Sex Differences in Energy Sensing, Inflammatory State, and Mitochondrial Biogenesis in Geriatric Post-COVID-19 Patients

Sex Differences in Energy Sensing, Inflammatory State, and Mitochondrial Biogenesis in Geriatric Post-COVID-19 Patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sex Differences in Energy Sensing, Inflammatory State, and Mitochondrial Biogenesis in Geriatric Post-COVID-19 Patients Olesya Vakhrusheva, Misael Estepa, Michael Nnaji, Louis Marx, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7257776/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Viral infections including, respiratory viruses such as the SARS-CoV-2, cause mitochondrial dysfunction and exacerbate systemic inflammation. In the present study, sex differences were investigated in patients who have recovered from the acute phase of infection regardless of whether they have ongoing symptoms (post-COVID-19 condition). Peripheral blood mononuclear cells (PBMCs) and plasma samples were collected from hospitalized geriatric patients following their PCR-confirmed negative result for SARS-CoV-2infection. The inflammatory state was assessed by measuring the expression of inflammatory markers using real-time PCR and ELISA. Furthermore, the expression of oxidative, and mitochondrial markers along with metabolic sensors, i.e., AMPK and Sirt1, was examined. The metabolic sensor AMPK was activated (pAMPK/AMPK ratio) only in post-COVID-19 women and was accompanied by women-specific elevation of circulating TNF-α levels in plasma and upregulation of pro-inflammatory mediators IL-1β and IL-18 in their PBMCs. The expression of the antioxidant SOD2 and its acetylation were reduced in PBMCs obtained from male post-COVID-19 patients, whereas SOD2 was hyperacetylated in women. The NRF1 expression was notably upregulated in post-COVID-19 female PBMCs. In addition, the mitochondrial genes cox1 and nd4 were upregulated in post-COVID-19 female patients, whereas, the expression of Sirt1, Sirt3, and TFAM remained unchanged in all studied groups. In summary, the present study revealed a women-specific elevation of inflammatory markers associated with increased AMPK activity and acetylation of mitochondrial SOD2. Health sciences/Biomarkers Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Biological sciences/Microbiology Biological sciences/Molecular biology post-COVID-19 mitochondrial homeostasis sex differences AMPK activity SOD acetylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Plain summary After recovering from COVID-19, older adults can still have changes in their body’s immune response and energy systems. This study looked at blood samples from men and women who had been hospitalized for COVID-19 but had since tested negative. We found that women had higher levels of certain inflammation markers and more activity in the protein AMPK that helps manage energy in cells. Their cells also showed more signs of stress protection. In men, the body’s natural defense against cell damage (the antioxidant SOD2) was weaker after COVID-19. Certain genes linked to cell energy were more active in women, but not in men. Overall, women showed more ongoing inflammation and changes in cell energy use after COVID-19, while men had less protection against cell stress. These differences could help doctors better understand and treat long-term effects of COVID-19 in both men and women. Highlights - In post-COVID-19 women, there was a unique activation of the metabolic sensor AMPK. - Mitochondrial genes (cox1 and nd4) and the transcription factor NRF1 were upregulated in women’s PBMCs. - Post-COVID − 19 men showed reduced expression and acetylation of the antioxidant SOD2 in their PBMCs 1. Introduction Several studies have addressed sex differences in the severity and outcome of SARS-CoV-2 (COVID-19) infection. Men have higher rates of hospitalization and mortality during acute infection, 1 whereas women are more frequently affected by post-COVID-19 syndrome (PCS) and report more often core symptoms. 2 Several studies have suggested that women may experience a persistent low-grade inflammation in post-COVID-19 con-dition. 3 This highlights an urgent need for further research exploring sex-specific issues of low-grade inflammation that leads to oxidative stress and results in tissue damage. 4 , 5 One hypothesis of post-COVID-19 pathology currently explored is a virally-induced, chronic and self-sustaining metabolic imbalance characterized by mitochondrial dysfunction. This condition maintains a non-resolving state where reactive oxygen species (ROS) continuously drive inflammation and promote a shift towards glycolysis. One key cellular energy sensor is the AMP-activated protein kinase (AMPK), which is directly activated by ROS accumulation and by ATP depletion. AMPK regulates metabolic pathways that affect the redox state and promote mitochondrial biogenesis. 6 Additionally, the NAD + -dependent deacetylase, Sirt1, plays a crucial role in regulating inflammation and ROS, and thus pre-venting oxidative stress. 7 , 8 Dysregulation of NAD metabolism has been observed in post-COVID-19 patients, and Sirt1 activity is often downregulated, which may contribute to prolonged inflammation and PCS. 9 Moreover, Sirt1 regulates NFκB by deacetylating the RelA/p65 protein at lysine 310. 10 This deacetylation process inhibits the transcriptional activity of NFκB, thereby suppressing NFκB-mediated inflammatory responses. 11 – 13 Mitochondrial impairment and inflammation are tightly interconnected and both have been implicated in the pathogenesis of different diseases. 14 In acute COVID-19 and post-COVID-19 phases, mitochondrial dysfunction, e.g. impaired energy production, in-creased ROS production and decreased antioxidant levels, lead to the release of proinflammatory cytokines. This heightens systemic inflammation and contributes to disease severity. 15 In addition, mitochondrial impairment affects immune cell function, potentially contributing to the prolonged symptoms of post-COVID-19. 16 , 17 Thus, mitochondrial dysfunction during acute COVID-19 and especially during post-COVID-19 stage may lead to dysregulation of the immune system and contribute to the development of PCS. 17 , 18 However, little is known about the inflammatory status, mitochondrial biogenesis, and energy sensing in older male and female patients recovering from COVID-19 disease. Therefore, in the present study, we evaluated those parameters in plasma and PBMCs samples collected during the early recovery stage after COVID-19 infection. 2. Materials and Methods 2.1. Patient Cohort 35 patients (women: n = 18, men: n = 17) treated at the Department of Geriatrics and Medical Gerontology, Charité - Universitätsmedizin Berlin were recruited for the present study. 17 patients had pneumonia, requiring hospitalization, and were initially PCR-positive for SARS-CoV-2. Peripheral EDTA-blood samples were collected within 4 weeks from post-COVID-19 patients being blood PCR-negative for SARS-CoV-2. The timing of sample collection relative to acute illness showed that patients in the COVID positive group were recruited after being tested negative (mean = 9.15, SD = 7.88). The control group comprised patients who were tested negative for SARS-CoV-2 and presented to the emergency department with acute inflammatory, infectious, or traumatic disorders (Suppl. table 1). Patients were between 57 and 95 years old (Table 1 ). Table 1 Characterization of patients with post-COVID-19. Men Women control (n = 9) post-COVID-19 (n = 8) control (n = 9) post-COVID-19 (n = 9) Age 82.00 (10.75) 78.00 (16.75) 82.00 (4.5) 80.00 (10) BMI 24.31 (8.87) 23.84 (4.33) 22.20 (4.00) 24.67 (7.96) CCI 9.00 (3.00) 7.00 (5.50) 5.00 (3.00) 7.00 (4.50) HbA1c (%) 5.90 (2.45) 6.00 (1.25) 5.80 (1.35) 6.60 (2.10) Leukocyte (/nl) 11.94 (8.66) 9.01 (5.07) 8.50 (5.55) 9.03 (4.75) CRP (mg/l) 74.05 (97.77) 36.50 (96.30) 47.20 (197.10) 56.30 (60.85) LDH (U/l) 252 (141) 403 (150)** 278 (189) 284 (224) Vitamin D (nmol/l) 72.90 (86.47) 28.00 (51.05) 30.95 (30.90) 32.55 (52.25) Albumin (g/dl) 29.00 (12.63) 28.30 (4.20) 33.55 (11.37) 29.40 (5.40) Vitamin B12 (ng/l) 655 (360) 382 (926) 426 (687) 506 (1253) Folic acid (µg/l) 11.80 (13.60) 4.80 (7.05)* 6.70 (15.08) 4.50 (4.45) Data are shown as median and IQR. n = 8–9. Control vs. post-COVID-19: *p < 0.05, **p < 0.01. Elisa analyses were performed to analysed CRP, LDH, Vitamin D, albumin, Vitamin B12 and folic acid. BMI: body mass index, CCI: Charlon’s Comorbidity Index, HbA1c: haemoglobin A1c, CRP: c reactive protein, LDH: lactate dehydrogenase, IQR: interquartile range. We obtained informed consent from all study participants. Sample collection and the experimental protocols were approved by the Scientific Board at the Charité – Universitätsmedizin Berlin (EA4/154/16 and EA1/037/22). All experiments were performed in accordance with the German regulations and the ethical standards as laid down in the Declaration of Helsinki. 