Hamsters are a model for COVID-19 alveolar regeneration mechanisms: an opportunity to understand post-acute sequelae of SARS-CoV-2 (preprint)

A relevant number of coronavirus disease 2019 (COVID-19) survivors suffers from post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC). Current evidence suggests a dysregulated alveolar regeneration in COVID-19 as a possible explanation for respiratory PASC symptoms, a phenomenon which deserves further investigation in a suitable animal model. This study investigates morphologic and transcriptomic features of alveolar regeneration in SARS-CoV-2 infected Syrian golden hamsters. We demonstrate that CK8+ alveolar differentiation intermediate (ADI) cells accumulate following SARS-CoV-2-induced diffuse alveolar damage. A subset of ADI cells shows nuclear accumulation of p53 at 6- and 14-days post infection (dpi), indicating a prolonged block in the ADI state. Transcriptome data shows the expression of gene signatures driving ADI cell senescence, epithelial-mesenchymal transition, and angiogenesis. Moreover, we show that multipotent CK14+ airway basal cell progenitors migrate out of terminal bronchioles, aiding alveolar regeneration. At 14 dpi, persistence of ADI cells, peribronchiolar proliferates, M2-type macrophages, and sub-pleural fibrosis is observed, indicating incomplete alveolar restoration. The results demonstrate that the hamster model reliably phenocopies indicators of a dysregulated alveolar regeneration of COVID-19 patients. The study provides a suitable translational model for future research on the pathomechanims of PASC and testing of prophylactic and therapeutical approaches.

Assessing and improving the validity of COVID-19 autopsy studies - a multi center approach to establish essential standards for immunohistochemical and ultrastructural analyses (preprint)

Background Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, e.g. by an overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. Methods We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 spike protein and nucleocapsid, dsRNA, and non-structural protein Nsp3 in cultured cell lines and COVID-19 autopsy tissues. In a multicenter study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 nucleocapsid staining. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. Findings Publications show high variability in the detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. In our study, we show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and also provide recommendations for optimized sampling and analysis. 116 of 122 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM and IHC sections as a reference and for training purposes. Interpretation Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2.

Replication of SARS-CoV-2 in Adipose Tissue Determines Organ and Systemic Lipid Metabolism in Hamsters and Humans (preprint)

Obesity increases the risk for poor outcome in patients with coronavirus disease-19 (COVID-19). However, the role of adipose tissue for viral propagation and potential metabolic implications are not understood. We detected SARS-CoV-2 in adipose tissue of overweight but not lean male COVID-19 patients. SARS-CoV-2 replicates to high titres in cultured mature adipocytes, a process depending on lipid accumulation and mobilization. After intranasal inoculation, we observed high viral replication in fat depots of Golden Syrian hamsters, demonstrating dissemination from the respiratory tract and subsequent propagation in adipose tissue. Following induction of pro-inflammatory responses, expression of de novo lipogenesis enzymes was suppressed in adipose tissue. This specific down-regulation was reflected by lipidomic alterations in plasma of SARS-CoV-2 infected hamsters as well as in hospitalized COVID-19 patients. Overall, our study highlights that adipose tissue is an important site of SARS-CoV-2 replication, contributing to dysregulation of systemic lipid metabolism.Funding: This study was supported by a rapid response grant from the Federal Ministry of Health (BMG; ZMV I 1-2520COR501 to GG), by DFG grants SCHE522/4-1 (LS) and SFB1328, project- ID:335447727 (JH). As part of the National Network University Medicine (NUM) funded by the Federal Ministry of Education and Research (BMBF, Germany), this work was funded within the research consortium DEFEAT PANDEMIcs, grant number 01KX2021 (FH, PL, KP, BO).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The Ethics Committee of the Hamburg Chamber of Physicians reviewed and approved the studies (PV7311, 2020-10353-BO-ff, WF-051/20, WF-053/20). For the preparation of primary human white adipocytes, biopsies of subcutaneous and visceral adipose tissues were taken during bariatric surgery at the Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf. All participants signed an informed consent and the study was approved by the Ethics Committee of the Hamburg Chamber of Physicians (PV4889).

