Fig.4:
MHV-1 infection causes diffuse alveolar damage (DAD) in the lungs of
C57BL/6N and C3H/HeJ mice.
Lungs of MHV-1 and mock-infected C3H/HeJ were collected for
histopathological analysis. H&E-stained images were captured at 20X,
scale bar = 100 um. Semi-quantitative histopathology scores showed
significant inflammation of the septa and alveoli, along with the
presence of type II pneumocyte hyperplasia, in MHV-1 infected compared
to mock-infected C57BL/6N (A-C) and C3H/HeJ (D-F) mice.
Statistical analysis was carried out
by 2way ANOVA. (G) Representative lung histopathology images
from C3H/HeJ mice depicting diffuse alveolar lung damage (DAD). Lungs
of MHV-1 infected mice show alveolar and septal inflammation, along with
type II pneumocyte hyperplasia compared to mock-infected mice. The level
of significance is represented as follows: p < 0.05 (*); p
< 0.001 (**); p = 0.001 to 0.0001 (***); p < 0.0001
(****), ns = non-significant. The error bars indicate SD of the mean.
Pathways and genes altered in obese C3H/HeJ mice exposed to
MHV-1 are similar to those identified in severe COVID-19 patients.COVID-19 is a complex multisystem disorder involving vast interactions
of immune and inflammatory pathways55. Identifying
potential disease-associated biomarkers may be useful to predict the
outcome of COVID-19 in humans to assess risk levels to enable timely
medical intervention. In this study, we hypothesized that blood would be
an easily accessible clinical sample to monitor the complex
transcriptional changes occurring early during the course of COVID-19.
Therefore, we carried out RNAseq on blood from MHV-1 and mock-infected
lean and obese C3H/HeJ mice at 2 dpi.
First, we carried out principal
component analysis (PCA) to assess the clustering of the samples. We
observed a clear separation between MHV-1 and mock-infected groups in
lean and obese C3H/HeJ mice; in contrast, we observed no obvious
clustering for lean and obese groups (Fig.5A), suggesting that
infection, but not diet, contributes more significantly to gene
expression changes between groups. Differential gene expression (DEG)
analysis revealed 2635 upregulated and 863 downregulated genes in MHV-1
vs mock-infected lean mice (Supplementary Fig.S1-A) and 2089 upregulated
and 681 downregulated genes in MHV-1 vs mock-infected obese mice
(Supplementary Fig.S1-B). Pathway analysis was initially carried out by
Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes
and Genomes (KEGG) for identifying canonical pathways that were up or
downregulated in MHV-1 infected vs mock lean (Supplementary Fig.S1-C)
and obese mice (Supplementary Fig.S1-D). To account for the effect of
mock infection on gene expression, we removed the genes expressed in
mock-infected lean or obese mice from their MHV-1 infected counterparts
and then identified unique DEGs in MHV-1 infected obese vs lean mice.
There were 177 upregulated and 334 downregulated genes that were unique
to MHV-1 infected obese mice compared to the infected lean mice, after
adjusting for the DEG expression in their respective mocks and this list
was used for generating pathways using the ShinyGO 0.77 analysis tool.
These pathways were then compared to those identified in severe vs mild
cases of COVID-19 (generated from data obtained from Wang et
al.56) and we found numerous upregulated (Fig.5B) and
downregulated (Fig.5C) pathways shared between MHV-1 infected obese vs
lean C3H/HeJ mice and human patients with severe vs mild COVID-19. The
common upregulated pathways included Tumor necrosis factor (TNF)
signaling, NOD-like receptor (NLR) signaling, platelet activation,
Neutrophil extracellular trap (NET) formation, and Rap1 signaling
pathways; while the common downregulated pathways included T cell
receptor signaling pathway, Th1, Th2, Th17 cell-differentiation,
inflammatory bowel disease, hematopoietic cell lineage, Natural Killer
(NK) cell mediated cytotoxicity and primary immunodeficiency pathways.