2.2. Clinical and Laboratory Parameters The patient’s clinical, laboratory, and demographic parameters were obtained from electronic medical health records (EMHR). These included age, biological sex, COVID-19 status, acute diagnosis, Body Mass Index (BMI), Charlson Comorbidity Index (CCI), pre-existing lung disease, hyperglycemic index (HbA1c), immune status, relevant medication in the context of COVID-19 infection and disease severity. 2.3. Preparation of Plasma Samples and Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Biocoll®, Bio&Sell, Germany) density centrifugation as previously described. Briefly, 10 to 20 ml of venous blood was collected by peripheral phlebotomy in EDTA-coated vials (BD, Germany) from both post-COVID-19 and non-COVID-19 patients. Thereafter, the blood was di-luted with an equal amount of PBS (Biochrom, Germany) and subjected to Ficoll-density centrifugation at 500 g for 20 min. The resulting supernatant was designated as plasma. Plasma samples were snap-frozen and stored at -80°C. PBMCs were collected from the interphase, washed with PBS and then pelleted at 200 g for 20 min. Cell pellets were subsequently incubated for 5 min in a red blood cell lysis buffer (Merck, Germany), washed with PBS and centrifuged at 300 g for 10 min. The cell pellets were promptly stored at -80°C. 2.4. RNA Extraction and Quantitative Real-Time PCR Total RNA from PBMCs was isolated in RNA-Bee reagent (Amsbio, UK). The reverse transcription of RNA into cDNA was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Germany) following the manufacturer’s instructions. Quantitative real-time PCR was performed using the Brilliant SYBR Green qPCR master mix (Applied Biosystems, USA). The relative amount of the mRNA was determined using the comparative threshold (Ct) method as previously described. Expression of the target genes was normalized to the expression of hypoxanthine phosphoribosyl transferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Suppl. table 2). In each case, the samples were plotted as technical duplicates. 2.5. Protein Extraction and Immunoblotting PBMCs were homogenized in Laemmli buffer as has been previously described. Proteins were quantified using the BCA Assay (Thermo Scientific Pierce Protein Biology, Germany). Equal protein amounts from each sample were separated using SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were immunoblotted overnight with the following primary antibodies: AMPK (1:2000, #2532L, Cell Signaling, USA), pAMPK (1:2000, Thr172, #2535L, Cell Signaling, USA), ac-lysine (1:500, #9411, Cell Signaling, USA), SOD2 (1:1000, #13194, Cell Signaling, USA), and acSOD2 (1:1000, ab13533, Abcam, UK). Equal sample loading was confirmed by an analysis of actin (1:1500, sc-1616-R, Santa Cruz, USA) and α-tubulin (1: 5000, #3873, Cell Signaling, USA) expression. All proteins were visualized using ECL Plus (GE Healthcare, Buckinghamshire, UK), detected with the ChemiDoc system (Bio-Rad Laboratories, USA) and quantified using ImageLab (version 5.2.1 build 11, Bio-Rad Laboratories, USA). 2.6. Detection of TNF-α, Extracellular SOD3 and DNA Damage in Plasma Samples by ELISA The concentration of circulating TNF-α was analyzed in plasma samples from post-COVID-19 patients and the control group using ELISA according to manufacturer's recommendations (BioLegend, Germany). The extracellular SOD3 content was also deter-mined in plasma from post-COVID-19 and non-COVID-19 patients using a Superoxide Dismutase (SOD) Colorimetric Activity Kit (Invitrogen, Germany). DNA damage was estimated utilizing the DNA damage Competitive ELISA Kit (Invitrogen, Germany). All samples were measured using a microplate reader (Tecan, Switzerland) as recommended by the manufacturer. In each case, the samples were plotted as technical duplicates. 2.7. Mitochondrial Mass Analysis The amount of nuclear and mitochondrial DNA (mt-DNA) was analyzed via quantitative real-time PCR utilizing the Brilliant SYBR Green qPCR master mix (Applied Biosystems, USA). The ratio of mt-DNA to nuclear DNA was built to quantify the mitochondrial mass content. In each case, the samples were plotted as technical duplicates. 2.8. Statistical Analysis The data are given as the mean ± standard error of the mean (SEM). The data were evaluated using the non-parametric test (Mann-Whitney test for two independent groups). Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, USA). Statistical significance was accepted when p < 0.05. 3. Results 3.1. Characterization of Post-COVID-19 Patients To characterize the study group, age, BMI, CCI as well as inflammatory and biochemical blood parameters were analyzed. The majority of the parameters show no differences between the analyzed groups (p > 0.05) (Table 1 ). Among post-COVID-19 patients, 78% of men experienced severe COVID-19 disease, compared to only 44% of women. In post-COVID-19 male patients, the lactate dehydrogenase (LDH) concentration was significantly increased compared to the control group (p < 0.01), while the folic acid concentration was significantly decreased in male post-COVID-19 patients (p < 0.05) (Table 1 ). We found that patients in the post-COVID-19 group were more likely to have short-ness of breath/malaise/fever, etc. (data not shown). In regard to the functional capacity of the patients – as defined by the clinically validated Barthel index – we found no statistical differences between both groups (p = 0.685). This might be due to the fact that our study was underpowered to detect any such differences, especially since post-COVID-19 has been shown to be associated with progressive functional decline in survivors. 19 Further-more, this could be due to confounding factors related to multimorbidity associated with ageing, especially in the light of the fact that both groups do not show any significant differences at baseline (p = 0.850). In addition, due to the lack of follow-up, we were not able to capture the duration of complaints since the study was conducted in an in-hospital setting (data not shown). 3.2. Upregulated AMPK Phosphorylation in Samples from Post-COVID-19 Women The expression and activity of the key metabolic sensor AMPK, a regulator of cellular energy homeostasis and multiple aspects of cell metabolism, have been reported to be dysregulated in several diseases and inflammatory conditions. 13 , 20 However, its role in the early recovery stage of COVID-19 is poorly understood. No alteration of the total AMPK protein expression in PBMCs samples between all groups investigated was found (Fig. 1 A, B). Phosphorylated, i.e., activated form of AMPK, was significantly upregulated only in post-COVID-19 women compared to non-COVID-19, control, patients (p = 0.0167) (Fig. 1 A, C). 3.3. Proinflammatory Mediators Are Increased in PBMC and Plasma Samples of Post-COVID-19 Women Activated AMPK and NF-κB play a crucial role in modulating inflammatory responses in monocytes and may have significant implications for the treatment of inflammatory conditions. 21 mRNA expression of neither NF-κB nor its inhibitor, IκBα was found to be modulated in post-COVID-19 female and male PBMC samples compared to the control (Fig. 2 A, B). Since the post-COVID-19 is associated with systemic inflammation, 18 , 22 inflammatory markers in plasma and PBMCs samples were analyzed. A significant women-specific upregulation of proinflammatory IL-1β and IL-18 mRNA expression in PBMCs samples of post-COVID-19 patients was observed (p = 0.0074 and p = 0.0057, respectively) (Fig. 2 C, D). No significant differences were observed in the mRNA expression of IL-1β downstream effectors IL-6, TLR4, and TNF-α in PBMCs samples in the investigated groups (Fig. 2 E-G). In addition, circulating TNF-α was significantly elevated in the plasma of post-COVID-19 women compared to the control group (p = 0.0422) (Fig. 2 H). 3.4. Expression of Mitochondrial Proteins and Genes in Post-COVID-19 Patients Mitochondria play an important role in regulating immune cell activity, inflammation, and oxidative stress. 23 , 24 The mRNA expression of the mitochondria-encoded genes, cox1 , and nd4 , was significantly upregulated in PBMC samples of post-COVID-19 women compared to the control group and the post-COVID men (p = 0.019 and p = 0.029; p = 0.029 and p = 0.007, respectively) (Fig. 3 A, B). In contrast, the expression of nucleus-encoded ndusf1 was similar in PBMC samples from post-COVID-19 and non-COVID-19 patients (Fig. 3 C). Mitochondrial mass, as indicated by the rnr2/β-globin ratio, remained un-changed in post-COVID-19 patients (Fig. 3 D). Similarly, no differences in the expression of Sirt3 and TFAM were found (Fig. 3 E, F). The expression of Sirt1 was not altered in post-COVID-19 patients compared to non-COVID-19 patients (Fig. 3 G). 3.5. Oxidative Stress and Expression of Antioxidative Enzyme in Post-COVID-19 Patients Inflammation is typically associated with the increased ROS formation, leading to oxidative stress. 