Fig.5: Similarities in gene expression and pathways in MHV-1 infected obese vs lean mice and severe vs mild COVID-19 patients.
RNAseq was performed on the RNA collected from blood of MHV-1 and mock-infected lean and obese C3H/HeJ mice (n = 8 per group) at 2 dpi.(A) Principal component analysis (PCA) of MHV-1 and mock-infected lean and obese mice. PC1 and PC2 were plotted to identify clustering of the MHV-1 and mock-infected samples. For identification of DEGs, genes with p-adjusted values < 0.1 and log2FoldChange > 1, were selected by integrated Differential Expression and Pathway (iDEP .96) analysis tool.Upregulated (B) and downregulated (C) pathways unique to MHV-1 infected obese vs lean mice. For this comparison, gene expression in mock-infected lean or obese mice was removed from their MHV-1 infected counterparts and the resulting data was compared between obese and lean mice to identify DEGs unique to obese infected mice. For pathway analysis, DEGs with p-adjusted values < 0.1 and log2FoldChange > 1 were selected and pathways were generated using the ShinyGO 0.77 analysis tool. The pathways obtained were then compared to data from the blood of COVID-19 patients with severe disease; orange arrows indicate common upregulated and blue arrows indicate common downregulated pathways between MHV-1 infected obese vs lean mice and severe vs mild COVID-19 patients.
TNF has been shown to damage the respiratory epithelium and stimulate the synthesis of fibrin and collagen in the respiratory system57,58. Although the direct role of TNF in severe COVID-19 has not yet been established, it has been associated with heart failure59, blood clotting60-63, and is elevated in patients with obesity64 and hypertension65, all of which are risk factors for severe COVID-19. The NLR signaling pathway could potentially be linked to COVID-19 related multiple sclerosis (MS)66considering that the NLRP3 inflammasome has been implicated in COVID-19 pathogenesis67. Platelet abnormalities in COVID-19 patients have been associated with disease severity and mortality68 related to organ failure69. The underlying mechanisms leading to severe COVID-19 symptoms, such as acute respiratory distress syndrome (ARDS), cytokine storm and thrombotic events, have been associated with neutrophils, particularly increased formation of neutrophil extracellular traps (NETs)70. Excessive NET formation in SARS-CoV-2 infection has been associated with the onset of acute lung injury (ALI)71. The Rap1 signaling pathway has been implicated in the pathogenesis of respiratory, cardiovascular, and nervous system and some studies have shown that activation of the Rap1 signaling pathway can improve the symptoms of pulmonary fibrosis72.
T cells are recognized as potent immune modulators against various infections by activating cytotoxic or humoral immune-mediated reactions and are mainly divided into T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17) and regulatory T (Treg) cells73. The equilibrium between Th1 and Th2 responses has been linked to COVID-19 outcomes, with an adequate Th1 response being associated with good prognosis and Th2 activation being linked to worse disease outcomes74,75. Th17 has been associated with chronic inflammation and autoimmune diseases and may be involved in SARS-CoV-2 induced pneumonia76. Similarly, the other downregulated pathways involved hematopoietic cell lineage, NK cell-mediated cytotoxicity and primary immunodeficiency, suggesting lowered lymphocyte immune responses in MHV-1-infected mice and COVID-19 patients.
We also compared expression of individual DEGs unique to MHV-1 infected obese vs. lean mice with expression patterns in severe vs mild COVID-19 patients and found 20 upregulated and 23 downregulated genes shared between MHV-1 and COVID-19 (Supplementary Table 1). In particular, MHV-1 infected obese mice exhibited upregulation of B4galt577, Flt178, Stx379, and Ass180 and downregulation of Crip277 and Rora81changes (Supplementary Fig.S2) which have also been associated with severe disease outcomes in COVID-19 patients. Strikingly, the similarities observed in the immune pathways and gene expression patterns in our MHV-1 infected obese vs lean mice suggest that our coronavirus infection model mimics COVID-19 in humans and could be a potentially relevant model to identify biomarkers of severe disease outcome in SARS-CoV-2 infected individuals. However, future mechanistic studies are needed to elucidate the precise roles of these biomarkers in coronavirus disease outcomes.
Gene Set Variation Analysis (GSVA) showed significant differences between MHV-1 infected lean and obese C3H/HeJ mice. We further utilized our RNAseq data to carry out Gene Set Variation Analysis (GSVA) and compare the impact of MHV-1 infection and obesity on differential enrichment of disease-relevant immune cell, inflammatory, and cellular pathway gene signatures12,33,82 (Fig.6A & B). Several immune cell type signatures associated with COVID-19 patient disease pathology were also changed in MHV-1 infected mice, including enrichment of inflammatory neutrophils, low density granulocytes (LDGs), monocytes, and activated B cells as well as de-enrichment of T cells (Fig.6A). Pathway gene signatures representing response to inflammatory cytokines, such as interferons (IFN) and TNF, as well as metabolic pathways, including glycolysis and lipid metabolism were also elevated in infected mice (Fig.6B). Gene expression changes associated with obesity in the context of infection were much more subtle. However, we did find that the unfolded protein and lipid metabolism gene signatures were significantly increased suggesting that dysregulated cell stress and metabolism pathways could be involved in exacerbating disease outcomes in obese mice (Fig.6B).
In order to relate disease pathology with changes in gene expression, we carried out linear regression analysis using the inflammatory neutrophil gene signature, which was derived from gene expression studies of severe COVID-19 patients83, and clinical features. This revealed a significant correlation between the inflammatory neutrophil GSVA enrichment score and weight loss as well as lung and liver tissue pathology scores (Fig.6C-E) suggesting inflammatory neutrophils may also be contributing to disease severity in MHV-1 infected mice. In contrast, the activated B cell gene signature was significantly correlated with overall weight loss (Fig.6F), but not tissue pathology scores (Fig.6G-H) indicating that inflammatory neutrophils and not B cells were associated with tissue damage in infected mice. Overall, we have demonstrated the utility of MHV-1 infected C3H/HeJ mice as a model for human COVID-19 as well as the effect of diet-induced obesity on disease pathogenesis.