PLASMA LIPIDS AND AMINO ACIDS LINKED TO LOW SARS-CoV-2 SUSCEPTIBILITY

Background: The mechanisms driving SARS-CoV-2 susceptibility remain poorly understood, especially the factors determining why a subset of unvaccinated individuals remain uninfected despite high-risk exposures. Method(s): We studied an exceptional group of unvaccinated healthcare workers heavily exposed to SARS-CoV-2 ('nonsusceptible') from April to June 2020, who were compared against 'susceptible' individuals to SARS-CoV-2, including uninfected subjects who became infected during the follow-up, and hospitalized patients with different disease severity providing samples at early disease stages. We analyzed plasma samples using different mass spectrometry technique and obtained metabolites and lipids profiles. Result(s): We found that the metabolite profiles were predictive of the selected study groups and identified lipids profiles and metabolites linked to SARS-CoV-2 susceptibility and COVID-19 severity. More importantly, we showed that non-susceptible individuals exhibited unique metabolomics and lipidomic patterns characterized by upregulation of most lipids -especially ceramides and sphingomyelin-and amino acids related to tricarboxylic acid cycle and mitochondrial metabolism, which could be interpreted as markers of low susceptibility to SARS-CoV-2 infection. Lipids and metabolites pathways analysis revealed that metabolites related to energy production, mitochondrial and tissue dysfunction, and lipids involved in membrane structure and virus infectivity were key markers of SARS-CoV-2 susceptibility. Conclusion(s): Lipid and metabolic profiles differ in 'nonsusceptible' compared to individuals susceptible to SARS-CoV-2. Our study suggests that lipid profiles are relevant actors during SARS-CoV-2 pathogenesis and highlight certain lipids relevant to understand SARS-CoV-2 pathogenesis. (Figure Presented).

Mitochondrial Dysfunction Is Associated with Post-Acute Sequelae of Covid-19

Background: Mitochondrial (mt) dysfunction has been described in acute severe SARS-CoV2 infection. It remains unclear whether the disturbances in mt are also present in post-acute sequelae of COVID-19 (PASC). Method(s): We analyzed cross-sectional data from participants without history of COVID and SARS-CoV2 antibody negative (COVID-), with documented prior COVID and full recovery (COVID+ PASC-), and with prior COVID with PASC as defined by the CDC (COVID+PASC+). Mt respiration was measured from peripheral blood mononuclear cells utilizing the Seahorse XFe96 analyzer. Generalized linear regression was used to compare estimates of mt and non-mt respirations, and unadjusted odds ratios using multinomial logistic regression to assess if mt respiration were associated with PASC. Result(s): For this analysis, 59 participants were enrolled, 71.19% (n=42) had a confirmed COVID-19 diagnosis. The overall mean age was 47.47 +/- 14.86 years, 69.49% (n=41) were females and 33.90% (n=20) were non-white race. There was no difference in demographics between participants with and without COVID (p>=0.72). Amongst all COVID+ participants, 19% (n=11) had hypertension and 8% (n=5) had diabetes. Among all COVID+, the median time between COVID diagnosis and study evaluation was 210 (IQR: 119, 453) days, and 50% (n=21) of COVID+ experienced persistent symptoms consistent with PASC. PASC participants had the highest observed values in non-mt respiration (21.57 +/- 10.77 pmol/min), basal respiration (38.95 +/- 17.58 pmol/min), proton leak (10.41 +/- 3.1), maximal respiration (103.91 +/- 58.63 pmol/min), spare respiratory capacity (64.96 +/- 41.82 pmol/min), and ATP production (28.55 +/-14.85 pmol/min). Basal respiration, ATP production, maximal respiration, and non-mt respiration were highest in PASC compared to COVID- (p<=0.02). There was marginal evidence (p=0.05) of a mean difference (8.09 pmol/min) in ATP production between COVID+PASC+ and COVID+PASC-, without differences in proton leak (p=0.23) or spare respiration capacity (p=0.07). Every unit increase in non-mt respiration, basal respiration, maximal respiration, and ATP production increased the predicted odds of PASC by 10.99, 5.6, 1.6 and 6.2%, respectively (Figure). Conclusion(s): Individuals with PASC are consuming more oxygen and producing more ATP in the PBMCs compared to controls. There also appears to be increased PBMC ATP production between PASC and COVID+. We hypothesize that this may reflect a crucial pathogenic mechanism in PASC that may be associated with ongoing inflammation. (Figure Presented).