25 Analysis of oxidative DNA damage, measured as 8-OHdG, revealed no differences in plasma concentrations among the investigated groups (Fig. 4 A). Additionally, two isoforms of SOD, i.e., the extracellular SOD3 and the intramitochondrial SOD2, were analyzed in plasma or PBMCs samples, respectively. No differences in the SOD3 concentration were determined between the plasma of post-COVID-19 female patients and the control group. In contrast, in post-COVID-19 male patients, SOD3 levels were significantly increased compared to the control group (p = 0.0468) (Fig. 4 B). The expression of intramitochondrial SOD2 was significantly downregulated in post-COVID-19 men and became significantly lower compared with post-COVID-19 female patients (Fig. 4 C, D). The acetylation state of SOD2 is an important regulator of its activity. 26 , 27 Analysis of SOD2 acetylation revealed its significant elevation in post-COVID-19 female patients (p = 0.0402) (Fig. 4 C, E), suggesting a potential down-regulation of SOD2 activity in this group. In contrast, SOD2 acetylation was markedly reduced in post-COVID-19 men. Additionally, the expression levels of NRF1 and NRF2 were upregulated in post-COVID-19 women, however only the upregulation of NRF1 demonstrated statistically significant differences (p = 0.0539) (Fig. 4 F, G). 4. Discussion Growing evidence suggests that PCS is a significant burden of COVID-19-related diseases. 18 , 28 However, little is known about post-COVID-19 patients, particularly for the geriatric population. The alterations in energy sensing, mitochondrial homeostasis, and inflammatory state, especially in circulating immune cells, may significantly contribute to the understanding of post-COVID-19. 29 It has been documented that female, but not male, patients are at increased risk of developing PSC.30 Studies have shown that COVID-19 male patients have higher rates of hospitalization and mortality during acute viral infection, 1 whereas women are prone to develop PSC and experience core symptoms more fre-quently. 2 Our findings suggest that women are likely to exhibit more pronounced systemic post-COVID-19 inflammation during early recovery stage as evidenced by increased circulated TNF-α levels in plasma and elevated proinflammatory interleukins IL-1β and IL-18 in PBMCs. The persistence of elevated inflammatory markers in women with ongoing symptoms suggests that prolonged inflammation may play a role in post-COVID-19 pathogenesis. To fully understand sex differences in post-COVID-19-related inflammation, more targeted studies that specifically compare inflammatory stage between men and women during the recovery phase are needed. The upregulation of proinflammatory pathways is usually accompanied by enhanced ROS formation. 25 , 31 Although no alteration in the concentration of the oxidative stress marker 8-OHdG was found, an enhanced anti-oxidative defense was observed in post-COVID-19 male patients compared to females, as indicated by the increased levels of extracellular SOD3 in plasma. Upregulation of SOD3 in post-COVID-19 male patients might indicate a compensatory response to inflammation, as SOD3 tightly regulates the tissue redox balance under physiological stress caused by viral infections. In contrast, expression of mitochondrial SOD2 was downregulated in post-COVID-19 men. It should be noted here that the activity of SOD2 strongly depends on the acetylation state of this protein. 32 Analysis of SOD2 acetylation revealed a notable sex difference: post-COVID-19 female patients exhibited significant hyperacetylation, whereas post-COVID-19 male patients showed a marked reduction in SOD2 acetylation, although this reduction did not reach statistical significance. The hyperacetylation of mitochondrial SOD2 in post-COVID-19 women may be due to reduced expression or activity of the main mitochondrial deacetylase Sirt3. Since its ex-pression in post-COVID-19 PBMC samples did not differ from the control group, Sirt3 activity likely declined in post-COVID-19 female patients. Several studies have demonstrated that Sirt3-dependent deacetylation of mitochondrial matrix proteins is associated with improved mitochondrial function. 33 – 35 and alleviation of inflammatory phenotype in immune cells, like macrophages. 36 In line with these findings, the present study documents an association of women-specific SOD2 hyperacetylation with increased levels of the pro-inflammatory markers in post-COVID-19 patients. Although the expression of mitochondrial SOD2, Sirt3, and the mitochondrial mass was not altered in the studied post-COVID-19 patients, the expression of the mitochondria-encoded genes was elevated in women. Mitochondrial dysfunction in immune cells has been implicated in the context of severe acute SARS-CoV2 infection 37 , 38 and in post-COVID-19 patients, 16 suggesting that it may be still affected in post-COVID-19. While mitochondrial respiration was not assessed in the current study, our data suggest an energy deficit in immune cells of post-COVID-19 female patients, leading to a compensatory upregulation of mitochondrial genes. This is further supported by elevated phosphorylation of AMPK, a key cellular energy sensor. Chronic AMPK activation may reflect energy stress in PBMC from both post-COVID-19 female and male patients. Consistent with our findings, a previous study by Ajas et al. has revealed compromised mitochondrial function and an energy deficit in PBMCs from COVID-19 patients. 39 Thus, activation of AMPK accompanied by the mitochondrial gene upregulation observed in women may be a natural compensatory mechanism to combat the aberrant inflammation. The absence of this mechanism in men may reflect a higher mortality and hospitalization rate in COVID-19 male than female patients. 1 Therefore, improving mitochondrial homeostasis through AMPK activation could be a promising therapy to alleviate the severity of COVID-19 in male patients. In conclusion, the current study analyzing plasma and PBMCs during post-COVID-19 condition revealed women-specific elevation of inflammatory markers accompanied by the hyperacetylation of mitochondrial SOD2 and upregulation of mitochondrial genes. These changes, together with activated AMPK, may indicate energetic stress, potentially explaining the higher prevalence of post-COVID-19 condition in women. Limitation of the Study In this study, a small cohort of patients was investigated due to the limited availability of hospitalized geriatric, untreated patients who had recovered from COVID-19 infection. Another major limitation of the present study is that it was conducted during the SARS-CoV-2 pandemic, which resulted in deviations from the intended recruitment protocol; specifically, patients were not consistently enrolled within first 4 weeks after symptom onset. In retrospect, this recruitment approach does not align with the current definitions of post-acute sequelae of SARS-CoV-2 infection, which were established subsequently. Nevertheless, this aspect may also be considered a strength, as it provides valuable in-sights into the early subcellular events following SARS-CoV-2 infection. Especially, the key effects observed in the study, such as alterations in the expression of mitochondrial genes and inflammatory markers in post-COVID-19 women compared to non-COVID-19 individuals, are robust enough to support the validity of the conclusions. Furthermore, the restriction of the statistical analyses in comparing different sample sizes across various assays. This was due to a reduced amount of PBMCs in some individuals, which prevented us from obtaining enough material to test in all experiments. Declarations Author Contributions: O.V. analyzed data, prepared figures and wrote the main manuscript text. M.E. analyzed data, prepared figures and wrote the main manuscript text. L.M. isolated PBMC and revised the manuscript. M.N. provided the blood samples and undertook patient characterization and revised the manuscript. J.T. wrote and revised the manuscript. Y.L. analyzed the data and wrote the main part of the manuscript, and M.B. conceived the project, analyzed the data, prepared the figures, and wrote the main manuscript text. All authors have read and agreed to the published version of the manuscript. Funding: We acknowledge support from the Open Access Publication Fund of Universitätsklinikum Tübingen. Ethics and Consent to Participate declaration: We obtained informed consent from all study participants. Sample collection and the experimental protocols were approved by the Scientific Board at the Charité – Universitätsmedizin Berlin (EA4/154/16 and EA1/037/22). All experiments were performed in accordance with the German regulations and the ethical standards as laid down in the Declaration of Helsinki. Data Availability Statement: The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author. Consent to publish declaration: not applicable. Acknowledgments: We thank Natalia Haritonow for the technical assistance. Conflicts of Interest: The authors declare no competing interests. References Gebhard, C. E. et al. Sex versus gender-related characteristics: which predicts clinical outcomes of acute COVID-19? Intensive Care Med. 48 , 1652–1655 (2022). Pela, G. et al. Sex-Related Differences in Long-COVID-19 Syndrome. J. Womens Health (Larchmt) . 