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Looking Into a COVID Lung Using Spatial Transcriptomics

As the early weeks of the pandemic unfolded and health care workers struggled to save so many lives, researchers began tracking the path of destruction of SARS-CoV-2, focusing at first on the tango between cells of the lung and of the immune system.

Just as mRNA vaccine technology was years in the making, so was a powerful way to illuminate cellular pathology: single-cell transcriptomics using single-cell RNA-Seq, aka simply RNA-Seq . It detects the abundance of all unique messenger RNA (mRNA) molecules in a cell, collectively called the transcriptome. RNA-Seq reveals the suite of proteins a cell produces in response to a stimulus – such as an influx of viruses. The technology is more than a decade old.

But cataloging the abundance of mRNAs in a lung cell, or in any cell, isn’t meaningful without the context of the organ of which it is a part. It’s a little like counting the number of times the words “the,” “a,” and “there” appear in a novel and trying to deduce the narrative.


In 2016, researchers at the Karolinska Institute improved upon RNA-Seq when they pioneered spatial transcriptomics, which, to borrow from Hamilton, provides “the room where it happens.” The technology places a cell’s entire mRNA repertoire at a given moment into the context of its surroundings by capturing images of the cell’s environs and interrogating them with visualization software.

Considering a cell’s surroundings provides a more meaningful view than picking out lung cells in sputum or collecting them from a bronchoscopy procedure and staining their surface features. Spatial transcriptomics even works on old tissue samples that have been preserved in formaldehyde and embedded in wax, thanks to a product called Visium from 10x Genomics, which gave me the idea for this post. It’s possible now to analyze the more than a billion such samples stored in hospital biopsy archives and biobanks for telltale gene expression patterns.

Spatial transcriptomics can counter bias from the researcher and even highlight cell types not known to be part of a particular organ. In short, it overcomes the human tendency to look for what we think we already know – or assume that we’ve seen it all.

A Cell-By-Cell Atlas

Two recent reports in Nature describe RNA-Seq analysis of cells harvested from the lungs of COVID patients within hours of death and flash-frozen, capturing in context the devastation of the acute respiratory distress syndrome that develops in about 15% of patients. Comparison of COVID lungs so soon after death to the lungs of healthy people, as well as to those with other respiratory conditions, reveals that the infection unfurls a profound impairment of the ability to repair damage.

RNA-Seq provides large data sets from just a few patients. For example, one of the recent reports in Nature, from researchers at Columbia University, analyzed RNA in 116,314 lung cells, from 19 adult COVID patients and 7 healthy controls. The researchers describe “a detrimental trifecta of runaway inflammation, direct destruction and impaired regeneration of lung cells involved in gas exchange, and accelerated lung scarring.” The second paper, from investigators at the Broad Institute, looked at 420 specimens from 17 adults, with similar findings.

Deadly and Different

What happens in a COVID lung is both different and deadly.

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

SARS-CoV-2 spreads quickly in cells lining the nasal passages, shoots down the trachea and bronchi, and traverses the narrowing bronchioles to the alveoli, stopping the vital exchange of gases.

RNA-Seq applied to cells from the fresh, autopsied lungs of COVID patients showed characteristic changes:

#1 “Pathological” fibroblasts unique to COVID rapidly produce collagen, burying the organ in scar tissue, like pouring thick glue or cement onto bubblewrap. The lungs stiffen, impairing gas exchange.

#2 B cells churn out antibodies against the virus, but the numbers of T cells, which activate B cells to produce antibodies, plummet. (These are white blood cells.)

#3 Levels of blood cells called monocytes skyrocket. Monocytes give rise to blobby macrophages, which engulf and dissolve pathogens, while also secreting cytokines, which drive the horrific “storm” of inflammation characteristic of COVID. Two cytokines – interleukin (IL)-1beta from the blood and IL-6 from the alveoli – are especially abundant.

#4 The lungs’ natural ability to regenerate halts. Normally, lining cells called alveolar type 2 (AT2) cells serve as caretakers, secreting the surfactant that bathes the air sacs. AT2 cells also boost regeneration by reverting to a stem-cell-like state, becoming able to divide and give rise to alveolar type 1 cells, which comprise the air sacs. The virus halts this vital transition, and the lung, at a microscopic level, can’t heal. So not only does the virus directly destroy alveoli, but it also attacks their ability to regenerate even if they survive. The lungs break down.

Higher Risks for People with Chronic Lung Disease

Another recent study, from The Translational Genomics Research Institute and the Human Cell Atlas Lung Biological Network and published in Nature Communications, used RNA-seq to discover what happens when COVID strikes people who already have difficulty breathing. “It was recognized early in the pandemic that patients with chronic lung diseases were at particularly high risk for severe COVID-19, and our goal was to gain insight into the cellular and molecular changes responsible for this,” explained co-author Jonathan Kropski.

Chronic lung diseases include chronic obstructive pulmonary disease and interstitial lung diseases such as idiopathic pulmonary fibrosis, which scars and stiffens lung tissue. These conditions may drive acute respiratory distress syndrome and multi-system organ failure and are worse in people with other risk factors for COVID, such as males, smokers, and those with hypertension, obesity, or diabetes.

The researchers analyzed mRNA sequences from 611,398 cells in various databases, representing healthy lungs and those from people with chronic lung disease. A “viral entry score” represented the expression of all genes associated with SARS-CoV-2 infection. Higher scores were seen in cells from patients with chronic lung disease.

“Our results suggest that patients with chronic lung disease are molecularly primed to be more susceptible to infection by SARS-CoV-2,” said co-author Nicholas Banovich.

Specifically, in people with COVID and chronic lung disease, patterns of gene expression in the all-important AT2 cells essentially “prime the pump,” easing the entry of the virus into lung lining cells. At the same time, localized inflammation and body-wide cytokine storms flare. This may make mechanical ventilation necessary to prevent drowning from the cascade of immune cells that flood the lungs, severely damaging organs. Often, the intervention isn’t enough.

The cell atlases of COVID lungs may suggest new drug targets or repurposing candidates, and may help to better understand what happens to the lungs of patients who recover from the distinctive pneumonia. It will take many years to unravel, illuminate, and understand the many effects on the human body of infection with this novel coronavirus.

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