Association of polymorphic variants in human genome with the COVID-2019 severity and post-COVID syndrome development

Background/Objectives: Despite intensive research of the novel coronavirus SARS-CoV-2 and COVID-2019 caused by it, factors affecting the severity of the disease remains poorly understood. Clinical manifestations of COVID-2019 may vary from asymptomatic form to pneumonia, acute respiratory distress syndrome (ARDS) and multiorgan failure. Features of individual genetic landscape of patients can play an important role in development of the pathological process of COVID-19. In this regard the purpose of this study was to investigate the influence of polymorphic variants in genes (ADD1, CAT, IL17F, IL23R, NOS3, IFNL3, IL6, F2, F13A1, ITGB3, HIF1A, MMP12, VEGFA), associated with cardiovascular, respiratory and autoimmune pathologies, on the severity of COVID-19 and post-COVID syndrome in patients from Russia. Method(s): The study included 200 patients recovered from COVID-19. Two groups of patients were formed in accordance with clinical manifestations: with mild and moderate forms of the disease. The polymorphic variants were analysed with real-time PCR using commercial kits (Syntol). Result(s): 13 SNPs (rs4961;rs1001179;rs612242;rs11209026;rs2070744;rs8099917;rs1800795;rs1799963;rs5985;rs5918;rs11549465;rs652438;rs699947) were genotyped and comparative analysis of allele frequency distribution was carried out in two groups of patients recovered from COVID-2019. Conclusion(s): Identification of polymorphic variants in genome associated with severity of pathological processes in patients infected with SARS-CoV-2 can contribute to the identification of individuals with an increased risk of severe infection process and can also serve as a basis for developing personalized therapeutic approaches to the treatment of post-COVID syndrome.

PLASMA FROM SARS-COV-2 PATIENTS INDUCES OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION VIA ANGIOTENSIN II TYPE 1 RECEPTOR (AT1R)/P38 MAPK PATHWAY

Background: Acute respiratory distress syndrome (ARDS) is a distinctive feature of severe COVID-19 infections that occurs mainly in patients with coexisting health problems, such as hypertension, atherosclerosis, and diabetes. Endothelial dysfunction is a major contributing factor during ARDS development in COVID- 19 patients with pre-existing comorbidities. Objective: Studying the mechanism by which endothelial activation and dysfunction could provide a therapeutic target for COVID-19 treatment. Design and method: The current study measured endothelial dysfunction and oxidative stress by incubating human umbilical vein endothelial cells (HUVECs) with plasma from patients with mild, moderate, severe and extremely severe COVID- 19. Using flow cytometry, wound-healing assays and phosphokinase arrays, Results: We detected increases in cell apoptosis;reactive oxygen species (ROS) formation;hypoxia-inducible factor-1 alpha (HIF-1 alpha), vascular cell adhesion molecule-1 (VCAM-1), and vascular endothelial growth factor receptor-1 (VEGFR-1) expression;viral entry;and inflammatory-related protein activity. We also found an impairment in the wound-healing process. Moreover, we found that AT1R blockade and P38 MAPK inhibition reversed all of these effects, especially in the severe group. Conclusions: These findings indicate that AT1R/P38 MAPK-mediated oxidative stress and endothelial dysfunction occur during COVID-19 infection.