31 , 620–630 (2022). Koczulla, A. et al. 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Acetylation of Mitochondrial Proteins in the Heart: The Role of SIRT3. Front. Physiol. 9 , 1094 (2018). Barcena, M. L. et al. Upregulation of Mitochondrial Sirt3 and Alleviation of the Inflammatory Phenotype in Macrophages by Estrogen. Cells 2024;13. Romao, P. R. et al. Viral load is associated with mitochondrial dysfunction and altered monocyte phenotype in acute severe SARS-CoV-2 infection. Int. Immunopharmacol. 108 , 108697 (2022). De la Cruz-Enriquez, J., Rojas-Morales, E., Ruiz-Garcia, M. G., Tobon-Velasco, J. C. & Jimenez-Ortega, J. C. SARS-CoV-2 induces mitochondrial dysfunction and cell death by oxidative stress/inflammation in leukocytes of COVID-19 patients. Free Radic Res. 55 , 982–995 (2021). Ajaz, S. et al. Mitochondrial metabolic manipulation by SARS-CoV-2 in peripheral blood mononuclear cells of patients with COVID-19. Am. J. Physiol. Cell. Physiol. 320 , C57–C65 (2021). Additional Declarations No competing interests reported. Supplementary Files Suppltable1.docx Suppl.table2.docx AMPKoriginalblots.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviews received at journal 08 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 17 Mar, 2026 Editor assigned by journal 26 Nov, 2025 Editor invited by journal 06 Aug, 2025 Submission checks completed at journal 05 Aug, 2025 First submitted to journal 05 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7257776","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":608463443,"identity":"f3ce7a76-97a8-4acc-8be4-7861640c38dd","order_by":0,"name":"Olesya Vakhrusheva","email":"","orcid":"","institution":"Eberhard Karls University of Tuebingen","correspondingAuthor":false,"prefix":"","firstName":"Olesya","middleName":"","lastName":"Vakhrusheva","suffix":""},{"id":608463444,"identity":"2fff6196-8a5e-46e4-919b-14278f905313","order_by":1,"name":"Misael Estepa","email":"","orcid":"","institution":"Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Misael","middleName":"","lastName":"Estepa","suffix":""},{"id":608463445,"identity":"5e42cd51-af26-4faa-88ab-806327457dd5","order_by":2,"name":"Michael Nnaji","email":"","orcid":"","institution":"Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Nnaji","suffix":""},{"id":608463446,"identity":"e4cd9cf1-679a-485a-a7eb-a7167c496f8d","order_by":3,"name":"Louis Marx","email":"","orcid":"","institution":"Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, Berlin Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Louis","middleName":"","lastName":"Marx","suffix":""},{"id":608463447,"identity":"9ab86ef3-f354-4fa3-b2bc-da7b781a4442","order_by":4,"name":"Julia Temp","email":"","orcid":"","institution":"Eberhard Karls University of Tuebingen","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Temp","suffix":""},{"id":608463448,"identity":"06c615e6-4dd7-4bbf-a77d-6ada63df7647","order_by":5,"name":"Yury Ladilov","email":"","orcid":"","institution":"University Hospital, Brandenburg Medical School Theodor Fontane","correspondingAuthor":false,"prefix":"","firstName":"Yury","middleName":"","lastName":"Ladilov","suffix":""},{"id":608463449,"identity":"6573dbae-625a-42be-a9f5-d271498a5016","order_by":6,"name":"Maria Luisa Barcena","email":"data:image/png;base64,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","orcid":"","institution":"Eberhard Karls University of Tuebingen","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"Luisa","lastName":"Barcena","suffix":""}],"badges":[],"createdAt":"2025-07-31 04:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7257776/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7257776/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104998804,"identity":"18411f3f-8458-4f1f-8db4-9e8ff19dcf17","added_by":"auto","created_at":"2026-03-19 16:31:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":239307,"visible":true,"origin":"","legend":"\u003cp\u003epAMPK/AMPK ratio is increased in PBMC samples from post-COVID-19 female patients. Representative western blot images (A) and statistical analysis of AMPK expression (B) and pAMPK/AMPK ratio (C) in PBMCs lysates of post-COVID-19 and non-COVID-19 women and men. All data were normalized to the male control and shown in relative units (r.u.), representing the means ± SEM (n = 8-9/group). All samples were run in the same gel.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/1762aa011c6f814a78badef1.png"},{"id":104998805,"identity":"1f624bdc-fdc9-43a2-acb9-d3f9da712875","added_by":"auto","created_at":"2026-03-19 16:31:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":353963,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of proinflammatory markers in plasma and PBMCs samples from post-COVID-19 women and men. Statistical quantification of mRNA expression of NF-κB (A), IκBα (B), IL-1β, (C) IL-18, (D) IL-6, (E), TLR4 (F), and TNF-α, (G) in PBMCs lysates of post-COVID-19 and control group. Elevated levels of circulating TNF-α (H) determined by ELISA in post-COVID-19 women plasma samples. Data are shown as the means ± SEM (n = 8-9/group). All data were normalized to the non-COVID-19 men control.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/e20541c0af3a0d59a3acf889.png"},{"id":104998809,"identity":"f6b8681f-dd91-40e3-aa18-9f34d0630a3f","added_by":"auto","created_at":"2026-03-19 16:31:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":258916,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations in the expression of mitochondria-encoded genes in post-COVID-19 patients. Relative mRNA expression of \u003cem\u003ecox1\u003c/em\u003e (A), \u003cem\u003end4\u003c/em\u003e (B) and \u003cem\u003endhsf1\u003c/em\u003e (C) in PBMC samples of post-COVID-19 women and men. Mitochondrial mass was analyzed as the mt-rnr2/β-globin DNA ratio (D). Relative Sirt3 (E), TFAM (F) and Sirt1 (G) transcript levels in lysates of PBMCs from post-COVID-19 and non-COVID-19 women and men. Data are shown as the means ± SEM (n = 4-10/group). All data were normalized to the male control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/0603b1b0e8fac00f7eacfdb1.png"},{"id":105036145,"identity":"06d641fb-f5f3-4e0d-b511-124f86e8d5b9","added_by":"auto","created_at":"2026-03-20 07:29:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":430331,"visible":true,"origin":"","legend":"\u003cp\u003eAlterations in oxidative stress markers in post-COVID-19 patients. Analysis of oxidative DNA damage by 8-OHdG (A) and extracellular SOD3 (B) tested in plasma samples from post- and non-COVID-19 women and men. Representative western blot images (C) and statistics of total SOD2 (D) and acetylated SOD2 (E) expression as well as quantification of NRF1 (F) and NRF2 (G) in PBMCs from post-COVID-19 and non-COVID-19 women and men. Data are shown as the means ± SEM (n = 4-10/group). All data were normalized to the men's control and expressed in relative units (r.u.).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/143ea92b3066c01a0fe9417c.png"},{"id":105037767,"identity":"6efec3f9-d778-42fa-bf75-83a4af1be66e","added_by":"auto","created_at":"2026-03-20 07:40:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2421756,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/08b5657f-f4f3-4902-8d4c-351a28b0db42.pdf"},{"id":105035770,"identity":"57e0c6e2-629e-4a22-87c5-2062f406cd06","added_by":"auto","created_at":"2026-03-20 07:26:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14513,"visible":true,"origin":"","legend":"","description":"","filename":"Suppltable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/cdc11384aa75c1c2bde787b1.docx"},{"id":105035777,"identity":"747152d9-c191-483a-b7b4-9328a9d6832b","added_by":"auto","created_at":"2026-03-20 07:26:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15429,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/0e54a13087a7099726549312.docx"},{"id":104998807,"identity":"d529d909-db45-4f95-b48e-04fa490d7b1f","added_by":"auto","created_at":"2026-03-19 16:31:29","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":296876,"visible":true,"origin":"","legend":"","description":"","filename":"AMPKoriginalblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7257776/v1/3d339fd659056d9e456b095a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSex Differences in Energy Sensing, Inflammatory State, and Mitochondrial Biogenesis in Geriatric Post-COVID-19 Patients\u003c/p\u003e","fulltext":[{"header":"Plain summary","content":"\u003cp\u003eAfter recovering from COVID-19, older adults can still have changes in their body\u0026rsquo;s immune response and energy systems. This study looked at blood samples from men and women who had been hospitalized for COVID-19 but had since tested negative. We found that women had higher levels of certain inflammation markers and more activity in the protein AMPK that helps manage energy in cells. Their cells also showed more signs of stress protection. In men, the body\u0026rsquo;s natural defense against cell damage (the antioxidant SOD2) was weaker after COVID-19. Certain genes linked to cell energy were more active in women, but not in men. Overall, women showed more ongoing inflammation and changes in cell energy use after COVID-19, while men had less protection against cell stress. These differences could help doctors better understand and treat long-term effects of COVID-19 in both men and women.