Using Spatial Transcriptomics to Examine a COVID-19 affected Lungs

Researchers began studying SARS-CoV-2 path of destruction as the pandemic unfolded, initially focusing on the interplay between lung and immune system cells as the early weeks of the pandemic unfolded and health-care staff battled to save so many lives.

A powerful tool for elucidating cellular disease, single-cell transcriptomics utilising single-cell RNA-Seq, commonly known as RNA-Seq. The transcriptome is a measurement of the total number of distinct messenger RNA (mRNA) molecules in a cell. The set of proteins that a cell produces in response to a stimulus, such as a viral invasion, is revealed by RNA-Seq. More than a decade has passed since the invention of the technology.

Documenting the number of mRNAs in a lung cell, or any cell for that matter, is useless unless it is done in the context of the organ in which it is found. It’s akin to attempting to deduce the storyline by counting the number of times the words “the,” “a,” and “there” appear in a novel.

RNA-Seq

Researchers at the Karolinska Institute enhanced RNA-Seq in 2016 by pioneering spatial transcriptomics, which delivers “the room where everything happens,” to paraphrase Hamilton. The method places a cell’s whole mRNA repertoire in the context of its surroundings by taking images of the cell’s surroundings and exploring them with visualisation software.

Picking out lung cells in sputum or collecting them using a bronchoscopy procedure and labelling their surface features provides a more meaningful perspective than taking into account a cell’s surroundings. Because of a gadget called Visium from 10x Genomics, which inspired this piece, spatial transcriptomics may even be performed on old tissue samples that have been formaldehyde-fixed and encased in wax. Indicative gene expression patterns can now be found in the more than a billion such samples stored in hospital biopsy archives and biobanks.

Researchers’ bias can be mitigated by using spatial transcriptomics, which could lead to the discovery of cell types that were previously unknown to be part of a given organ. In a word, it overcomes the human proclivity to seek out what we already know – or that we’ve seen everything.

Anatomy of a Cell

RNA-Seq analysis of cells collected and flash-frozen from the lungs of COVID patients within hours of death was revealed in two new Nature publications, documenting in context the destruction of the acute respiratory distress syndrome that arises in around 15% of patients. When COVID lungs are compared to healthy people’s lungs and those with other respiratory illnesses so soon after death, it’s evident that the infection reduces the ability to repair damage.

RNA-Seq produces large data sets from a small number of people. For example, Columbia University researchers studied RNA in 116,314 lung cells from 19 adult COVID patients and 7 healthy controls in a recent Nature paper. There is a “dangerous trifecta of excessive inflammation, direct death and impeded regeneration of lung cells engaged in gas exchange, and accelerated lung scarring,” according to the researchers. The second study, undertaken by academics at the Broad Institute, looked at 420 specimens from 17 people and came up with similar findings.

A lung consists of three basic types of tissues: a connective tissue scaffold made up of fibroblasts and the collagen they secrete; white blood cells that aid in the immune response; and lining tissue (epithelium) that forms microscopic air sacs (alveoli) where inhaled oxygen is exchanged for carbon dioxide, which is then exhaled.

SARS-CoV-2 spreads quickly in nasal passage cells, moves via the trachea and bronchi, and enters the alveoli through the narrowing bronchioles, interrupting the vital gas exchange.

The following modifications were discovered by RNA-Seq study of cells from COVID patients’ fresh, autopsied lungs:

1 COVID-specific “pathological” fibroblasts produce collagen quickly, burying the organ in scar tissue like thick glue or cement on bubblewrap. As the lungs thicken, gas exchange becomes more difficult.

2 B cells produce antiviral antibodies, whereas T cells, which trigger B cells to produce antibodies, are in decline. (These are white blood cells, or WBCs.)

3 The amount of monocytes, or white blood cells, grows rapidly. Monocytes give rise to blobby macrophages, which absorb and destroy infections while also secreting cytokines that feed COVID’s awful “storm” of inflammation. Interleukin (IL)-1beta from the blood and interleukin (IL)-6 from the alveoli are particularly plentiful.

4 The lungs’ natural ability to mend breaks down. Alveolar type 2 (AT2) cells normally operate as carers by secreting the surfactant that keeps the air sacs clean. AT2 cells also aid regeneration by reverting to a stem-cell-like condition in which they can divide and produce alveolar type 1 cells, which make up the air sacs. This crucial transition is halted by the virus, and the lung cannot heal itself on a microscopic level. As a result, the virus not only kills alveoli but also hinders their ability to regenerate, even if they survive. The condition of the lungs deteriorates.

