Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response. The mechanisms of SARS-CoV-2 induced excessive inflammatory response associated with disease severity needs to be critically studied for therapeutic intervention. Cytokine storm is typical of macrophage activation; which leads to tissue damage, lung injury, and acute respiratory distress syndrome. Lung epithelial cells; a primary host for SARS coronavirus infection, can generate a cytokine response leading to an expanded pathology. Our present study was designed to understand SARS-CoV-2 and human epithelial cell interactions, by delineating their contribution to inflammatory cytokine systems with special emphasis on ectopic expression of the viral spike protein. We observed that SARS-CoV-2 infection or spike protein expression in human epithelial cells inhibits ACE2 expression leading to the induction of AT1 signaling, and promotion of IL-6/soluble IL-6R release. IL-6 is one of the major mediators of the hyper-inflammatory response, and can exert signaling through two main pathways referred to as classical or trans- signaling to coordinate prominent pro-inflammatory properties. Our experimental observations suggest the potential for IL-6 trans-signaling during SARS-CoV-2 infection of epithelial cells. These observations will aid in the development of new therapeutic modalities for combating COVID-19 associated disease severity. Aggressive tissue damage via increased IL-6 secretion and other cytokines leading to a hyper-inflammatory response are in line with COVID-19 disease severity. We propose that SARS-CoV-2 infection in pulmonary epithelial cells induces IL-6 trans-signaling for secretion of chemokines; like MCP-1, from pulmonary vascular endothelial cells to attract monocytes/macrophages and create a hyper-inflammatory state leading to enhanced disease severity and acute respiratory distress syndrome.

The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM).

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae , the causative agent of cholera. In this study, activation of the genes edd and eda , encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae . Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes ( ctxA and tcpA ) and their regulator ( toxT ). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δ edd mutation [Δ edd _N16961 strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

Long-chain fatty acids (LCFAs) are used as a rich source of metabolic energy by several bacteria including important pathogens. Because LCFAs also induce oxidative stress, which may be detrimental to bacterial growth, it is imperative to understand the strategies employed by bacteria to counteract such stresses. Here, we performed a genetic screen in Escherichia coli on the LCFA, oleate, and compared our results with published genome-wide screens of multiple non-fermentable carbon sources. This large-scale analysis revealed that among components of the aerobic electron transport chain (ETC), only genes involved in the biosynthesis of ubiquinone, an electron carrier in the ETC, are highly required for growth in LCFAs when compared with other carbon sources. Using genetic and biochemical approaches, we show that this increased requirement of ubiquinone is to mitigate elevated levels of reactive oxygen species generated by LCFA degradation. Intriguingly, we find that unlike other ETC components whose requirement for growth is inversely correlated with the energy yield of non-fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results therefore suggest that a mechanism in addition to the known electron carrier function of ubiquinone is required to explain its antioxidant role in LCFA metabolism. Importantly, among the various oxidative stress combat players in E. coli, ubiquinone acts as the cell's first line of defense against LCFA-induced oxidative stress. Taken together, our results emphasize that ubiquinone is a key antioxidant during LCFA metabolism and therefore provides a rationale for investigating its role in LCFA-utilizing pathogenic bacteria.

Hepatitis C virus (HCV) is characterized by a high number of chronic cases due to an impairment of protective innate and adaptive immune responses. Here, we examined the contribution of the individual ectodomains of E1, E2, or a modified E2 with reduced CD81 binding and an inserted N-linked glycosylation site in combination as vaccine antigen mRNA-lipid nanoparticles (LNPs). The induction of a protective immune response to surrogate recombinant vaccinia virus (VV) expressing homologous HCV glycoprotein(s) challenge infection in a BALB/c mouse model was observed. Vaccination with a mRNA-LNP expressing soluble E1 (sE1) significantly reduced vv/HCV titer in the mouse ovary. However, the addition of sE2 mRNA-LNP for immunization impaired the efficacy of the sE1 construct. Further analysis showed that Th1 related cytokine responses to the sE1 mRNA-LNP were significantly altered in the presence of sE2 following co-immunization. Evaluation of immunogenicity revealed that the use of modified sE2_F442NYT nucleoside mRNA-LNP vaccine results in an improved cellular immune response, IgG2a isotype switching, enhanced total IgG, and an increase in the neutralizing antibody response against HCV pseudotype virus. HCV cross genotype specific reactivity to peptides representing conserved E2 specific linear epitopes were enhanced in modified E2 vaccinated animal sera. In the absence of a suitable immunocompetent small animal model for HCV infection, protection from surrogate HCV vaccinia challenge infection model was observed in the immunized mice as compared to sE1 alone or an unmodified sE2 mRNA-LNP vaccine. Inclusion of sE1 with modified sE2_F442NYT as mRNA-LNP vaccine candidate appeared to be beneficial for protection.