\u003c/p\u003e\n"},{"header":"Highlights","content":"\u003cp\u003e- In post-COVID-19 women, there was a unique activation of the metabolic sensor AMPK.\u003c/p\u003e\u003cp\u003e- Mitochondrial genes (cox1 and nd4) and the transcription factor NRF1 were upregulated in women\u0026rsquo;s PBMCs.\u003c/p\u003e\u003cp\u003e- Post-COVID \u0026minus;\u0026thinsp;19 men showed reduced expression and acetylation of the antioxidant SOD2 in their PBMCs\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eSeveral studies have addressed sex differences in the severity and outcome of SARS-CoV-2 (COVID-19) infection. Men have higher rates of hospitalization and mortality during acute infection,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e whereas women are more frequently affected by post-COVID-19 syndrome (PCS) and report more often core symptoms.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Several studies have suggested that women may experience a persistent low-grade inflammation in post-COVID-19 con-dition.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This highlights an urgent need for further research exploring sex-specific issues of low-grade inflammation that leads to oxidative stress and results in tissue damage.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOne hypothesis of post-COVID-19 pathology currently explored is a virally-induced, chronic and self-sustaining metabolic imbalance characterized by mitochondrial dysfunction. This condition maintains a non-resolving state where reactive oxygen species (ROS) continuously drive inflammation and promote a shift towards glycolysis. One key cellular energy sensor is the AMP-activated protein kinase (AMPK), which is directly activated by ROS accumulation and by ATP depletion. AMPK regulates metabolic pathways that affect the redox state and promote mitochondrial biogenesis.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Additionally, the NAD\u003csup\u003e+\u003c/sup\u003e-dependent deacetylase, Sirt1, plays a crucial role in regulating inflammation and ROS, and thus pre-venting oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Dysregulation of NAD metabolism has been observed in post-COVID-19 patients, and Sirt1 activity is often downregulated, which may contribute to prolonged inflammation and PCS.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Moreover, Sirt1 regulates NFκB by deacetylating the RelA/p65 protein at lysine 310.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e This deacetylation process inhibits the transcriptional activity of NFκB, thereby suppressing NFκB-mediated inflammatory responses.\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMitochondrial impairment and inflammation are tightly interconnected and both have been implicated in the pathogenesis of different diseases.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e In acute COVID-19 and post-COVID-19 phases, mitochondrial dysfunction, e.g. impaired energy production, in-creased ROS production and decreased antioxidant levels, lead to the release of proinflammatory cytokines. This heightens systemic inflammation and contributes to disease severity.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In addition, mitochondrial impairment affects immune cell function, potentially contributing to the prolonged symptoms of post-COVID-19.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Thus, mitochondrial dysfunction during acute COVID-19 and especially during post-COVID-19 stage may lead to dysregulation of the immune system and contribute to the development of PCS.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, little is known about the inflammatory status, mitochondrial biogenesis, and energy sensing in older male and female patients recovering from COVID-19 disease. Therefore, in the present study, we evaluated those parameters in plasma and PBMCs samples collected during the early recovery stage after COVID-19 infection.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Patient Cohort\u003c/h2\u003e \u003cp\u003e35 patients (women: n\u0026thinsp;=\u0026thinsp;18, men: n\u0026thinsp;=\u0026thinsp;17) treated at the Department of Geriatrics and Medical Gerontology, Charit\u0026eacute; - Universit\u0026auml;tsmedizin Berlin were recruited for the present study. 17 patients had pneumonia, requiring hospitalization, and were initially PCR-positive for SARS-CoV-2. Peripheral EDTA-blood samples were collected within 4 weeks from post-COVID-19 patients being blood PCR-negative for SARS-CoV-2. The timing of sample collection relative to acute illness showed that patients in the COVID positive group were recruited after being tested negative (mean\u0026thinsp;=\u0026thinsp;9.15, SD\u0026thinsp;=\u0026thinsp;7.88). The control group comprised patients who were tested negative for SARS-CoV-2 and presented to the emergency department with acute inflammatory, infectious, or traumatic disorders (Suppl. table 1). Patients were between 57 and 95 years old (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of patients with post-COVID-19.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eWomen\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epost-COVID-19 (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epost-COVID-19\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.00 (10.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.00 (16.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.00 (4.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.00 (10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.31 (8.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.84 (4.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.20 (4.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.67 (7.96)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.00 (3.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.00 (5.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.00 (3.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.00 (4.50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbA1c (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.90 (2.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.00 (1.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.80 (1.35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.60 (2.10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeukocyte (/nl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.94 (8.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.01 (5.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.50 (5.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.03 (4.75)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRP (mg/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.05 (97.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.50 (96.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.20 (197.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.30 (60.85)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDH (U/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e252 (141)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e403 (150)**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e278 (189)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e284 (224)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin D (nmol/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.90 (86.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.00 (51.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.95 (30.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.55 (52.25)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.00 (12.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.30 (4.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.55 (11.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.40 (5.40)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin B12 (ng/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e655 (360)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e382 (926)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e426 (687)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e506 (1253)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFolic acid (\u0026micro;g/l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.80 (13.