A Network-Based Analysis Reveals the Mechanism Underlying Vitamin D in Suppressing Cytokine Storm and Virus in SARS-CoV-2 Infection

Abstract

Background: SARS-CoV-2 causes ongoing pandemic coronavirus disease of 2019 (COVID-19), infects the cells of the lower respiratory tract that leads to a cytokine storm in a significant number of patients resulting in severe pneumonia, shortness of breathing, respiratory and organ failure. Extensive studies suggested the role of Vitamin D in suppressing cytokine storm in COVID-19 and reducing viral infection; however, the precise molecular mechanism is not clearly known. In this work, bioinformatics and systems biology approaches were used to understand SARS-CoV-2 induced cytokine pathways and the potential mechanism of Vitamin D in suppressing cytokine storm and enhancing antiviral response.

Results: This study used transcriptome data and identified 108 differentially expressed host genes (DEHGs) in SARS-CoV-2 infected normal human bronchial epithelial (NHBE) cells compared to control. Then, the DEHGs was integrated with the human protein-protein interaction data to generate a SARS-CoV-2 induced host gene regulatory network (SiHgrn). Analysis of SiHgrn identified a sub-network "Cluster 1" with the highest MCODE score, 31 up-regulated genes, and predominantly associated immune and inflammatory response. Interestingly, the iRegulone tool identified that "Cluster 1" is under the regulation of transcription factors STAT1, STAT2, STAT3, POU2F2, and NFkB1, collectively referred to as "host response signature network". Functional enrichment analysis with NDEx revealed that the "host response signature network" is predominantly associated with critical pathways, including "cytokines and inflammatory response", "non-genomic action of Vitamin D", "the human immune response to tuberculosis", and "lung fibrosis". Finally, in-depth analysis and literature mining revealed that Vitamin D binds with its receptor and could work through two different pathways: (i) it inhibits the expression of pro-inflammatory cytokines through blocking the TNF induced NFkB1 signaling pathway; and (ii) it initiates the expression of interferon-stimulating genes (ISGs) for antiviral defense program through activating the IFN-α induced Jak-STAT signaling pathway.

Conclusion: This comprehensive study identified the pathways associated with cytokine storm in SARS-CoV-2 infection. The proposed underlying mechanism of Vitamin D could be promising in suppressing the cytokine storm and inducing a robust antiviral response in severe COVID-19 patients. The finding in this study urgently needs further experimental validations for the suitability of Vitamin D in combination with IFN-α to control severe COVID-19.

Keywords: COVID-19; RNA sequencing; SARS-CoV-2; bioinformatics; cytokine storm; lung fibrosis; regulatory network; vitamin D.

Figure 1

Schematic diagram of present study workflow.

Figure 2

Functional enrichment of host genes in cells infected with SARS-CoV-2 and their biological network. (A) GO and KEGG pathways functional enrichment of up-regulated host genes. (B) SARS-CoV-2 induced the Host gene regulatory network (SiHgrn). (C) MCODE clusters extracted from the SiHgrn network. MCODE score is given in the bracket. A red node and a green node represent an up-regulated and down-regulated gene, respectively, in DEHGs, while the blue node represents without expression value. GO, Gene Ontology; BP, Biological Processes; MF, Molecular Function; CC, Cell Component; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

Functional enrichment of Cluster 1 and its potential upstream regulators. (A) GO and KEGG pathways functional enrichment. (B) MSigDB Hallmark and positional gene sets enrichment. (C) Potential upstream regulators of Cluster 1 genes. Each column indicates the gene of Cluster 1, while each row indicates TF identified by iRegulone. Up-regulated DEHGs in the cluster are red with positive log2FC. TF binding with the mRNA is in purple, while non-binding in cyan.

Figure 4

Biological pathways enrichment of “host response signature network”: (A) “Cytokines and Inflammatory Response” and (B) “Non-genomic actions of 1,25 dihydroxyvitamin D3”. Gene from the “host response signature network” of the SARS-CoV-2 infected cell is in the red box. The IRF9 and CYP27B1 genes are up-regulated in DEHGs. CYP27B1 in the purple box is not associated with the “host response signature network”.

Figure 5

Biological pathways enrichment of “host signature network”: (A) “The human immune response to tuberculosis” and (B) “Lung fibrosis.” Gene from the “host response signature network” of the SARS-CoV-2 infected cell is in the red box.

Figure 6

Host response pathways using differentially expressed host genes (DEHGs^COVID-19) in the high viral load compared to the low viral load of lungs autopsy of COVID-19 patients. (A) “Cytokines and Inflammatory Response” and (B) “Non-genomic actions of 1,25 dihydroxyvitamin D3.” A red box indicates an up-regulated gene, while a blue box represents a down-regulated gene in the high viral load compared to the low viral load of COVID-19 lung samples, respectively.

Figure 7

The proposed model of the non-genomic actions of 1,25 dihydroxyvitamin D3 (1,25 D) in the lungs infected with SARS-CoV-2. The 1,25 D is a biologically active form of vitamin D that blocks the TNF induced NFkB1 activation. The 1,25 D binds with VDR and enhances the interaction between VDR-IKKβ, which prevents phosphorylation of IKKβ and formation of active IKK. Therefore, the degradation of IkB is blocked, resulting in preventing the translocation of NFkB1 to the nucleus. Consequently, the transcription and expression of NFkB1 target genes responsible for the cytokine storm are suppressed. In addition, 1,25 D enhances the IFN-α induced Jak-STAT signaling pathway. IFN-α activates the JAK1 and TYK2 signaling, which subsequently phosphorylate and activate the downstream targets STAT1 and STAT2. The 1,25 D binds with VDR and induces the dissociation between VDR-STAT1; thus, STAT1 is available for phosphorylation and formation of active TF complex ISGF3. The translocation of ISGF3 to the nucleus activates the transcription of interferon-stimulated genes (ISGs), which provide antiviral activity and reduce the SARS-CoV-2 load in cells. The figure was adapted from the WikiPathways (WP4341) www.wikipathways.org/instance/WP4341.