INTERFERON-A MEDIATED THERAPEUTIC RESISTANCE in EARLY RA IMPLICATES EPIGENETIC REPROGRAMMING

Background: An interferon gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients. We previously demonstrated it negatively impacts on initial disease outcomes. Objectives: To 1) reproduce previous fndings demonstrating the harmful effects of the IGS on early RA clinical outcomes, 2) identify which IFN class is responsible for the IGS and 3) seek evidence that IFN-a exposure contributes to harmful epigenetic footprint at disease onset. Methods: In a large multicentre inception cohort (n=190) of eRA patients (RA-MAP TACERA) whole blood transcriptome, IGS (MxA, IFI44L, OAS1, ISG15, IFI6) and circulating interferons (IFN)-a,-β,-y and-), was examined at baseline and 6 months in conjunction with disease activity and clinical characteristics. A separate eRA cohort of paired methylome and transcriptome from CD4 T and CD19 B cells (n=41 for each) was used to explore any epigenetic influence of the IGS. Results: The baseline IGS reproducibly and signifcantly negatively impacts on 6-month clinical outcomes. In the high IGS cohort there was increased DAS-28 (p=0.025) and reduced probability of achieving a good EULAR response (p=0.034) at 6-months. In addition, the IGS in eRA is shown for the frst time to predominantly refect raised circulating IFN-a protein, not other classes of IFN and examination of whole blood upstream nucleic acid sensors expression suggest a RNA trigger. Both the IGS and IFN-a signifcantly fell in parallel at 6 months (p<0.0001), whereas other classes of IFN remained statistically static. There was a signifcant association with IFN-a and RF titre but not ACPA. Comparison of CD4 T and CD19 B cells between IGS high and low eRA patients demonstrated differentially methylated CPG sites and altered transcript expression of disease relevant genes e.g. PARP9, STAT1, EPTSI1 which was similarly, and persistently altered 6 months in the separate TACERA cohort. Differentially methylated CPGs implicated altered transcription factor binding in B cells (GATA3, ETSI, NFATC2, EZH2) and T cells (p300, HIF1a) which cumulatively suggested IFN-a induced epigenetic changes promoting increased, and sustained, lymphocyte activation, proliferation and loss of anergy in the IGS high cohort. Conclusion: We validate that the IGS is a robust prognostic biomarker in eRA predicting poor therapeutic response. Its persistent harmful effects may be driven via epigenetic modifcations. These data have relevance for other IFN-a states, such as COVID-19, but also provide a rationale for the initial therapeutic targeting of IFN-a signalling, such as with JAKi, at disease onset in stratifed eRA subsets.

EVIDENCE of BRAIN HYPOXIA in NEUROPATHOLOGY of SARS-CoV-2-INFECTED NONHUMAN PRIMATES

Background: Neurological manifestations are a major complication of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and likely contribute to symptoms of "long COVID". Elucidating the mechanisms that underlie neuropathogenesis in infection is critical for identifying or developing viable therapeutic strategies. While neurological injury in infection is varied, cerebrovascular disease is seen at a high frequency among patients over 50 years of age. Additionally, microhemorrhages and hypoxic-ischemic injury are often described in brain autopsy series of human subjects who died from COVID-19. Here, we report neuropathology in aged SARS-CoV-2 infected non-human primates (NHPs) is consistent with that observed in aged human subjects and provide insight into the underlying cause. Methods: Four adult Rhesus macaques and four African green monkeys were inoculated with the 2019-nCoV/USA-WA1/2020strain of SARS-CoV-2 via a multi-route mucosal or aerosol challenge. Two of each species were included as age-matched controls. Frontal, parietal, occipital, and temporal lobes, basal ganglia, cerebellum, and brainstem were interrogated through histopathological and immunohistochemical techniques to identify and characterize the observed pathology. Results: Like humans, pathology was variable but included wide-spread inflammation with nodular lesions, neuronal injury, and microhemorrhages. Neuronal degeneration and apoptosis were confirmed with FluoroJade C and cleaved caspase 3 IHC, which showed foci of positivity, particularly among cerebellar Purkinje cells. This was seen even among infected animals that did not develop severe respiratory disease but was not seen in age-matched controls. Significant upregulation of the alpha subunit of hypoxia inducible factor 1 (HIF1-α), indicative of tissue hypoxia, was observed in brain of all infected animals, regardless of disease severity. Sparse virus was detected in brain endothelial cells but did not associate with the severity of CNS injury. Conclusion: SARS-CoV-2 infected NHPs are a viable animal model for advancing our current understanding of infection-associated neuropathogenesis. Upregulation of HIF1-α in brain of infected animals suggests cerebral hypoxia may underlie or contribute to neuroinflammation and neuronal injury/death and may provide some insight into neurological manifestations observed among asymptomatic patients or those only suffering mild disease.

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.