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.80 (7.05)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.70 (15.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.50 (4.45)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are shown as median and IQR. n\u0026thinsp;=\u0026thinsp;8\u0026ndash;9. Control vs. post-COVID-19: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eElisa analyses were performed to analysed CRP, LDH, Vitamin D, albumin, Vitamin B12 and folic acid. BMI: body mass index, CCI: Charlon\u0026rsquo;s Comorbidity Index, HbA1c: haemoglobin A1c, CRP: c reactive protein, LDH: lactate dehydrogenase, IQR: interquartile range.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e We obtained informed consent from all study participants. Sample collection and the experimental protocols were approved by the Scientific Board at the Charit\u0026eacute; \u0026ndash; Universit\u0026auml;tsmedizin Berlin (EA4/154/16 and EA1/037/22). All experiments were performed in accordance with the German regulations and the ethical standards as laid down in the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Clinical and Laboratory Parameters\u003c/h2\u003e \u003cp\u003eThe patient\u0026rsquo;s clinical, laboratory, and demographic parameters were obtained from electronic medical health records (EMHR). These included age, biological sex, COVID-19 status, acute diagnosis, Body Mass Index (BMI), Charlson Comorbidity Index (CCI), pre-existing lung disease, hyperglycemic index (HbA1c), immune status, relevant medication in the context of COVID-19 infection and disease severity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of Plasma Samples and Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)\u003c/h2\u003e \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Biocoll\u0026reg;, Bio\u0026amp;Sell, Germany) density centrifugation as previously described. Briefly, 10 to 20 ml of venous blood was collected by peripheral phlebotomy in EDTA-coated vials (BD, Germany) from both post-COVID-19 and non-COVID-19 patients. Thereafter, the blood was di-luted with an equal amount of PBS (Biochrom, Germany) and subjected to Ficoll-density centrifugation at 500 g for 20 min. The resulting supernatant was designated as plasma. Plasma samples were snap-frozen and stored at -80\u0026deg;C. PBMCs were collected from the interphase, washed with PBS and then pelleted at 200 g for 20 min. Cell pellets were subsequently incubated for 5 min in a red blood cell lysis buffer (Merck, Germany), washed with PBS and centrifuged at 300 g for 10 min. The cell pellets were promptly stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. RNA Extraction and Quantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA from PBMCs was isolated in RNA-Bee reagent (Amsbio, UK). The reverse transcription of RNA into cDNA was carried out using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Germany) following the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR was performed using the Brilliant SYBR Green qPCR master mix (Applied Biosystems, USA). The relative amount of the mRNA was determined using the comparative threshold (Ct) method as previously described. Expression of the target genes was normalized to the expression of hypoxanthine phosphoribosyl transferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Suppl. table 2). In each case, the samples were plotted as technical duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Protein Extraction and Immunoblotting\u003c/h2\u003e \u003cp\u003ePBMCs were homogenized in Laemmli buffer as has been previously described. Proteins were quantified using the BCA Assay (Thermo Scientific Pierce Protein Biology, Germany). Equal protein amounts from each sample were separated using SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were immunoblotted overnight with the following primary antibodies: AMPK (1:2000, #2532L, Cell Signaling, USA), pAMPK (1:2000, Thr172, #2535L, Cell Signaling, USA), ac-lysine (1:500, #9411, Cell Signaling, USA), SOD2 (1:1000, #13194, Cell Signaling, USA), and acSOD2 (1:1000, ab13533, Abcam, UK). Equal sample loading was confirmed by an analysis of actin (1:1500, sc-1616-R, Santa Cruz, USA) and α-tubulin (1: 5000, #3873, Cell Signaling, USA) expression. All proteins were visualized using ECL Plus (GE Healthcare, Buckinghamshire, UK), detected with the ChemiDoc system (Bio-Rad Laboratories, USA) and quantified using ImageLab (version 5.2.1 build 11, Bio-Rad Laboratories, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Detection of TNF-α, Extracellular SOD3 and DNA Damage in Plasma Samples by ELISA\u003c/h2\u003e \u003cp\u003e The concentration of circulating TNF-α was analyzed in plasma samples from post-COVID-19 patients and the control group using ELISA according to manufacturer's recommendations (BioLegend, Germany). The extracellular SOD3 content was also deter-mined in plasma from post-COVID-19 and non-COVID-19 patients using a Superoxide Dismutase (SOD) Colorimetric Activity Kit (Invitrogen, Germany). DNA damage was estimated utilizing the DNA damage Competitive ELISA Kit (Invitrogen, Germany). All samples were measured using a microplate reader (Tecan, Switzerland) as recommended by the manufacturer. In each case, the samples were plotted as technical duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Mitochondrial Mass Analysis\u003c/h2\u003e \u003cp\u003eThe amount of nuclear and mitochondrial DNA (mt-DNA) was analyzed via quantitative real-time PCR utilizing the Brilliant SYBR Green qPCR master mix (Applied Biosystems, USA). The ratio of mt-DNA to nuclear DNA was built to quantify the mitochondrial mass content. In each case, the samples were plotted as technical duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe data are given as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The data were evaluated using the non-parametric test (Mann-Whitney test for two independent groups). Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, San Diego, USA). Statistical significance was accepted when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of Post-COVID-19 Patients\u003c/h2\u003e \u003cp\u003eTo characterize the study group, age, BMI, CCI as well as inflammatory and biochemical blood parameters were analyzed. The majority of the parameters show no differences between the analyzed groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among post-COVID-19 patients, 78% of men experienced severe COVID-19 disease, compared to only 44% of women. In post-COVID-19 male patients, the lactate dehydrogenase (LDH) concentration was significantly increased compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the folic acid concentration was significantly decreased in male post-COVID-19 patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe found that patients in the post-COVID-19 group were more likely to have short-ness of breath/malaise/fever, etc. (data not shown). In regard to the functional capacity of the patients \u0026ndash; as defined by the clinically validated Barthel index \u0026ndash; we found no statistical differences between both groups (p\u0026thinsp;=\u0026thinsp;0.685). This might be due to the fact that our study was underpowered to detect any such differences, especially since post-COVID-19 has been shown to be associated with progressive functional decline in survivors. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Further-more, this could be due to confounding factors related to multimorbidity associated with ageing, especially in the light of the fact that both groups do not show any significant differences at baseline (p\u0026thinsp;=\u0026thinsp;0.850). In addition, due to the lack of follow-up, we were not able to capture the duration of complaints since the study was conducted in an in-hospital setting (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Upregulated AMPK Phosphorylation in Samples from Post-COVID-19 Women\u003c/h2\u003e \u003cp\u003eThe expression and activity of the key metabolic sensor AMPK, a regulator of cellular energy homeostasis and multiple aspects of cell metabolism, have been reported to be dysregulated in several diseases and inflammatory conditions.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e However, its role in the early recovery stage of COVID-19 is poorly understood. No alteration of the total AMPK protein expression in PBMCs samples between all groups investigated was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Phosphorylated, i.e., activated form of AMPK, was significantly upregulated only in post-COVID-19 women compared to non-COVID-19, control, patients (p\u0026thinsp;=\u0026thinsp;0.0167) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Proinflammatory Mediators Are Increased in PBMC and Plasma Samples of Post-COVID-19 Women\u003c/h2\u003e \u003cp\u003eActivated AMPK and NF-κB play a crucial role in modulating inflammatory responses in monocytes and may have significant implications for the treatment of inflammatory conditions.