Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has brought about more than 623 million infections along with more than 6.56 million deaths as reported by the World Health Organization as of 1st December 2021. Approximately 80% of COVID-19 patients were estimated to experience asymptomatic, moderate, or mild symptoms, while 20% experienced severe disease and death. However, mechanisms and molecular events of how SARS-CoV-2 infection can lead to severe pneumonia are still unknown.

Background

Several studies have highlighted that dysfunctional immune response plays an important role in severe COVID-19 symptoms. Patients with severe COVID-19 were observed to show abnormal peripheral immune activities along with a cytokine storm resulting from the upregulation of proinflammatory chemokines and cytokines as well as calprotectin. Moreover, high levels of hyperinflammatory monocyte-derived macrophages have been discovered in the bronchoalveolar lavage fluid (BALF) of severe COVID-19 patients. Also, highly clonal expanded CD8+ T cells were observed in moderate patients.

Furthermore, cellular metabolism is known to be important for the proper functioning of immune cells under both healthy and diseased conditions. Many recent clinical studies have indicated that COVID-19 causes alteration to plasma metabolites. Drop in blood nutrients, and dysregulation of metabolic components have been observed in patients with severe COVID-19.

Additionally, higher levels of T cells expressing voltage-dependent anion channel 1 were observed in the peripheral blood of acute COVID-19 patients. However, the immunometabolic regulation of leukocytes in the lungs, which is the most affected organ, and its association with disease severity and immune function is still unclear.

A new study to be published in the iScience aimed to analyze the immunometalobism BALF cells from mild to severe COVID-19 patients using computational algorithms that quantified metabolic fluxes and metabolic pathways.

About the study

The study involved the collection of single-cell RNA sequencing (scRNA-seq) from BALF of severe and mild COVID-19 patients as well as healthy controls, followed by cell clustering and annotation. Comparison of activities of metabolic pathways between various cell subsets was carried out using data imputation and normalization along with calculation of metabolic pathway activity. After that, differential gene expression analysis, correlation analysis, trajectory analysis, single-cell flux balance analysis, visualization of gene expressions, and score calculation of certain metabolic pathways were carried out.

Plasma metabolome data was collected from COVID-19 patients and healthy controls, followed by stimulation of primary macrophages by SARS-CoV-2 or TLR7/8 agonist resiquimod (R848). Phagocytosis and fatty acid uptake assay measured macrophages and fatty acids. Preparation of SARS-CoV-2 viral stocks was carried out in Vero-E6 cells. Human monocyte-derived macrophages were obtained from peripheral human blood mononuclear cells (PBMCs) and stimulated by SARS-CoV-2. Finally, RNA isolation, real-time PCR, and enzyme-linked immunosorbent assay (ELISA) were carried out.

Study findings

The results reported 9 major cell populations involving neutrophils, macrophages, plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC), natural killer (NK) cells, plasma cells, B and T lymphocytes, as well as epithelial cells in the immune cells from BALF of the COVID-19 patients. Cells from BALF were found to be segregated into mild, healthy, and severe based on 1526 metabolic gene expression levels. Moreover, most metabolic pathways such as oxidative phosphorylation, citrate cycle (TCA cycle), and glycolysis were unregulated in mDC, B cells, pDC, NK cells, macrophages, and T cells from BALF of mild COVID-19 patients. However, most of these pathways were downregulated for severe patients compared to mild patients.

71 out of 85 metabolic pathways were upregulated in macrophages from BALF of mild COVID-19 patients. However, only 8 pathways were reported to be upregulated in macrophages from BALF of severe COVID-19 patients, while 65 were down-regulated. Only fatty acid biosynthesis pathways and glycolysis pathways and their genes were observed to be upregulated in severe COVID-19 patients, while most of the metabolic pathways and their genes were observed to be upregulated in mild COVID-19 patients.

Out of the 294 detectable metabolic fluxes, 223 were decreased in the macrophages from BALF of severe COVID-19 patients compared to mild patients. Moreover, out of the 18 clusters of macrophages discovered, 8 comprised macrophages from mild COVID-19 patients, and 9 comprised macrophages from severe patients. CD14+ macrophage derived from lung (BALF-MCs) from mild patients were observed to show high levels of metabolic-related gene expression and metabolic pathway activity compared to those from severe patients. Also, metabolic activity increased along the CD14+ monocytes from PBMC (PBMC-MCs) to BALF-MCs trajectory for mild patients.

Furthermore, genes that encoded proinflammatory chemokines and cytokines were higher in severe patients, while genes for endocytosis and antigen presentation were downregulated. Expression of enzymes involved in pyruvate and glutamate metabolism was lower in severe COVID-19 patients, leading to high proinflammatory chemokines and cytokines. Macrophages with inhibited glycolysis showed reduced levels of these chemokines and cytokines. Moreover, lipid metabolism was also lower for severe COVID-19 patients. 

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathway that plays an important role in lipid metabolism and maintenance of metabolic homeostasis was also inhibited in macrophages derived from severe COVID-19 patients. Inhibition of the PPARγ signaling pathway was also associated with increased inflammatory cytokine and chemokines production. Finally, treatment with rosiglitazone, an FDA-approved antidiabetic drug, was reported to activate the PPARγ pathway in the macrophages. This further led to a reduction in proinflammatory cytokine production and an increase in metabolic gene expression.

Therefore, the current study demonstrated that the metabolic imbalance of bronchoalveolar macrophages could lead to hyperinflammation in severe COVID-19 patients. Regulation of metabolism by drugs such as rosiglitazone can help to prevent cytokine storms in such patients. Moreover, the study also identified immunometabolism dysregulation as an important part of COVID-19 pathogenesis, which could be used to develop novel COVID-19 therapeutics.

Limitations

The current study has certain limitations. First, the computational algorithms used in the study did not consider post-translational modulation and post-transcriptional regulation. Second, all major conclusions of the study were carried out in vitro. Third, the impact of rosiglitazone on PPARγ needs to be confirmed using PPARγ genetically knock-out cells. Fourth, viral biomass objective function (VBOF) was not added in the study to determine the metabolic requirement of the virus from the host. Finally, only human macrophages from healthy donors were used to test the effect of rosiglitazone, while its effect remains to be tested in cases of severe COVID-19 patients with type 2 diabetes.

Written by

Suchandrima Bhowmik

Suchandrima has a Bachelor of Science (B.Sc.) degree in Microbiology and a Master of Science (M.Sc.) degree in Microbiology from the University of Calcutta, India. The study of health and diseases was always very important to her. In addition to Microbiology, she also gained extensive knowledge in Biochemistry, Immunology, Medical Microbiology, Metabolism, and Biotechnology as part of her master's degree.