An airway organoid-based screen identifies a role for the HIF1α-glycolysis axis in SARS-CoV-2 infection.

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.

Effects of moderate-intensity intermittent hypoxic training on health outcomes of patients recovered from COVID-19: the AEROBICOVID study protocol for a randomized controlled trial.

BACKGROUND: Recent studies point to a lower number and reduced severity of cases in higher altitude cities with decreased oxygen concentration. Specific literature has shown several benefits of physical training, so, in this sense, physical training with hypoxic stimulus appears as an alternative that supports the conventional treatments of the COVID-19 patient's recovery. Thus, this study's primary aim is to analyze the effects of moderate-intensity intermittent hypoxic training on health outcomes in COVID-19 recovered patients. METHODS: A clinical trial controlled double-blind study was designed. Participants (30-69 years old) will be recruited among those with moderate to severe COVID-19 symptoms, approximately 30 days after recovery. They will be included in groups according to the training (T) and recovery (R) association with hypoxia (H) or normoxia (N): (a) TH:RH, (b) TN:RH, (c) TN:RN, and last (d) the control group. The 8-week exercise bike intervention will be carried out with a gradual load increase according to the established periods, three times a week in sets of 5 min, 90 to 100% of the anaerobic threshold (AT), and a 2.5-min break. Blood will be collected for genotyping. First, after 4 weeks (partial), after 8 weeks, and later, 4 weeks after the end of the physical training intervention, participants will perform assessments. The primary outcome is the maximum oxygen consumption (VO2peak). The secondary outcomes include lung function, inflammatory mediators, hematological, autonomic parameters, AT, body composition analysis, quality of life, mental health, anthropometric measurements, and physical fitness. The statistical analysis will be executed using the linear regression model with mixed effects at a 5% significance level. DISCUSSION: This study is designed to provide evidence to support the clinical benefits of moderate-intensity intermittent hypoxic training as a part of the treatment of patients recovered from COVID-19. It may also provide evidence on the efficacy and safety of intermittent hypoxic training in different health conditions. Lastly, this study presents an innovative strategy enabling up to 16 participants in the same training session. TRIAL REGISTRATION: ClinicalTrials.gov RBR-5d7hkv. Registered after the start of inclusion on 3 November 2020 with the Brazilian Clinical Trials Registry.

SARS-CoV-2 and Glutamine: SARS-CoV-2 Triggered Pathogenesis via Metabolic Reprograming of Glutamine in Host Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as coronavirus disease 2019 (COVID-19) pandemic, has killed more than a million people worldwide, and researchers are constantly working to develop therapeutics in the treatment and prevention of this new viral infection. To infect and induced pathogenesis as observed in other viral infections, we postulated that SARS-CoV-2 may also require an escalation in the anabolic metabolism, such as glucose and glutamine, to support its energy and biosynthetic requirements during the infection cycle. Recently, the requirement of altered glucose metabolism in SARS-CoV-2 pathogenesis was demonstrated, but the role of dysregulated glutamine metabolism is not yet mentioned for its infection. In this perspective, we have attempted to provide a summary of possible biochemical events on putative metabolic reprograming of glutamine in host cells upon SARS-CoV-2 infection by comparison to other viral infections/cancer metabolism and available clinical data or research on SARS-CoV-2 pathogenesis. This systematic hypothesis concluded the vital role of glutaminase-1 (GLS1), phosphoserine aminotransferase (PSAT1), hypoxia-inducible factor-1 alpha (HIF-1α), mammalian target of rapamycin complex 1 (mTORC1), glutamine-fructose amidotransferase 1/2 (GFAT1/2), and transcription factor Myc as key cellular factors to mediate and promote the glutamine metabolic reprogramming in SARS-CoV-2 infected cells. In absence of concrete data available for SARS-CoV-2 induced metabolic reprogramming of glutamine, this study efforts to connect the gaps with available clinical shreds of evidence in SARS-CoV-2 infection with altered glutamine metabolism and hopefully could be beneficial in the designing of strategic methods for therapeutic development with elucidation using in vitro or in vivo approaches.