Short-term effect of molecular hydrogen on mitochondrial respiration and hydrogen peroxide production in permeabilized HEK 293T cells

Molecular hydrogen H2 has been reported to be an antioxidative, anti-inflammatory, and antiapoptotic agent with therapeutic potential for various diseases such as cardiac arrest, asthma, chronic obstructive pulmonary disease (COPD), and, most recently, COVID-19 [1]. In previous studies, H2 is typically administered repeatedly or over longer periods of time (hours to days) via inhalation of H2 gas, drinking H2-rich water, or injection of H2 saline, wherefore the observed effects, e.g. on mitochondrial metabolism [2], might be either directly or indirectly related to H2. To investigate a direct short-term effect of H2 on mitochondrial function, we measured mitochondrial respiration and H2O2 production in permeabilized HEK 293T cells upon sequential changes of H2 concentration cH2 in the experimental medium. O2 and H2O2 flux were measured simultaneously in the O2k with the Fluo-Module (Oroboros Instruments). Increase of cH2 was accomplished by injecting H2 into the gas phase of the open O2k-chamber. This causes not only an increase of cH2 but also a decrease of oxygen concentration cO2. As mitochondrial ROS production is a continuous function of cO2, we used the conventionally applied N2 gas as a control to distinguish between cO2- and cH2-dependent effects. Measurements were started near air saturation (~160 muM of oxygen). The plasma membrane was permeabilized with digitonin and the NADH-linked substrates pyruvate & malate were titrated to measure O2 and H2O2 flux in the LEAK state (without ADP). Upon transition of cO2 from ~160 to ~25 muM, a decrease in O2 and H2O2 flux was observed. This was comparable between regimes with increased cH2 or cN2. Further transitions by re-oxygenation and injection of H2 or N2 yielded the same results. Similarly, cO2-dependent changes in mitochondrial respiration and H2O2 production in the OXPHOS state (kinetically saturating [ADP]) were independent of the increase in cH2 or cN2. These results indicate that short-term exposure to increased cH2 does not affect mitochondrial respiration or H2O2 production. [1] Y. Tian, Y. Zhang, Y. Wang, Y. Chen, Hydrogen, a Novel Therapeutic Molecule, Regulates Oxidative Stress, Inflammation, and Apoptosis, Frontiers in Physiology, 12 (2021) 1-14 [2] A. Gvozdjakova, J. Kucharska, B. Kura, O. Vancova O, A new insight into the molecular hydrogen effect on coenzyme Q and mitochondrial function of rats, J Physiol Pharmacol., 1 (2020) 29-34 Copyright © 2022

The Efficient Antiviral Response of A549 Cells Is Enhanced When Mitochondrial Respiration Is Promoted.

When exposed to a viral infection, the attacked cells promptly set up defense mechanisms. As part of the antiviral responses, the innate immune interferon pathway and associated interferon-stimulated genes notably allow the production of proteins bearing antiviral activity. Numerous viruses are able to evade the interferon response, highlighting the importance of controlling this pathway to ensure their efficient replication. Several viruses are also known to manipulate the metabolism of infected cells to optimize the availability of amino acids, nucleotides, and lipids. They then benefit from a reprogramming of the metabolism that favors glycolysis instead of mitochondrial respiration. Given the increasingly discussed crosstalk between metabolism and innate immunity, we wondered whether this switch from glycolysis to mitochondrial respiration would be beneficial or deleterious for an efficient antiviral response. We used a cell-based model of metabolic reprogramming. Interestingly, we showed that increased mitochondrial respiration was associated with an enhanced interferon response following polyriboinosinic:polyribocytidylic acid (poly:IC) stimulation. This suggests that during viral infection, the metabolic reprogramming towards glycolysis is also part of the virus' strategies to inhibit the antiviral response.