Sex hormone dysregulations are associated with disease severity in critically ill male COVID-19 patients - a retrospective analysis (preprint)

BACKGROUNDMale sex was repeatedly identified as a risk factor for death and intensive care admission. However, it is yet unclear whether sex hormones are associated with disease severity in COVID-19 patients. We sought to characterize sex differences in hormone levels and cytokine responses in critically ill COVID-19 patients.METHODSWe performed a retrospective cohort study of critically ill COVID-19 patients. Males and females were compared. Multivariate regression was performed to assess the association between sex hormones, cytokine responses and the requirement for extracorporeal membrane oxygenation (ECMO) treatment.RESULTSWe analyzed sex hormone levels (estradiol and testosterone) of n=181 male and female individuals. These consisted of n=50 critically ill COVID-19 patients (n=39 males, n=11 females), n=42 critically ill non-COVID-19 patients (n=27 males, n=15 females), n=39 non-COVID-19 patients with coronary heart diseases (CHD) (n=25 males, n=14 females) and n=50 healthy individuals (n=30 males, n=20 females). We detected highest estradiol levels in critically ill male COVID-19 patients compared to non-COVID-19 patients (p=0.0123), patients with CHD (p=0.0002) or healthy individuals (p=0.0007). Lowest testosterone levels were detected in critically ill male COVID-19 patients compared to non-COVID-19 patients (p=0.0094), patients with CHD (p=0.0068) or healthy individuals (p<0.0001). No statistically significant differences in sex hormone levels were detected in critically ill female COVID-19 patients, albeit similar trends in estradiol levels were observed. In critically ill male COVID-19 patients, cytokine and chemokine responses (IFN-γ, p=0.0301; IL-1RA, p=0.0160; IL-6, p=0.0145; MCP-1, p=0.0052; MIP-1α, p=0.0134) were significantly elevated in those with higher Sequential Organ Failure Assessment (SOFA) scores (8-11). Linear regression analysis revealed that herein IFN-γ levels correlate with estradiol levels in male and female COVID-19 patients (R2=0.216, =0.0009). Male COVID-19 patients with elevated estradiol levels were more likely to receive ECMO treatment in the course of their ICU stay (p=0.0009). CONCLUSIONS We identified high estradiol and low testosterone levels as a hallmark of critically ill male COVID-19 patients. Elevated estradiol levels in critically ill male COVID-19 patients were positively associated with IFN-γ levels and increased risk for ECMO requirement. 

The majority of male patients with COVID-19 present low testosterone levels on admission to Intensive Care in Hamburg, Germany: a retrospective cohort study. (preprint)

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide and pose a major public health burden. There is increasing evidence that men are more likely to die from SARS-CoV-2 infection than women. However, underlying factors that mediate the observed sex bias in coronavirus disease 2019 (COVID-19) remain unknown. Methods. In this retrospective cohort, we included COVID-19 patients who were admitted to an Intensive Care Unit at the University Hospital Hamburg-Eppendorf, Germany. We obtained demographic data of all patients who were discharged or had died by 29th April 2020. We systematically analyzed sex hormones as well as cytokine and chemokine responses in male and female patients with laboratory-confirmed SARS-CoV-2 infections upon hospital admission. We used uni- and multivariable linear regression methods to identify potential risk factors for disease severity in males and females. Findings. All enrolled patients (n=45; n=35 males and n=10 females) presented comorbidities with hypertension being the most common (45.7% in males; 40% in females), followed by cancer (35% in males; 40% in females), obesity (34.3% in males and 30% in females), type II diabetes (25.7% in males and 20% in females) and chronic heart diseases (8.6% in males and 0% in females). We detected that the vast majority of male COVID-19 patients present low testosterone (68.6%) and low dihydrotestosterone (48.6%) levels. In contrast, most female COVID-19 patients have elevated testosterone levels (60%) without alterations in dihydrotestosterone levels. Both, female and male COVID-19 patients may present elevated estradiol levels (45.7% in males and 40% in females). Disease severity defined by SOFA score correlates with elevated cytokine responses (e.g. IL-6) in males and IL-2 in females. In male COVID-19 patients, testosterone levels negatively correlate with inflammatory IL-2 and IFN-{gamma}, whereas estradiol levels positively correlate with the inflammatory cytokine IL-6. Vice versa, in female COVID-19 patients, testosterone levels positively correlate with inflammatory cytokines (e.g. IL-6). Interpretation. We here show that critically ill male COVID-19 patients suffer from severe testosterone and dihydrotestosterone deficiencies. Both androgens are required to mount antiviral immune responses to combat infection in males.