Spatial map of the lung reveals cellular mechanisms driving damage and fibrosis in severe COVID-19

A lung affected by deadly forms of COVID-19 not only loses essential cells for breathing but also undergoes profound changes in its internal organization and the way its cells communicate with each other. A study involving the Sant Pau Research Institute (IR Sant Pau), published in Nature Communications, has created a spatial map of this transformation and identified some of the molecular mechanisms that accompany the progression of lung damage and fibrosis.

The study was led by Dr. Manel Esteller, head of the Cancer Epigenetics group at IR Sant Pau, ICREA research professor and professor of genetics at the University of Barcelona (UB), and included researchers from different Spanish and European institutions.

The authors analyzed lung tissue from 19 patients who died from COVID-19, seven with acute diffuse alveolar damage (DAD) and 12 with proliferative DAD, along with control lung samples. DAD is a lung injury that appears in severe forms of acute respiratory distress syndrome and is one of the main changes observed in fatal cases of COVID-19.

In total, the researchers studied 91,068 microscopic regions of lung tissue and used the Human Lung Cell Atlas as a reference to identify and locate 45 cell types.

Unlike conventional transcriptomic analyses, which require breaking the tissue apart and cause its original organization to be lost, spatial transcriptomics preserves the position of each analyzed region and allows gene expression to be related to the architecture of the lung.

“What we see is that severe COVID-19 does not only cause cellular damage, but a profound reorganization of the lung tissue. The populations of cells present, their distribution and the signals they exchange with each other change, and this combination helps us better understand how the damage progresses and why such intense fibrosis can end up occurring,” explains Esteller.

A profound reorganization of lung tissue

The analysis shows that the progression from normal lung tissue to acute DAD and subsequently proliferative DAD is accompanied by a profound transformation of the tissue’s cellular composition.

Among the epithelial cells, the researchers observed a decrease in alveolar type 1 pneumocytes (AT1), responsible for much of the exchange of oxygen and carbon dioxide between the lung and the blood. In contrast, type 2 pneumocytes (AT2) increased in proliferative DAD. These cells can proliferate and act as AT1 progenitors after injury, so this increase reflects the activation of repair mechanisms in the alveolar epithelium.

“As the damage progresses, we see that cells essential for normal lung function are lost, such as capillary endothelial cells or natural killer cells, while certain populations of macrophages and, in a particularly marked way, peribronchial fibroblasts increase,” explains Esteller. “These fibroblasts not only become much more abundant, but they also reorganize themselves to form specific niches within the tissue, a change that is closely related to the fibrotic response that we observe in the most advanced stages of the disease.”

A molecular pathway that drives fibrosis

Spatial information allowed the researchers to analyze not only which cells were present but also how they communicated with each other through interactions between ligands and receptors. The analysis identified a cellular communication signature particularly associated with proliferative DAD and enriched in the transforming growth factor beta (TGF-β) pathway, one of the main mechanisms involved in tissue repair and in the development of fibrosis when its activation is excessive or persistent.

Within this pathway, the study reveals an imbalance between two regulators with opposing functions. In proliferative DAD, SMAD3 activity increases, favoring the production of extracellular matrix, tissue repair and the formation of fibrosis. Meanwhile, the activity of SMAD7, which acts as a brake on profibrotic TGF-β/SMAD3 signaling, decreases.

“What we see is that the lung sets in motion mechanisms to repair the damage, but in the most severe cases this response can end up favoring the formation of fibrous tissue and making it difficult to recover its normal structure,” says Esteller. “Identifying the signals that push this process helps us better understand why such intense fibrosis can develop and points to specific targets that could be studied in the future to try to stop it.”

The researchers also identified the TIMP1-CD63 interaction as one of the main components of this profibrotic signature. Based on these data, they reconstructed a signaling network within peribronchial fibroblasts in which activation of the ITGB1 receptor is related to changes in SMAD3 and SMAD7 and to the activation of kinases such as ILK, AKT1, MAP2K4 and MAPK14. Some of these components could become targets for future research aimed at limiting pulmonary fibrosis.

The lung tries to regenerate the alveoli

The same approach has allowed us to reconstruct another process that occurs simultaneously with inflammation and fibrosis: the lung’s attempt to repair the destroyed alveolar epithelium. The researchers analyzed the transition from AT2 pneumocytes to AT1 and, by combining gene expression with the position of the cells within the tissue, were able to identify different states of differentiation.

Some regions showed cells still in intermediate stages, while others showed markers of more advanced maturation, such as CAV1, characteristic of mature AT1 pneumocytes. The results indicate that even in lungs with very advanced disease, programs aimed at regenerating the alveolar epithelium remain active, although the process does not occur uniformly throughout the tissue and may be incomplete.

“The lung does not remain passive in the face of injury. We were able to follow the attempt of type 2 pneumocytes to generate new type 1 cells and rebuild the alveolar epithelium within the same tissue,” explains Esteller. “The problem is that, in these patients, this regeneration process coexists with a profound alteration of cell communication and a very intense fibrotic response, so that the normal architecture of the lung is not recovered.”

Changes different from those in other lung diseases

To find out to what extent the observed changes were specific to severe COVID-19 or were part of a general response of the lung to injury, the researchers compared their results with eight cases of diffuse alveolar damage unrelated to COVID-19 and with samples of idiopathic pulmonary fibrosis.

The differences were especially clear in fibroblasts. In proliferative DAD associated with COVID-19, peribronchial fibroblasts predominated and formed specific niches within the tissue. In alveolar damage from other causes, alveolar fibroblasts predominated, while in idiopathic pulmonary fibrosis, peribronchial fibroblasts were not organized in the same way as in COVID-19.

“Comparison with other lung diseases allows us to see that we are not facing identical fibrosis in all cases,” explains Esteller. “In severe COVID-19 we find a particular combination of cell types, molecular signals and forms of tissue organization. This is important because it helps us distinguish which changes are part of a general response to damage and which seem to be more closely related to this disease.”

This conclusion was also strengthened by comparing gene regulation programs with Human Lung Cell Atlas data from more than 1.4 million cells from healthy lungs and from patients with COVID-19, non-COVID-19 pneumonia, idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease.

In this analysis, some of the mechanisms identified in the study, including the pattern of higher SMAD3 activity and lower SMAD7 activity, showed a particularly marked association with COVID-19.

The study was carried out mainly with postmortem samples, and the samples representing different phases of lung damage came from different patients, so it does not allow us to directly follow the progression of the disease in the same person.

The authors also point out that the clinical differences between the groups make it difficult to completely separate the specific effects of the infection from other factors such as comorbidities, duration of the disease or secondary lung infections.

“This work gives us a much more complete view of what happens inside the lung in the most severe forms of COVID-19, because we can simultaneously observe which cells are present, where they are located and how they communicate,” concludes Esteller. “But, beyond this specific disease, it demonstrates the potential of spatial transcriptomics to study how a diseased tissue is organized and to identify mechanisms that may be hidden when we analyze cells in isolation.”

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Sadie Harley

BSc Life Sciences & Ecology. Microbiology lab background with pharmaceutical news experience in oil, gas, and renewable industries.

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Robert Egan

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