X1R), an ATP-gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT-B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) and nondenaturing (such as 1% Triton X-100) solubilization conditions were compared, resulting in about 30% of the P2X1 receptors being solubilized (S1). However, at pH 13 most of the H-mP2X1R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H-mP2X1Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., ~90% vs. ~75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8-azido-ATP-γ-32P than the receptors from S1. The binding of 8-azido-ATP-γ-32P to H-mP2X1R was inhibited by ATP-γ-S, α,β-me-ATP, and PPADS, but not by a nucleoside analog (N6-methyl-2′-deoxy-adenosine). In the presence of 2 mM Ca2+ or Mg2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8-azido-ATP-γ-32P or were phosphorylated at 4°C in the presence of 2 mM Mg2+. These data suggest that the decrease in potency of ATP in the presence of Ca2+ and Mg2+, as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H-mP2X1R. Autoradiographic analysis of the cleavage products showed that 8-azido-ATP-γ-32P was crosslinked to the carboxyl side of the extracellular domain of the receptor.
ion channels; nucleotides; affinity chromatography; agonist; binding site
A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) — a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1–/– mice. Comprehensive examination of adenosine receptor–knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.
The A2A adenosine receptor (A2AR)-mediated immunosuppression is firmly implicated in the life-saving down-regulation of collateral tissue damage during the anti-pathogen immune response and in highly undesirable protection of cancerous tissues during anti-tumor immune response. Therefore, depending on specific clinical situation there is a need to either weaken or strengthen the intensity of A2AR signal. While the A2AR-mediated immunosuppression was shown to be T cell autonomous in studies of effector T cells, it was not clear how A2AR stimulation affects regulatory T cells (Treg). Here we show in parallel assays that while A2AR stimulation on T cells directly inhibits their activation, there is also indirect and longer-lasting T cell inhibitory effect through modulation of Treg. A2AR stimulation expanded CD4+ CD25hi FoxP3+ cells, which also express CD39, CD73, and CTLA-4. Treg cultured with A2AR agonist showed increased expression of CTLA-4 and stronger immunosuppressive activity. There was a significant increase of Treg cell number after A2AR stimulation. The CD4+ FoxP3+ population contained those induced from CD4+ CD25− cells, but CD4+ FoxP3+ cells predominantly derived from CD4+ CD25+ natural Treg. Thus, A2AR stimulation numerically and functionally enhanced Treg-mediated immunosuppressive mechanism. These data suggest that the A2AR-mediated stimulation of lymphocytes using A2AR agonists should be considered in protocols for ex vivo expansion of Treg before the transfer to patients in different medical applications.
regulatory T cells; adenosine; immunosuppression; A2A adenosine receptor; cancer; autoimmune; transplantation
The purine nucleoside adenosine is an important anti-inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor-mediated mechanisms. Invariant natural killer T (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN-γ production by iNKT cells.
We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR, and A3R, on mouse iNKT cells. We show that IL-4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL-4. These data are supported by A2aR-deficient mice, which exhibit largely decreased levels of IL-4, IL-10 and TGF-β concomitantly with an increase of IFN-γ upon α-GalCer administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL-4 and IL-10 by iNKT cells. These data suggest adenosine:A2aR-mediated mechanisms can control the cytokine secretion pattern of iNKT cells.
NKT cells; Cellular activation; Immune regulation
Hypoxia inducible factor –1α (HIF-1α) plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that HIF-1α gene deficiency caused abnormal B cell development and autoimmunity. Here we show that HIF-1α-enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1α deficient B220+ bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3 (pfkfb3), was greatly reduced in HIF-1α deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory-chain related genes and TCA-cycle related genes in HIF-1α deficient cells than in wild-type cells. In agreement with these findings, HIF-1α deficient cells utilized pyruvate more efficiently than wild-type cells. The key role of HIF-1α-enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1α plays important role in the B cell development.
Rodent; B Cells; Cell Differentiation
Activation of immune cells is under control of immunological and physiological regulatory mechanisms to ensure adequate destruction of pathogens with the minimum collateral damage to “innocent” bystander cells. The concept of physiological negative regulation of immune response has been advocated based on the finding of the critical immunoregulatory role of extracellular adenosine. Local tissue oxygen tension was proposed to function as one of such physiological regulatory mechanisms of immune responses. In the current study, we utilized in vivo marker of local tissue hypoxia pimonidazole hydrochloride (Hypoxyprobe-1) in the flowcytometric analysis of oxygen levels to which lymphocytes are exposed in vivo. The level of exposure to hypoxia in vivo was low in B cells and the levels increased in the following order: T cells < NKT cells < NK cells. The thymus was more hypoxic than the spleen and lymph nodes, suggesting the variation in the degree of oxygenation among lymphoid organs and cell types in normal mice. Based on in vitro studies, tissue hypoxia has been assumed to be suppressive to T cell activation in vivo, but there was no direct evidence demonstrating that T cells exposed to hypoxic environment in vivo are less activated. We tested whether the state of activation of T cells in vivo changes due to their exposure to hypoxic tissue microenvironments. The parallel analysis of more hypoxic and less hypoxic T cells in the same mouse revealed that the degree of T cell activation was significantly stronger in better-oxygenated T cells. These observations suggest that the extent of T cell activation in vivo is dependent on their localization and is decreased in environment with low oxygen tension.