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e mRNA expression of neither NF-κB nor its inhibitor, IκBα was found to be modulated in post-COVID-19 female and male PBMC samples compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Since the post-COVID-19 is associated with systemic inflammation,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e inflammatory markers in plasma and PBMCs samples were analyzed. A significant women-specific upregulation of proinflammatory IL-1β and IL-18 mRNA expression in PBMCs samples of post-COVID-19 patients was observed (p\u0026thinsp;=\u0026thinsp;0.0074 and p\u0026thinsp;=\u0026thinsp;0.0057, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). No significant differences were observed in the mRNA expression of IL-1β downstream effectors IL-6, TLR4, and TNF-α in PBMCs samples in the investigated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). In addition, circulating TNF-α was significantly elevated in the plasma of post-COVID-19 women compared to the control group (p\u0026thinsp;=\u0026thinsp;0.0422) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Expression of Mitochondrial Proteins and Genes in Post-COVID-19 Patients\u003c/h2\u003e \u003cp\u003eMitochondria play an important role in regulating immune cell activity, inflammation, and oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The mRNA expression of the mitochondria-encoded genes, \u003cem\u003ecox1\u003c/em\u003e, and \u003cem\u003end4\u003c/em\u003e, was significantly upregulated in PBMC samples of post-COVID-19 women compared to the control group and the post-COVID men (p\u0026thinsp;=\u0026thinsp;0.019 and p\u0026thinsp;=\u0026thinsp;0.029; p\u0026thinsp;=\u0026thinsp;0.029 and p\u0026thinsp;=\u0026thinsp;0.007, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). In contrast, the expression of nucleus-encoded ndusf1 was similar in PBMC samples from post-COVID-19 and non-COVID-19 patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Mitochondrial mass, as indicated by the rnr2/β-globin ratio, remained un-changed in post-COVID-19 patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Similarly, no differences in the expression of Sirt3 and TFAM were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). The expression of Sirt1 was not altered in post-COVID-19 patients compared to non-COVID-19 patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Oxidative Stress and Expression of Antioxidative Enzyme in Post-COVID-19 Patients\u003c/h2\u003e \u003cp\u003eInflammation is typically associated with the increased ROS formation, leading to oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Analysis of oxidative DNA damage, measured as 8-OHdG, revealed no differences in plasma concentrations among the investigated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, two isoforms of SOD, i.e., the extracellular SOD3 and the intramitochondrial SOD2, were analyzed in plasma or PBMCs samples, respectively. No differences in the SOD3 concentration were determined between the plasma of post-COVID-19 female patients and the control group. In contrast, in post-COVID-19 male patients, SOD3 levels were significantly increased compared to the control group (p\u0026thinsp;=\u0026thinsp;0.0468) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The expression of intramitochondrial SOD2 was significantly downregulated in post-COVID-19 men and became significantly lower compared with post-COVID-19 female patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). The acetylation state of SOD2 is an important regulator of its activity.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Analysis of SOD2 acetylation revealed its significant elevation in post-COVID-19 female patients (p\u0026thinsp;=\u0026thinsp;0.0402) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, E), suggesting a potential down-regulation of SOD2 activity in this group. In contrast, SOD2 acetylation was markedly reduced in post-COVID-19 men. Additionally, the expression levels of NRF1 and NRF2 were upregulated in post-COVID-19 women, however only the upregulation of NRF1 demonstrated statistically significant differences (p\u0026thinsp;=\u0026thinsp;0.0539) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGrowing evidence suggests that PCS is a significant burden of COVID-19-related diseases.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e However, little is known about post-COVID-19 patients, particularly for the geriatric population. The alterations in energy sensing, mitochondrial homeostasis, and inflammatory state, especially in circulating immune cells, may significantly contribute to the understanding of post-COVID-19.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e It has been documented that female, but not male, patients are at increased risk of developing PSC.30 Studies have shown that COVID-19 male patients have higher rates of hospitalization and mortality during acute viral infection,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e whereas women are prone to develop PSC and experience core symptoms more fre-quently.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Our findings suggest that women are likely to exhibit more pronounced systemic post-COVID-19 inflammation during early recovery stage as evidenced by increased circulated TNF-α levels in plasma and elevated proinflammatory interleukins IL-1β and IL-18 in PBMCs. The persistence of elevated inflammatory markers in women with ongoing symptoms suggests that prolonged inflammation may play a role in post-COVID-19 pathogenesis. To fully understand sex differences in post-COVID-19-related inflammation, more targeted studies that specifically compare inflammatory stage between men and women during the recovery phase are needed.\u003c/p\u003e \u003cp\u003eThe upregulation of proinflammatory pathways is usually accompanied by enhanced ROS formation.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Although no alteration in the concentration of the oxidative stress marker 8-OHdG was found, an enhanced anti-oxidative defense was observed in post-COVID-19 male patients compared to females, as indicated by the increased levels of extracellular SOD3 in plasma. Upregulation of SOD3 in post-COVID-19 male patients might indicate a compensatory response to inflammation, as SOD3 tightly regulates the tissue redox balance under physiological stress caused by viral infections.\u003c/p\u003e \u003cp\u003eIn contrast, expression of mitochondrial SOD2 was downregulated in post-COVID-19 men. It should be noted here that the activity of SOD2 strongly depends on the acetylation state of this protein.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Analysis of SOD2 acetylation revealed a notable sex difference: post-COVID-19 female patients exhibited significant hyperacetylation, whereas post-COVID-19 male patients showed a marked reduction in SOD2 acetylation, although this reduction did not reach statistical significance.\u003c/p\u003e \u003cp\u003eThe hyperacetylation of mitochondrial SOD2 in post-COVID-19 women may be due to reduced expression or activity of the main mitochondrial deacetylase Sirt3. Since its ex-pression in post-COVID-19 PBMC samples did not differ from the control group, Sirt3 activity likely declined in post-COVID-19 female patients. Several studies have demonstrated that Sirt3-dependent deacetylation of mitochondrial matrix proteins is associated with improved mitochondrial function.\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and alleviation of inflammatory phenotype in immune cells, like macrophages.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e In line with these findings, the present study documents an association of women-specific SOD2 hyperacetylation with increased levels of the pro-inflammatory markers in post-COVID-19 patients.\u003c/p\u003e \u003cp\u003eAlthough the expression of mitochondrial SOD2, Sirt3, and the mitochondrial mass was not altered in the studied post-COVID-19 patients, the expression of the mitochondria-encoded genes was elevated in women. Mitochondrial dysfunction in immune cells has been implicated in the context of severe acute SARS-CoV2 infection\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and in post-COVID-19 patients,\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e suggesting that it may be still affected in post-COVID-19. While mitochondrial respiration was not assessed in the current study, our data suggest an energy deficit in immune cells of post-COVID-19 female patients, leading to a compensatory upregulation of mitochondrial genes. This is further supported by elevated phosphorylation of AMPK, a key cellular energy sensor. Chronic AMPK activation may reflect energy stress in PBMC from both post-COVID-19 female and male patients. Consistent with our findings, a previous study by Ajas et al. has revealed compromised mitochondrial function and an energy deficit in PBMCs from COVID-19 patients.