Opposite Effect of Thyroid Hormones on Oxidative Stress and on Mitochondrial Respiration in COVID-19 Patients.

BACKGROUND: Thyroid hormones (TH)s are master regulators of mitochondrial activity and biogenesis. Nonthyroidal illness syndrome (NTIS) is generally considered an adaptative response to reduced energy that is secondary to critical illness, including COVID-19. COVID-19 has been associated with profound changes in the cell energy metabolism, especially in the cells of the immune system, with a central role played by the mitochondria, considered the power units of every cell. Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects and alters mitochondrial functions, both to influence its intracellular survival and to evade host immunity. AIM OF THE STUDY: This study was undertaken to analyze the oxidative balance and mitochondrial respiration in COVID-19 patients with and without NTIS to elucidate the role that thyroid hormones (TH)s play in this context. METHODS: In our cohort of 54 COVID-19 patients, admitted to our University Hospital during the COVID-19 pandemic, we evaluated the generation of reactive oxygen species (ROS) by measuring the serum levels of derivatives of reactive oxygen metabolites (dROMs), and we analyzed the antioxidant capacity by measuring the serum biological antioxidant potential (BAP). We then analyzed the mitochondrial respiration in peripheral blood mononuclear cells (PBMC)s of 28 of our COVID-19 patients, using the seahorse instrument (Agilent). Results were correlated with the serum levels of THs and, in particular, of FT3. In addition, the role of T3 on bioelectrical impedance analysis (BIA) and mitochondrial respiration parameters was directly evaluated in two COVID-19 patients with NTIS, in which treatment with synthetic liothyronine (LT3) was given both in vivo and in vitro. RESULTS: In our COVID-19 patients with NTIS, the dROMs values were significantly lower and the BAP values were significantly higher. Consequently, the oxidative stress index (OSi), measured as BAP/dROMs ratio was reduced compared to that observed in COVID-19 patients without NTIS, indicating a protective role exerted by NTIS on oxidative stress. In our COVID-19 patients, the mitochondrial respiration, measured in PBMCs, was reduced compared to healthy controls. Those with NTIS showed a reduced maximal respiratory capacity and a reduced proton leak, compared to those with normal FT3 serum values. Such lowered mitochondrial respiratory capacity makes the cells more vulnerable to bioenergetic exhaustion. In a pilot study involving two COVID-19 patients with NTIS, we could reinforce our previous observation regarding the role of T3 in the maintenance of adequate peripheral hydroelectrolytic balance. In addition, in these two patients, we demonstrated that by treating their PBMCs with LT3, both in vitro and in vivo, all mitochondrial respiration parameters significantly increased. CONCLUSIONS: Our results regarding the reduction in the serum levels of the reactive oxygen species (ROS) of COVID-19 patients with NTIS support the hypothesis that NTIS could represent an adaptative response to severe COVID-19. However, beside this beneficial effect, we demonstrate that, in the presence of an acute reduction of FT3 serum levels, the mitochondrial respiration is greatly impaired, with a consequent establishment of a hypoenergetic state of the immune cells that may hamper their capacity to react to massive viral infection.

Measurement of Mitochondrial Respiration in Cryopreserved Human Peripheral Blood Mononuclear Cells (PBMCs).

Inflammatory diseases caused by infectious agents such as the SARS-CoV-2 virus can lead to impaired reductive-oxidative (REDOX) balance and disrupted mitochondrial function. Peripheral blood mononuclear cells (PBMCs) provide a useful model for studying the effects of inflammatory diseases on mitochondrial function but can be limited by the need to store these cells by cryopreservation prior to assay. Here, we describe a method for improving and determining PBMC viability with normalization of values to number of living cells. The approach can be applied not only to PBMC samples derived from patients with diseases marked by an altered inflammatory response such as viral infections.

In Vivo and Ex Vivo Mitochondrial Function in COVID-19 Patients on the Intensive Care Unit.

