Study identifies the essential genetic and immunologic perturbations occurring in patients with long COVID lung fibrosis

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Mar 3 2023

In a recent study published in PNAS, researchers elucidated immunologic and genetic perturbations in long coronavirus disease (COVID) patients with pulmonary fibrosis.

Background

Long COVID presents a spectrum of clinical manifestations, among which lung fibrosis is among the most severe. Studies have reported elevated expression of the cluster of differentiation 47 (CD47), interleukin-6 (IL-6), and the Jun proto-oncogene, AP-1 Transcription Factor Subunit (JUN) expression among patients with COVID 2019 (COVID-19)-induced lung fibrosis. However, the exact pathophysiological mechanisms of pulmonary fibrosis development in long COVID patients are unclear.

About the study

In the present study, researchers modeled pulmonary fibrosis in long COVID and profiled the immunological responses using JUN mice by single-cell mass cytometry.

The team analyzed the single-cell transcriptomic profiles of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected pulmonary tissues to detect the innate immunological mediators of COVID-induced pulmonary fibrosis. Using mass spectrometry and histopathological analyses, innate immunological alterations concerning the progression of lung fibrosis were assessed.

Pulmonary tissues of patient groups published in GEO GSE122960, GSE149878, GSE158127, and GSE163919, were analyzed. A total of 251,612 single-cell transcriptomes from five long COVID patients and 20 controls were analyzed using uniform manifold approximation and projection (UMAP), and differential gene expression among the two groups was assessed.

Differentially expressed biomarkers in the single-cell ribonucleic acid sequencing (scRNA-seq) analysis were validated by immunofluorescence analyses of long COVID pulmonary tissues with COVID-19 markers. Immunofluorescence markers included SARS-CoV-2 nucleocapsid (N) protein, neutrophil elastase (NE), CD3, CD11b, CD20, CD31, CD68, transcription termination factor-1 (TTF-1), collagen I, mesothelin, fibroblast-specific protein-1 (FSP-1), and smooth muscle actin (SMA).

For recapitulating pulmonary fibrosis pathophysiology IN long COVID, the team generated a murine model that developed pulmonary fibrosis as a response to SARS-CoV-2 pseudotype transduction. Further, the therapeutic efficacy of IL-6/CD47 inhibition in managing lung fibrosis post-COVID-19 was evaluated in the humanized SARS-CoV-2-positive murine model.

Results

The chronic immunological activation was reminiscent of post-COVID-19 conditions or long COVID among humans, characterized by elevated IL-6, CD47, and phosphorylated-JUN (pJUN) levels, correlating with the severity of lung fibrosis, and the number of pathogenic fibroblasts. Subsequent treatment of humanized mice with COVID-19-induced lung fibrosis, by combined inhibition of fibrosis and inflammation, eliminated fibrosis, restoring the innate immunological equilibrium.  

The findings indicated that CD47/IL-6 inhibitors could treat lung fibrosis among long COVID patients. Matured neutrophil and monocyte-derived alveolar-type macrophages (MoAM) were markedly elevated with greater IL-6 expression in SARS-CoV-2-positive tissues. COVID-19 modulated profibrotic genetic signatures, including CD47 and JUN, among fibroblasts. In lung fibrosis tissues, elevated CD68 and CD163 levels, indicating profibrotic macrophages, and elevated myeloperoxidase and CD15 levels, indicating activated neutrophils, were observed in fibrotic areas rich in collagen.

Among humans, pathogenic pulmonary fibrosis in the post-acute COVID-19 phase was recapitulated in the COVID-19-induced pulmonary fibrosis murine model and the human lung organ model. White blood cell counts, comprising neutrophils, T lymphocytes, and macrophages, were significantly altered in the pulmonary tissues and fibroblasts of long COVID patients with lung fibrosis, alveolar bronchiolization, and fibrosis-induced interstitial tissue expansion.

The findings indicated that the macrophage leukocyte compartment is especially affected by long COVID, with loss of the majority of pulmonary macrophages and the differentiation of monocytes into alveolar macrophages during pulmonary fibrosis. The team observed the enrichment of the activator protein-1 (AP-1) genes and of those involved in neutrophil maturation-associated cytokine and chemokine among long COVID patients with pulmonary fibrosis.

In addition, Elsevier pathway database analysis showed upregulation of neutrophil activation-associated genes in the long COVID group. The findings highlighted the key roles of elevated MoAM, mature neutrophils, and injured tissue-resident alveolar macrophages (TRAM) in pulmonary tissues during the post-acute phase of COVID-19. SARS-CoV-2-induced inflammation involved IL-6 secretion by MOAM and mature neutrophils and elevated AP-1 levels among active fibroblasts for the progression of inflammation and fibrosis in long COVID patients via innate immunological interactions.

Therefore, the onset of lung fibrosis in long COVID occurred following extensive immunological infiltration and the activation of pro-fibrotic fibroblasts. The findings indicated that anti-CD47/IL-6 antibody treatment could effectively treat pulmonary fibrosis among long COVID patients, confirmed by cytometry by the time of flight (CyTOF) analysis, and lower SMA and FSP-1 levels post-therapy. Histopathological examination after 14 days of therapy showed significant restoration of normal pulmonary tissue morphology with less extracellular matrix/collagen deposition.

The findings elucidated lung fibrosis immunopathological alterations and the CD47-IL-6-JUN axis-mediated increase in lung fibrosis in long COVID. Neutrophils, macrophages, and monocytes were the most commonly observed innate immune system cells in the long COVID group lungs, and the association between the immunological cell counts and COVID-19-associated fibrosis progression indicated that the aforementioned innate immunological cells are the immediate-type effector cells accountable for chronic pulmonary inflammation in long COVID patients.

Conclusion

Overall, the study findings highlighted pulmonary fibrosis pathophysiology among long COVID patients using systems biological and mechanistic, therapeutic, and histopathological analyses in vivo utilizing humanized mice with COVID-19-induced pulmonary fibrosis. The findings indicated that anti-CD47/IL-6 inhibitors could treat lung fibrosis among long COVID patients.