Chronic obstructive pulmonary disease (COPD) patients are at a higher risk.

Another study, published in Nature Communications by The Translational Genomics Research Institute and the Human Cell Atlas Lung Biological Network, used RNA-seq to figure out what happens when COVID assaults people who already have breathing problems. “Early in the pandemic, it became clear that people with chronic lung diseases were at a very high risk for severe COVID-19, and our goal was to learn more about the cellular and molecular alterations that caused this,” said co-author Jonathan Kropski.

Chronic lung diseases include chronic obstructive pulmonary disease (COPD) and interstitial lung diseases, such as idiopathic pulmonary fibrosis, which scar and stiffen lung tissue. These problems can lead to acute respiratory distress syndrome and multi-system organ failure, and they are aggravated in people who have other COVID risk factors, such as men, smokers, and people who have hypertension, obesity, or diabetes.

The researchers analysed mRNA sequences from 611,398 cells from different databases, which represented both healthy and chronically ill lungs. A “viral entrance score” was used to indicate the expression of all genes associated with SARS-CoV-2 infection. Patients with persistent pulmonary disease exhibited higher-scoring cells.

Co-author Nicholas Banovich noted, “Our findings suggest that patients with chronic lung disease are molecularly prone to SARS-CoV-2 infection.”

Patterns of gene expression in the critical AT2 cells essentially “prime the pump,” allowing the virus to reach lung lining cells more easily in people with COVID and chronic lung disease. Both localised inflammation and systemic cytokine storms erupt at the same moment. To avoid drowning from the immune cell cascade that floods the lungs, severely damaging organs, artificial breathing may be required. Intervention is frequently insufficient.

Another new study from researchers at the University of Alabama at Birmingham is shining a light on the similarities and differences between COVID-19 and H1N1 influenza, and how they both affect the lungs in severe cases.

Like COVID-19, influenza has resulted in a number of global pandemic outbreaks, and the most severe cases of flu require the same degree of treatment as severe COVID-19 patients. Gaining a better knowledge of the distinctions and similarities between these two viral infections may help enhance patient care and find novel therapeutic targets.”

Researchers addressed this question using a cutting-edge technology called spatial transcriptomics, which enables the identification of genetic responses while preserving the tissue architecture, minimising the danger of processing-induced alterations.

COVID-19 individuals exhibited a reaction that resulted in lung tissue remodelling, making the organ more rigid and hence more difficult to breathe. These alterations were reflected in the adaptation of the lung’s various cell types: epithelium, vasculature, and macrophages, a type of immune cell found in the lungs that coordinates the inflammatory response.

These innovative findings identify a distinct tissue response to SARS-CoV-2 infection and may pave the way for the development of novel targeted therapeutic approaches for both COVID-19 and influenza patient groups. Additionally, they give a novel platform for studying other prevalent severe disorders that include organ injury.

From a technical aspect, this novel technique produced extremely accurate and high-resolution data, which when paired with information about tissue architecture exceeds other comparable techniques such as RNA sequencing. Despite the fact that these two patient groups received extremely similar clinical care, there were remarkable variations on a molecular level. Additionally, they discovered a patient who was co-infected with SARS-CoV-2 and H1N1, and whose lung response paralleled that of influenza patients. While this study only involved one patient who was infected with both viruses, it illustrates a scenario that may become more widespread in the future.

COVID lung cell atlases may lead to the discovery of novel therapeutic targets or repurposing possibilities, as well as a better understanding of what occurs to the lungs of those who recover from the rare illness. The multiple ramifications of infection with this particular coronavirus on the human body will take many years to disentangle, illuminate, and comprehend.

Reference
1.Melms, J. C., Biermann, J., Huang, H., Wang, Y., Nair, A., Tagore, S., … & Izar, B. (2021). A molecular single-cell lung atlas of lethal COVID-19. Nature, 1-6.
2.Bui, L. T., Winters, N. I., Chung, M. I., Joseph, C., Gutierrez, A. J., Habermann, A. C., … & Network, H. C. A. L. B. (2021). Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity. bioRxiv.
3.Margaroli, C., Benson, P., Sharma, N. S., Madison, M. C., Robison, S. W., Arora, N., … & Gaggar, A. (2021). Spatial mapping of SARS-CoV-2 and H1N1 lung injury identifies differential transcriptional signatures. Cell Reports Medicine, 2(4), 100242.