Mitochondrial dysfunction has been linked to disease progression in COVID-19 patients. This observational pilot study aimed to assess mitochondrial function in COVID-19 patients at intensive care unit (ICU) admission (T1), seven days thereafter (T2), and in healthy controls and a general anesthesia group. Measurements consisted of in vivo mitochondrial oxygenation and oxygen consumption, in vitro assessment of mitochondrial respiration in platelet-rich plasma (PRP) and peripheral blood mononuclear cells (PBMCs), and the ex vivo quantity of circulating cell-free mitochondrial DNA (mtDNA). The median mitoVO2 of COVID-19 patients on T1 and T2 was similar and tended to be lower than the mitoVO2 in the healthy controls, whilst the mitoVO2 in the general anesthesia group was significantly lower than that of all other groups. Basal platelet (PLT) respiration did not differ substantially between the measurements. PBMC basal respiration was increased by approximately 80% in the T1 group when contrasted to T2 and the healthy controls. Cell-free mtDNA was eight times higher in the COVID-T1 samples when compared to the healthy controls samples. In the COVID-T2 samples, mtDNA was twofold lower when compared to the COVID-T1 samples. mtDNA levels were increased in COVID-19 patients but were not associated with decreased mitochondrial O2 consumption in vivo in the skin, and ex vivo in PLT or PBMC. This suggests the presence of increased metabolism and mitochondrial damage.

Analysis of mitochondrial function in COVID-19 patients using in vivo and ex vivo techniques

Introduction: Mitochondrial dysfunction has been linked to the persistent hypoxia and altered aerobic glycolytic metabolism seen in COVID-19 patients. This observational pilot study assessed mitochondrial function in COVID-19 patients and healthy controls (HC) utilizing in vivo and ex vivo techniques. Methods: This single center observational study examined COVID-19 patients on two time points, the first within 72 h after intensive care admission (T1), and the second seven days after T1 (T2). HC were age and sex matched to the included COVID-19 patients. In vivo epidermal mitochondrial oxygen utilization was analyzed using the COMET (Cellular Oxygen METabolism) monitor, which employs the protoporphyrin- IX triplet state technique. Ex vivo measurements consisted of in vitro mitochondrial respiration analyzed by the Oroboros O2k respirometer and free mitochondrial DNA (fMtDNA) which was isolated from plasma and quantified by qPCR. Results: 16 COVID-19 sepsis patients and 16 HC were included. The median MitoVO2 of COVID-19 patients on T1 was 4.6 mmHg s-1 [IQR;3.6-6.0], 4.6 mmHg s-1 [IQR;3.9-5.8] on T2 and 5.3 mmHg s-1 [IQR;4.5-6.3] in the HC. Basal platelet respiration did not differ substantially between the three groups, whilst PBMC basal respiration was increased by approximately 80% in the T1 group when contrasted to T2 and the HC. fMtDNA was 14 times higher in the T1 group and 5 times higher in the T2 group when compared to the HC. Conclusions: fMtDNA levels were increased in COVID-19 patients, but were not associated with decreased mitochondrial O2 consumption in vivo in the skin, and ex vivo in platelets or PBMC. This suggests the presence of mitochondrial stress, with concurrent preservation of mitochondrial respiration and function. It must be noted that due to the timing of T1, the optimal measurement window could have been missed. Therefore, the role of mitochondrial dysfunction in COVID-19 should be further evaluated at different time points.

Off-Target In Vitro Profiling Demonstrates that Remdesivir Is a Highly Selective Antiviral Agent.

Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family, such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess the potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Last, the active 5'-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5 to 14 days of continuous exposure, the 50% cytotoxic concentration (CC50) values of RDV ranged from 1.7 to >20 µM, resulting in selectivity indices (SI, CC50/EC50) from >170 to 20,000, with respect to RDV anti-SARS-CoV-2 activity (50% effective concentration [EC50] of 9.9 nM in human airway epithelial cells). Overall, the cellular and biochemical assays demonstrated a low potential for RDV to elicit off-target toxicity, including mitochondria-specific toxicity, consistent with the reported clinical safety profile.