Multiplying viruses disrupting lung tissue do not directly cause severe outcomes of COVID-19 disease, instead inflammatory immune processes affecting the endothelial lining of the airways are responsible, reports a new study. Cellular mechanisms at early stages of lung endothelial inflammation in SARS-CoV-2 infection are still poorly understood.

The new study uses Syrian hamster models of moderate COVID-19 disease to better understand systemic and lung cellular responses and match these to severity outcomes in datasets from COVID-19 patients. Pathogen-engulfing macrophages in the lung are the first and strongest responders to infection, the study shows. Even while cells lining lung passages show mild changes in gene expression, macrophages revamp their transcriptional machinery immediately following infection, and pump up the production of pro-inflammatory gene products.

In the absence of the release of viral particles from infected cells (productive infection), the response of endothelial cells in the lungs depends on the cell-subtype and involves robust, early induction of pro-inflammatory genes and genes that attract T cells to organize an immune response against the infection. Before the virus is eliminated from the body at around five days after infection, killer T cells—that kill virus-infected cells—and immunoglobulin M antibodies are recruited to the lungs.

Collaborating scientists from several research institutes in Germany including scientists at the Charité—Universitätsmedizin, Max Delbrück Center (MDC) for Molecular Medicine and Freie Universität, Berlin, report these findings in the Nature Communications article, “Temporal omics analysis in Syrian hamsters unravel cellular effector responses to moderate COVID-19.” The study identifies cell type-specific effector functions, providing detailed insights into pathological mechanisms of COVID-19 and informing future therapeutic strategies. Financial support for this study was provided by the German Research Foundation and the Berlin Institute of Health.

As early as the second day after infection, SARS-CoV-2 (red) invades nearly all areas of the lungs (infected lung tissue, image A). The virus invades and destroys both the cells lining the respiratory tract (B, arrowhead) and the cells responsible for gas exchange (arrow pointers). Numerous virus particles can be detected within macrophages (E, arrowhead icon) [Dietert Gruber/ Freie Universität Berlin]
The authors emphasize, the Syrian hamster is an ideal model for investigating SARS-CoV-2 infections. Patient-centered studies in identifying therapeutic targets for COVID-19 and understanding underlying mechanisms of the disease particularly in early stages of infection, are limited in their scope. Biological samples needed for this type of research can usually be obtained only after a patient has been admitted to hospital. Also, it is virtually impossible to obtain lung tissue samples from patients with mild or moderate disease and pneumonia, as the biopsy procedure would place patients at undue risk. Therefore, majority of patient-centered studies are based on the analyses of tissues obtained post-mortem.

Researchers in the current study have used available patient samples to better understand both disease mechanisms progressions but they also searched for a suitable animal model that would enable them to study compartments of the lungs important in the early phase of the disease that are not easily accessible in patients.

“We wanted to know whether we could use these models to develop new treatment options and tried to replicate findings from patient samples. We were remarkably successful in this regard,” says Martin Witzenrath, PhD, Deputy Head and Professor at Charité’s Department of Infectious Diseases and Respiratory Medicine, the study’s co-corresponding author.

“We were primarily interested in the lung’s endothelial cells, which line the pulmonary blood vessels and form a barrier there. In severe COVID-19 cases, this barrier becomes dysfunctional, a development which eventually results in lung failure.”

With the aim of identifying the ideal, non-transgenic animal model for research on COVID-19 treatments, the researchers describe the detailed characteristics of SARS-CoV-2 infection in the Syrian hamster model and corroborate their findings in data sets from patient samples.

Hamsters contract the same virus variants as humans. They also develop similar disease symptoms, and severe disease damages their lungs. Symptoms and progression of COVID-19, however, vary between different species of hamster. While symptoms usually remain moderate in Syrian hamsters, Roborovski hamsters develop severe disease. The authors conduct single-cell analyses to uncover the underlying mechanisms responsible for the differences in severity outcomes in the hamster models focusing on processes in the lung endothelial cells.

“We were able to observe how certain cells involved in lung immunity – namely monocytes and monocyte-derived macrophages – ingest the virus and subsequently show a very pronounced response. They send out biological messengers which then elicit a very strong inflammatory response. In our model, this is quickly brought under control by T cells, another type of immune cell which is dispatched for this very purpose. In severe COVID-19, however, this does not happen,” says Geraldine Nouailles, PhD, researcher at Charité’s Department of Infectious Diseases and Respiratory Medicine and co-first author on the study. “A fast and efficient T cell response is crucial to successful recovery from COVID-19.”

In the early stages of infection, SARS-CoV-2 replicates slowly in the lungs and respiratory tract while the immune response goes into overdrive.

Endothelial cells that line the blood vessels (arrowhead), are not infected but their strong response to the virus triggers an influx of inflammatory cells, primarily T cells (arrow pointer). Bars: 50µm. [Dietert Gruber/ Freie Universität Berlin]
“The destruction of lung tissue seen in severe COVID-19 is not a direct result of viral propagation inside cells, but of the strong inflammatory response,” says Emanuel Wyler, a researcher at the MDC, and co-first author on the study. “This also appears to apply to the cells of the vasculature, in particular the lung’s endothelial cells. They show a very strong response to the virus but are neither infected by it nor destroyed in the process.”

If the disease is severe, blood vessels can become obstructed and vessel walls unstable, resulting in acute lung failure. It does not appear likely, however, that this blood vessel damage also plays a part in moderate COVID-19.

“That COVID-19 activates the endothelium—a type of protective barrier lining blood vessels which, amongst other things, also controls a range of processes in the lung’s micro blood vessels – did not come as a surprise. What did come as a surprise, however, was that these cells are also the active driver of inflammation,” says Witzenrath.

Endothelial cells may be therapeutically targeted by using substances that either sealing or calm the endothelial barrier.

“One of these is already the target of research conducted in our Collaborative Research Center SFB-TR84, where we were able to show that it is effective in pneumonia and ventilated patients,” says Witzenrath. Other anti-inflammatory drugs currently being tested as treatments for COVID-19 target the immune response itself. They are also effective against monocytes and macrophages and temper their activity.

The researchers hope to use the Syrian hamster model to develop safe and effective treatments for patients with COVID-19. The multidisciplinary team of researchers are currently analyzing the responses of different cell types observed in Roborovski dwarf hamsters. The researchers want to find out why the infection produces severe disease in these animals, and why it is not self-limiting as in Syrian hamsters.

“We hope this will guide us to a possible explanation for why some people develop severe COVID-19 but others do not,” says Nouailles. The researchers will need to first decipher the dwarf hamster’s genome to begin to fill the knowledge-gap in this relatively exotic model.

“Information from our current study has enabled us to close some of these gaps. This represents major progress, including in terms of a more conscious and targeted approach to the use of animals in medical research,” says Jakob Trimpert, PhD, virologist and veterinary surgeon from Freie Universität Berlin and co-senior author on the paper.

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