Hypoxia induces dichotomous and reversible attenuation of T cell responses through reactive oxygen species-dependent phenotype redistribution and delay in lymphoblast proliferation.

As T cells transit between blood, lymphoid organs, and peripheral tissues, they experience varied levels of oxygen/hypoxia in inflamed tissues, skin, intestinal lining, and secondary lymphoid organs. Critical illness among COVID-19 patients is also associated with transient hypoxia and attenuation of T cell responses. Hypoxia is the fulcrum of altered metabolism, impaired functions, and cessation of growth of a subset of T cells. However, the restoration of normal T cell functions following transient hypoxia and kinetics of their phenotype-redistribution is not completely understood. Here, we sought to understand kinetics and reversibility of dichotomous T cell responses under sustained and transient hypoxia. We found that a subset of activated T cells accumulated as lymphoblasts under hypoxia. Further, T cells showed the normal expression of activation markers CD25 and CD69 and inflammatory cytokine secretion but a subset exhibited delayed cell proliferation under hypoxia. Increased levels of reactive oxygen species (ROS) in cytosol and mitochondria were seen during dichotomous and reversible attenuation of T cell response under hypoxia. Cell cycle analysis revealed maximum levels of cytosolic and mitochondrial ROS in dividing T cells (in S, G2, or M phase). Hypoxic T cells also showed specific attenuation of activation induced memory phenotype conversion without affecting naïve and activated T cells. Hypoxia-related attenuation of T cell proliferation was also found to be reversible in an allogeneic leukocyte specific mixed lymphocyte reaction assay. In summary, our results show that hypoxia induces a reversible delay in proliferation of a subset of T cells which is associated with obliteration of memory phenotype and specific increase in cytosolic/mitochondrial ROS levels in actively dividing subpopulation. Thus, the transient reoxygenation of hypoxic patients may restore normal T cell responses.

Effects of oxygen saturation on the hypoxia-inducible factor-1α, subfatin, asprosin, irisin, c-reactive protein, maresin-1, and diamine oxidase in diabetic patients with COVID-19.

OBJECTIVE: Oxygen is essential for living organisms that perform aerobic respiration since cells begin to die when humans and animals are deprived of oxygen. Oxygen saturation decreases and shortness of breath occurs in coronavirus (COVID-19) disease. Therefore, in this study, we aimed to determine the changes in hypoxia-inducible factor-1α (HIF-1α), subfatin, asprosin, irisin, C-reactive protein (C-RP), Maresin-1 (MaR-1), and diamine oxidase (DAO) molecules in diabetic patients with coronavirus according to their oxygen saturations. PATIENTS AND METHODS: Participants were classified into 4 Groups of 22, including patients with oxygen saturation between 95% and 100% (Group I, control), between 80% and 85% (Group II), between 75% and 79% (Group III), and between 70% and 74% (Group IV). COVID-19 was diagnosed with PCR testing and 5 mL of blood was taken following the diagnosis. HIF-1α, subfatin, asprosin, irisin, MaR-1, and DAO values of the participants were measured with ELISA. Other parameters used in the study were obtained from the records of the patients. RESULTS: When Group I was compared to Groups II, there was no significant change in Group II while HIF-1α, subfatin, asprosin, irisin, C-RP, and DAO counts had increased significantly in Groups III and IV. When the MaR-1 values were examined, they were reported to have decreased significantly in Groups III and IV (p < 0.05). Similarly, when Group II and Group IV were compared, HIF-1α, subfatin, asprosin, irisin, C-RP, and DAO values of the participants in Group IV had significantly increased while MaR-1 values had significantly decreased (p < 0.05). In the case of oxygen saturation decreasing below the critical value (70-74%) in patients with coronavirus, the release of HIF-1HIF-1α, subfatin, asprosin, irisin, C-RP, and DAO increased while the MaR-1 values decreased (p < 0.05). CONCLUSIONS: Changes in these molecules in patients with coronavirus and diabetes according to their oxygen saturation suggested that they functioned as the "metabolic oxygen sensors" of the metabolism. Therefore, according to these data, it was predicted that these molecules had the potential to be used in the diagnosis and follow-up of diseases related to oxygen (such as asthma, and critical intensive care patients) in clinics in the future.