T cell; oxygen; hypoxia; hyperoxia; Hypoxyprobe-1; cytometry; tumor
Hypoxia-driven increase of extracellular adenosine in local tissue microenvironments of inflamed and cancerous tissues plays a critical role in the regulation of tissue destruction by activated immune cells. Accumulated data suggest that injection or consumption of A2A adenosine receptor (A2AR) antagonists may represent a drug treatment that diminishes adenosine-mediated immunosuppression. Since this, in turn, enhances the immune response, inhibition of adenosine-A2AR signaling may be a promising approach to enhance anti-tumor or anti-pathogen immune response. Patients with disorders characterized by excessive inflammation may be at risk to A2AR antagonists (e.g. caffeine) because of the effect to increase inflammatory damage secondary to enhanced immunity. On the other hand, enhancement of hypoxia-adenosinergic immunomodulatory pathways may be beneficial to prevent inflammatory tissue destruction.
Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-inducible factor 1 (HIF-1) regulates several cellular processes, including apoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific I.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of I.3 isoform of hif-1α in LNCaP cells allows poly(I:C)-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.
Hypoxia-Inducible Factor 1α (HIF-1α) is critical not only in the regulation of oxygen homeostasis, but also in the regulation of innate and adaptive immune systems. We previously reported that TCR-mediated activation of T cells in mice leads to the expression of an alternative isoform of HIF-1α that inhibits activated T cells in a delayed negative feed-back manner. In this paper, we describe a novel mRNA isoform of human HIF-1α that is upregulated in peripheral T lymphocytes after TCR stimulation. This activation-inducible isoform is expressed using the alternative first exon I.3, and it encodes a protein that is 24 amino acids longer than the ubiquitous HIF-1α isoform. This mRNA isoform I.3 of HIF-1α is expressed in a tissue-specific manner with the highest expression found in peripheral blood leukocytes and the thymus.
T cells; TCR; PBMC; HIF-1; hypoxia-inducible factor
Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine → A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This “hypoxic” treatment resulted in significantly reduced hepatocellular damage and attenuated levels of serum cytokines in mice with acute liver inflammation. The anti-inflammatory effects of hypoxia were not observed in the absence of A2AR in studies of A2AR gene-deficient mice or when A2AR have been pharmacologically antagonized with synthetic antagonist. The presented data demonstrate that total body hypoxia-triggered pathway provides protection in acute hepatitis and that hypoxia (upstream) and A2AR (downstream) function in the same immunosuppressive and liver tissue-protecting pathway.
Sepsis patients may die either from an overwhelming systemic immune response and/or from an immunoparalysis-associated lack of anti-bacterial immune defence. We hypothesized that bacterial superantigen-activated T cells may be prevented from contribution into anti-bacterial response due to the inhibition of their effector functions by the hypoxia inducible transcription factor (HIF-1α) in inflamed and hypoxic areas.
Using the Cre-lox-P-system we generated mice with a T–cell targeted deletion of the HIF-1α gene and analysed them in an in vivo model of bacterial sepsis. We show that deletion of the HIF-1α gene leads to higher levels of pro-inflammatory cytokines, stronger anti-bacterial effects and much better survival of mice. These effects can be at least partially explained by significantly increased NF-κB activation in TCR activated HIF-1 α deficient T cells.
T cells can be recruited to powerfully contribute to anti-bacterial response if they are relieved from inhibition by HIF-1α in inflamed and hypoxic areas. Our experiments uncovered the before unappreciated reserve of anti-bacterial capacity of T cells and suggest novel therapeutic anti-pathogen strategies based on targeted deletion or inhibition of HIF-1 α in T cells.
Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA–/–) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA–/– mice. T cell apoptosis was abundant in thymi of ADA–/– mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA–/– mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA–/– T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA–/– mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.
J. Clin. Invest. 108:131–141 (2001). DOI:10.1172/JCI200110360.
Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure.
A mouse model suggests that oxygen therapy may exacerbate lung injury by weakening the anti-inflammatory mechanisms driven by hypoxia.