Differentially expressed genes in Diffuse Alveolar Damage (DAD) patterns of COVID-19

Severe COVID-19 induces DAD, a condition with temporal-spatial heterogeneity. We determined the differentially expressed genes (DEGs) in the histological patterns of DAD. Twelve fatal COVID-19 cases were classified in acute DAD (n=5) and intermediate/advanced (IA) DAD (n=7). Autopsy lung RNA was extracted from COVID-19 and 4 control cases. RNA sequencing was performed on the Illumina NovaSeq 6000. Enrichment analysis was performed with clusterProfiler using Genome-wide annotation for Human R package. GO terms and KEGG pathways were considered enriched if adjusted p<=0.05. Principal component analysis showed that IA-DAD samples were grouped, while acute DAD samples were scattered. The differential expression analysis between these two groups and the control cases revealed: 261 DEGs in the acute DAD (143 Up- and 53 Down-regulated), 244 DEGs in the IA- DAD tissues (67 Up- and 116 Down-regulated), and 61 DEGs were shared between them (45 Up- and 16 Downregulated). Patients with acute DAD had up-regulated genes related to oxidative phosphorylation, blood coagulation, megakaryocytes differentiation/regulation, and platelet degranulation/activation. Patients with IA-DAD had DEGs related to immunoglobulins and extracellular matrix. The shared up-regulated DEGs between both patterns are involved in innate and adaptive immune responses. We selected 3 DEGs in each DAD pattern for validation by realtime PCR. There were no differences in acute DAD DEGs, but DEGs overexpressed in intermediate DAD (COL3A1, IGLV3-19, IGHV1-58) were significantly higher. Genes related to thrombotic events occur at the acute stage of DAD, whereas immunoglobulin production and remodeling occur at later stages of DAD.

Correlation between lung inflammatory cells, viral load and cytokines in fatal COVID-19

SARS-CoV2 infection induces a complex interaction between virus and host immune system, activating multiple inflammatory pathways and leading to hyperinflammation, diffuse alveolar damage (DAD), ARDS, and multiorgan failure. We aimed to correlate the quantification of viral load, inflammatory cells and cytokines in lung tissue of fatal COVID-19. We assessed inflammatory cells by multiplex immunohistochemistry, cytokines by Luminex xMAP Assay and viral load by real time PCR in autopsy lung tissue of 18 COVID-19 patients. Correlations were considered statistically significant if p<0.05. Macrophages correlated with IL-1beta (r=0.54), IL-10 (r=0.5), IFN-alpha2 (r=0.72), IFN-gamma (r=0.6), CCL20 (r=0.5), TGF-beta1 (r=0.6), TGF-beta2 (r=0.6). CD4+T cells correlated with CCL20 (r=0.6), MDC/CCL2 (r=0.53), CCL17 (r=0.5), IP-10 (r=0.6), CXCL9 (r=0.6). CD8+T cells correlated with IL-1beta (r=0.54), IL-4 (r=0.63), IL-6 (r=0.7), IL-8 (r=0.63), IL-10 (r=0.6), TNF-alpha (r=0.6), IFN-gamma (r=0.74), CCL20 (r=0.7), TGF-beta1 (r=0.7), TGF-beta2 (r=0.56), TGF-beta3 (r=0.54), MDC/CCL2 (r=0.7), CCL17 (r=0.64). Langerin dendritic cells (DC) correlated with symptom onset to death interval (r=0.6), hospitalization length (r=0.65), mechanical ventilation (MV) length (r=0.6), ICU stay (r=0.6), exudative DAD (r=-0.5), viral load (r=-0.6). Myeloid DC correlated with symptom onset to death interval (r=0.8), hospitalization length (r=0.8), MV length (r=0.8), ICU stay (r=0.8), exudative DAD (r=-0.5), viral load (r=-0.7). Viral load correlated with symptom onset to death interval (r=-0.7), hospitalization length (r=-0.8), MV length (r=-0.7), ICU stay (r=-0.8), exudative DAD (r=0.6). There is a complex temporal inflammatory modulation in severe COVID-19.