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Thus, activation of AMPK accompanied by the mitochondrial gene upregulation observed in women may be a natural compensatory mechanism to combat the aberrant inflammation. The absence of this mechanism in men may reflect a higher mortality and hospitalization rate in COVID-19 male than female patients.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Therefore, improving mitochondrial homeostasis through AMPK activation could be a promising therapy to alleviate the severity of COVID-19 in male patients.\u003c/p\u003e \u003cp\u003eIn conclusion, the current study analyzing plasma and PBMCs during post-COVID-19 condition revealed women-specific elevation of inflammatory markers accompanied by the hyperacetylation of mitochondrial SOD2 and upregulation of mitochondrial genes. These changes, together with activated AMPK, may indicate energetic stress, potentially explaining the higher prevalence of post-COVID-19 condition in women.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimitation of the Study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, a small cohort of patients was investigated due to the limited availability of hospitalized geriatric, untreated patients who had recovered from COVID-19 infection. Another major limitation of the present study is that it was conducted during the SARS-CoV-2 pandemic, which resulted in deviations from the intended recruitment protocol; specifically, patients were not consistently enrolled within first 4 weeks after symptom onset. In retrospect, this recruitment approach does not align with the current definitions of post-acute sequelae of SARS-CoV-2 infection, which were established subsequently. Nevertheless, this aspect may also be considered a strength, as it provides valuable in-sights into the early subcellular events following SARS-CoV-2 infection. Especially, the key effects observed in the study, such as alterations in the expression of mitochondrial genes and inflammatory markers in post-COVID-19 women compared to non-COVID-19 individuals, are robust enough to support the validity of the conclusions.\u003c/p\u003e \u003cp\u003eFurthermore, the restriction of the statistical analyses in comparing different sample sizes across various assays. This was due to a reduced amount of PBMCs in some individuals, which prevented us from obtaining enough material to test in all experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e O.V. analyzed data, prepared figures and wrote the main manuscript text. M.E. analyzed data, prepared figures and wrote the main manuscript text. L.M. isolated PBMC and revised the manuscript. M.N. provided the blood samples and undertook patient characterization and revised the manuscript. J.T. wrote and revised the manuscript. Y.L. analyzed the data and wrote the main part of the manuscript, and M.B. conceived the project, analyzed the data, prepared the figures, and wrote the main manuscript text. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e We acknowledge support from the Open Access Publication Fund of Universit\u0026auml;tsklinikum T\u0026uuml;bingen.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declaration: \u003c/strong\u003eWe obtained informed consent from all study participants. Sample collection and the experimental protocols were approved by the Scientific Board at the Charit\u0026eacute; \u0026ndash; Universit\u0026auml;tsmedizin Berlin (EA4/154/16 and EA1/037/22). All experiments were performed in accordance with the German regulations and the ethical standards as laid down in the Declaration of Helsinki. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish declaration: \u003c/strong\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Natalia Haritonow for the technical assistance.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGebhard, C. E. et al. 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Immunopharmacol.\u003c/em\u003e \u003cb\u003e108\u003c/b\u003e, 108697 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe la Cruz-Enriquez, J., Rojas-Morales, E., Ruiz-Garcia, M. G., Tobon-Velasco, J. C. \u0026amp; Jimenez-Ortega, J. C. SARS-CoV-2 induces mitochondrial dysfunction and cell death by oxidative stress/inflammation in leukocytes of COVID-19 patients. \u003cem\u003eFree Radic Res.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e, 982\u0026ndash;995 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjaz, S. et al. Mitochondrial metabolic manipulation by SARS-CoV-2 in peripheral blood mononuclear cells of patients with COVID-19. \u003cem\u003eAm. J. Physiol. Cell. Physiol.\u003c/em\u003e \u003cb\u003e320\u003c/b\u003e, C57\u0026ndash;C65 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"post-COVID-19, mitochondrial homeostasis, sex differences, AMPK activity, SOD acetylation","lastPublishedDoi":"10.21203/rs.3.rs-7257776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7257776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eViral infections including, respiratory viruses such as the SARS-CoV-2, cause mitochondrial dysfunction and exacerbate systemic inflammation. In the present study, sex differences were investigated in patients who have recovered from the acute phase of infection regardless of whether they have ongoing symptoms (post-COVID-19 condition). Peripheral blood mononuclear cells (PBMCs) and plasma samples were collected from hospitalized geriatric patients following their PCR-confirmed negative result for SARS-CoV-2infection. The inflammatory state was assessed by measuring the expression of inflammatory markers using real-time PCR and ELISA. Furthermore, the expression of oxidative, and mitochondrial markers along with metabolic sensors, i.e., AMPK and Sirt1, was examined. The metabolic sensor AMPK was activated (pAMPK/AMPK ratio) only in post-COVID-19 women and was accompanied by women-specific elevation of circulating TNF-α levels in plasma and upregulation of pro-inflammatory mediators IL-1β and IL-18 in their PBMCs. The expression of the antioxidant SOD2 and its acetylation were reduced in PBMCs obtained from male post-COVID-19 patients, whereas SOD2 was hyperacetylated in women. The NRF1 expression was notably upregulated in post-COVID-19 female PBMCs. In addition, the mitochondrial genes \u003cem\u003ecox1\u003c/em\u003e and \u003cem\u003end4\u003c/em\u003e were upregulated in post-COVID-19 female patients, whereas, the expression of Sirt1, Sirt3, and TFAM remained unchanged in all studied groups. In summary, the present study revealed a women-specific elevation of inflammatory markers associated with increased AMPK activity and acetylation of mitochondrial SOD2.\u003c/p\u003e","manuscriptTitle":"Sex Differences in Energy Sensing, Inflammatory State, and Mitochondrial Biogenesis in Geriatric Post-COVID-19 Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 16:31:09","doi":"10.21203/rs.3.rs-7257776/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T06:11:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T17:21:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T22:35:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134085034454803490429601722745460904476","date":"2026-04-14T04:20:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T15:33:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307371485591420641513353327652019172636","date":"2026-04-08T14:40:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34929214776932883619590326143858357319","date":"2026-03-17T13:17:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T11:07:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T09:53:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-06T21:21:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T01:06:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-06T01:03:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5547d1b5-8c66-4d81-9d39-d3f39eefb35a","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64784250,"name":"Health sciences/Biomarkers"},{"id":64784251,"name":"Health sciences/Diseases"},{"id":64784252,"name":"Biological sciences/Immunology"},{"id":64784253,"name":"Health sciences/Medical research"},{"id":64784254,"name":"Biological sciences/Microbiology"},{"id":64784255,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-12T10:19:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 16:31:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7257776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7257776","identity":"rs-7257776","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}