Induction of Hypoxic Response in Caco-2 Cells Promote the Expression of Genes Involved in SARS-CoV-2 Endocytosis and Transcytosis.

In the present manuscript we analyzed the influence of hypoxic response in Caco-2 cells on the expression of genes and miRNAs involved in the mechanisms of intracellular transport of SARS-CoV-2 viral particles, especially endocytosis and transcytosis. With the use of RNA sequencing of Caco-2 cells treated with hypoxia-inducing oxyquinoline derivative, we showed two-fold increase in the expression of the main SARS-CoV-2 receptor ACE2. Expression of the non-canonical receptor TFRC was also elevated. We also observed a significant increase in the expression levels of genes from the low-density lipoprotein (LDL) receptor family, which play a crucial role in the transcytosis: LDLR, LRP1, LRP4, and LRP5. Upregulation of LDLR was coupled with the downregulation of hsa-miR-148a-3p, which can directly bind to LDLR mRNA. Thus, the hypoxic response in Caco-2 cells includes upregulation of genes involved in the mechanisms of endocytosis and transcytosis of SARS-CoV-2 viral particles.

Cathepsin L, a Target of Hypoxia-Inducible Factor-1-α, Is Involved in Melanosome Degradation in Melanocytes.

Hypoxic conditions induce the activation of hypoxia-inducible factor-1α (HIF-1α) to restore the supply of oxygen to tissues and cells. Activated HIF-1α translocates into the nucleus and binds to hypoxia response elements to promote the transcription of target genes. Cathepsin L (CTSL) is a lysosomal protease that degrades cellular proteins via the endolysosomal pathway. In this study, we attempted to determine if CTSL is a hypoxia responsive target gene of HIF-1α, and decipher its role in melanocytes in association with the autophagic pathway. The results of our luciferase reporter assay showed that the expression of CTSL is transcriptionally activated through the binding of HIF1-α at its promoter. Under autophagy-inducing starvation conditions, HIF-1α and CTSL expression is highly upregulated in melan-a cells. The mature form of CTSL is closely involved in melanosome degradation through lysosomal activity upon autophagosome-lysosome fusion. The inhibition of conversion of pro-CTSL to mature CTSL leads to the accumulation of gp100 and tyrosinase in addition to microtubule-associated protein 1 light chain 3 (LC3) II, due to decreased lysosomal activity in the autophagic pathway. In conclusion, we have identified that CTSL, a novel target of HIF-1α, participates in melanosome degradation in melanocytes through lysosomal activity during autophagosome-lysosome fusion.

Hypoxia alters the expression of ACE2 and TMPRSS2 SARS-CoV-2 cell entry mediators in hCMEC/D3 brain endothelial cells.

The mechanisms by which the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induces neurological complications remain to be elucidated. We aimed to identify possible effects of hypoxia on the expression of SARS-CoV-2 cell entry mediators, angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) protein, in human brain endothelial cells, in vitro. hCMEC/D3 cells were exposed to different oxygen tensions: 20% (Control group), 8% or 2% O2 (Hypoxia groups). Cells were harvested 6-, 24- and 48 h following hypoxic challenge for assessment of mRNA and protein, using qPCR and Western Blot. The response of the brain endothelial cells to hypoxia was replicated using modular incubator chambers. We observed an acute increase (6 h, p < 0.05), followed by a longer-term decrease (48 h, p < 0.05) in ACE2 mRNA and protein expression, accompanied by reduced expression of TMPRSS2 protein levels (48 h, p < 0.05) under the more severe hypoxic condition (2% O2). No changes in levels of von Willebrand Factor (vWF - an endothelial cell damage marker) or interleukin 6 (IL-6 - a pro-inflammatory cytokine) mRNA were observed. We conclude that hypoxia regulates brain endothelial cell ACE2 and TMPRSS2 expression in vitro, which may indicate human brain endothelial susceptibility to SARS-CoV-2 infection and subsequent brain sequelae.

Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells.

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1ß, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.

HIF-1α is a negative regulator of interferon regulatory factors: Implications for interferon production by hypoxic monocytes.

Patients with severe COVID-19 infection exhibit a low level of oxygen in affected tissue and blood. To understand the pathophysiology of COVID-19 infection, it is therefore necessary to understand cell function during hypoxia. We investigated aspects of human monocyte activation under hypoxic conditions. HMGB1 is an alarmin released by stressed cells. Under normoxic conditions, HMGB1 activates interferon regulatory factor (IRF)5 and nuclear factor-κB in monocytes, leading to expression of type I interferon (IFN) and inflammatory cytokines including tumor necrosis factor α, and interleukin 1ß, respectively. When hypoxic monocytes are activated by HMGB1, they produce proinflammatory cytokines but fail to produce type I IFN. Hypoxia-inducible factor-1α, induced by hypoxia, functions as a direct transcriptional repressor of IRF5 and IRF3. As hypoxia is a stressor that induces secretion of HMGB1 by epithelial cells, hypoxia establishes a microenvironment that favors monocyte production of inflammatory cytokines but not IFN. These findings have implications for the pathogenesis of COVID-19.

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate.

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.

C-Reactive Protein Triggers Cell Death in Ischemic Cells.

C-reactive protein (CRP) is the best-known acute phase protein. In humans, almost every type of inflammation is accompanied by an increase of CRP concentration. Until recently, the only known physiological function of CRP was the marking of cells to initiate their phagocytosis. This triggers the classical complement pathway up to C4, which helps to eliminate pathogens and dead cells. However, vital cells with reduced energy supply are also marked, which is useful in the case of a classical external wound because an important substrate for pathogens is disposed of, but is counterproductive at internal wounds (e.g., heart attack or stroke). This mechanism negatively affects clinical outcomes since it is established that CRP levels correlate with the prognosis of these indications. Here, we summarize what we can learn from a clinical study in which CRP was adsorbed from the bloodstream by CRP-apheresis. Recently, it was shown that CRP can have a direct effect on blood pressure in rabbits. This is interesting in regard to patients with high inflammation, as they often become tachycardic and need catecholamines. These two physiological effects of CRP apparently also occur in COVID-19. Parts of the lung become ischemic due to intra-alveolar edema and hemorrhage and in parallel CRP increases dramatically, hence it is assumed that CRP is also involved in this ischemic condition. It is meanwhile considered that most of the damage in COVID-19 is caused by the immune system. The high amounts of CRP could have an additional influence on blood pressure in severe COVID-19.

HPLC-MS/MS Shows That the Cellular Uptake of All-Trans-Retinoic Acid under Hypoxia Is Downregulated by the Novel Active Agent 5-Methoxyleoligin.

All-trans-retinoic acid (atRA) is the essential derivative of vitamin A and is of interest due to its various biological key functions. As shown in the recent literature, atRA also plays a role in the failing heart during myocardial infarction, the leading cause of death globally. To date insufficient mechanistic information has been available on related hypoxia-induced cell damage and reperfusion injuries. However, it has been demonstrated that a reduction in cellular atRA uptake abrogates hypoxia-mediated cell and tissue damage, which may offer a new route for intervention. Consequently, in this study, the effect of the novel cardio-protective compound 5-methoxyleoligin (5ML) on cellular atRA uptake was tested in human umbilical-vein endothelial cells (HUVECs). For this purpose, a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to assess intra-cellular levels of the active substance and corresponding levels of vitamin A and its derivatives, including potential cis/trans isomers. This work also focused on light-induced isomerization and the stability of biological sample material to ensure sample integrity and avoid biased conclusions. This study provides evidence of the inhibitory effect of 5ML on cellular atRA uptake, a promising step toward a novel therapy for myocardial infarction.