Abstract
Dysautonomia has substantially impacted acute COVID-19 severity as well as symptom burden after recovery from COVID-19 (long COVID), yet the underlying causes remain unknown. Here, we hypothesized that vagus nerves are affected in COVID-19 which might contribute to autonomic dysfunction. We performed a histopathological characterization of postmortem vagus nerves from COVID-19 patients and controls, and detected SARS-CoV-2 RNA together with inflammatory cell infiltration composed primarily of monocytes. Furthermore, we performed RNA sequencing which revealed a strong inflammatory response of neurons, endothelial cells, and Schwann cells which correlated with SARS-CoV-2 RNA load. Lastly, we screened a clinical cohort of 323 patients to detect a clinical phenotype of vagus nerve affection and found a decreased respiratory rate in non-survivors of critical COVID-19. Our data suggest that SARS-CoV-2 induces vagus nerve inflammation followed by autonomic dysfunction which contributes to critical disease courses and might contribute to dysautonomia observed in long COVID.
Introduction
COVID-19 is a systemic disease affecting multiple organ systems with a wide range of acute and chronic symptoms. It is estimated that 65 million people suffer from persisting symptoms after recovery from acute COVID-19 (long COVID) with a steadily increasing prevalence. Dysautonomia is frequently reported in long COVID which can last for years or is even expected to be lifelong. Additionally, critically ill COVID-19 patients often show rapid and unpredictable vegetative deterioration which substantially contribute to the disease’s severity and lethality. However, the cause and mechanism of dysautonomia in COVID-19 are largely unexplored and remain elusive.
The vagus nerve is an essential component of the autonomic nervous system (ANS) and regulates critical body functions such as heart rate, digestion, and respiratory rate. Dysautonomia is observed in long COVID and other neurological diseases which might be explained by vagus nerve impairment. However, the molecular underpinnings of the clinical observations of autonomic dysfunction are largely unknown. Notably, a recent histopathological study showed that the known SARS-CoV-2 entry receptors on host cells, angiotensin-converting enzyme 2 (ACE2), neuropilin 1 (NRP1), and transmembrane protease serine subtype 2 (TMPRSS2) are expressed on the vagus nerve . Therefore, we hypothesized that SARS-CoV-2 infects the vagus nerves resulting in an inflammatory response which might subsequently lead to dysautonomia during acute COVID-19 and long COVID.
Materials and methods
Human samples
Autopsies were performed at the Institute of Legal Medicine of the University Medical Center Hamburg-Eppendorf, Germany. Utilization of postmortem human tissue after conclusion of diagnostic procedures was reviewed and approved by the institutional review board of the independent ethics committee of the Hamburg Chamber of Physicians (protocol nos. PV7311, 2020-10353-BO-ff, and PV5034). Post-ganglionic cervical vagal nerve tissues were used for this study. For COVID-19, postmortem tissue samples were used; for non-COVID-19 controls, we used samples from individuals with other cause of death. Real-time quantitative PCR (RT-qPC) to determine SARS-CoV-2 viral load was performed as previously described. A summary of included samples is provided...
Statistical analysis
All statistical analyses and visualizations were performed within the R environment (v4.0.3). The detailed analyses of RNA-sequencing data are described above. For immunohistochemical quantification, we compare the two groups by Student’s t-test. For clinical data, we first performed one-way ANOVA to analyze variance on group levels and subsequently t-test with FDR correction for multiple comparisons. Modularity between controls and SARS-CoV-2-positive samples were compared for each identified module by Mann–Whitney U test. Odds ratios for lethal disease outcome were calculated in patients with critical COVID-19 by generalized mixed models with correction for age and sex. For correlation analyses, we first performed z-scaling to account for outliers and used Spearman correlation for testing significance. P values < 0.05 were considered as significant.
Results and discussion
Vagus nerve inflammation is commonly observed in COVID-19
To test our hypothesis, we first analyzed the vagal nuclei in the brain stem where we found an increased number of HLA-DR+ monocytes and CD8+ T cells. Notably, this is in line with a recent study that demonstrated a strong inflammatory response in brain stems of deceased COVID-19 patients . Next, we investigated whether the vagus nerve is directly affected by COVID-19. Therefore, we collected vagus nerves of 27 deceased patients with COVID-19 and 5 deceased controls without COVID-19 and performed mRNA sequencing. We identified 2031 differentially up- and 42 down-regulated genes with an absolute log2 foldchange above 1. Gene ontology (GO) term analysis revealed a strong enrichment of genes regulating antiviral responses and interferon signaling, supporting the notion that vagus nerve inflammation is commonly observed in COVID-19. Next, we aimed to determine whether SARS-CoV-2 is directly detectable in vagus nerves. We performed quantitative PCR in 23 samples of deceased COVID-19 patients and detected SARS-CoV-2 RNA in all specimens which was not the case for control samples. To investigate whether SARS-CoV-2 RNA load correlates with vagus nerve dysfunction, we grouped the 23 vagus nerves with available SARS-CoV-2 RNA quantification into low (n = 6), intermediate (n = 9), and high (n = 8) SARS-CoV-2 load groups. We separately tested differential expression of all groups against controls (Fig. 1g–i) and performed GO term enrichment analyses. By correlating the GO terms with SARS-CoV-2 RNA load, we identified GO terms that were enriched independent of SARS-CoV-2 load and GO terms that correlated with SARS-CoV-2 RNA load. Antigen processing, antiviral response, and interferon signaling were up-regulated independent of SARS-CoV-2 RNA load together with the coagulation cascade and leukocyte migration. Notably, complement activation and regulation of apoptotic cell clearance showed a direct correlation with viral RNA load. In contrast, we found that genes regulating trans-synaptic signaling or assembly of the neuronal transport machinery had an inverse correlation with SARS-CoV-2 RNA load suggesting dose-dependent axonal dysfunction...
Acknowledgements
We like to thank members of the Glatzel and Friese laboratories for helpful discussion. Graphical abstract and summary were generated with Biorender.