To Örebro University

Flaviviruses are usually transmitted to humans via mosquito or tick bites, whose infections may lead to severe diseases and fatality. During intracellular infection, they remodel the endoplasmic reticulum (ER) membrane to generate compartments scaffolding the replication complex (RC) where replication of the viral genome takes place. In this study, we purified the ER membrane fraction of virus infected cells to identify the proteins that were enriched during flavivirus infection. We found that tripartite motif-containing proteins (TRIMs) including TRIM38, TRIM21, and TRIM14 were significantly enriched during infection with mosquito-borne (West Nile virus strain Kunjin and Zika virus (ZIKV)) and tick-borne (Langat virus (LGTV)) flaviviruses. Further characterizations showed that TRIM21 and TRIM14 act as restriction factors against ZIKV and LGTV, while TRIM38 hinders ZIKV infection. These TRIMs worked as interferon-stimulated genes to mediate IFN-I response against LGTV and ZIKV infections. Restriction of ZIKV by TRIM14 and TRIM38 coincides with their colocalization with ZIKV NS3. TRIM14-mediated LGTV restriction coincides with its colocalization with LGTV NS3 and NS5 proteins. However, TRIM21 did not colocalize with ZIKV and LGTV NS3 or NS5 protein suggesting its antiviral activity is not dependent on direct targeting the viral enzyme. Finally, we demonstrated that overexpression of TRIM21 and TRIM14 restricted LGTV replication.

Interferon gamma (IFN-γ) regulates the expression of numerous genes, many with known immunomodulatory, angiostatic, and antiviral functions. However, the functions of many of these genes in vascular cells and their implications for atherosclerosis remain to be fully understood.

In this thesis, we investigate the functions of IFN-γ-regulated genes in vascular cells to understand their implications for atherosclerosis. In Paper I, we demonstrate RSAD2, a gene strongly induced by IFN-γ, to regulate the expression of CXCR3 chemokines in vascular smooth muscle cells (VSMCs), which are crucial for monocyte migration in vitro. Both VSMCs and macrophages express RSAD2 within human carotid atherosclerotic lesions where RSAD2 also correlates with CXCL10 and CXCL11. In Paper II, we generated a mousemodel of atherosclerosis lacking one or both alleles for RSAD2 and report that RSAD2-deficient mice exhibit reduced body weight without significant differences in plaque size. In Paper III, we observed tumour necrosis factor alpha (TNF-α) to upregulate the expression of genes encoding laminin-332 (LN332). These genes are downregulated by IFN-γ (unpublished results included in this thesis). Endothelial cells, when cultured on LN332, acquire an atherogenic phenotype and the levels of two of the genes encoding LN332 are elevated in human carotid atherosclerotic lesions where they also correlate with TNF-α. In Paper IV we demonstrate IFN-γ to alter the expression of genes involved in glutamine metabolism in VSMCs. Among these, a significant downregulation is observed for the enzyme, glutaminase, which converts glutamine to glutamate, a precursor required for de novo glutathione synthesis, a molecule that counters oxidative damage. Notably, glutaminase expression is significantly reduced in human carotid atherosclerotic lesions. Furthermore, the expression pattern of genes associated with glutamine metabolism in IFN-γ-stimulated VSMCs closely resembles that observed in human carotid lesions. In summary, this thesis investigates the effects of IFN-γ-regulated genes on vascular cells and demonstrates that IFN-γ may exert both pro- and anti-atherogenic effects in vascular cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r =  0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Laminins are essential components of the basement membranes, expressed in a tissue- and cell-specific manner under physiological conditions. During inflammatory circumstances, such as atherosclerosis, alterations in laminin composition within vessels have been observed. Our study aimed to assess the influence of tumor necrosis factor-alpha (TNF), a proinflammatory cytokine abundantly found in atherosclerotic lesions, on endothelial laminin gene expression and the effects of laminin-332 (LN332) on endothelial cells' behavior. We also evaluated the expression of LN332-encoding genes in human carotid atherosclerotic plaques. Our findings demonstrate that TNF induces upregulation of LAMB3 and LAMC2, which, along with LAMA3, encode the LN332 isoform. Endothelial cells cultured on recombinant LN332 exhibit decreased claudin-5 expression and display a loosely connected phenotype, with an elevated expression of chemokines and leukocyte adhesion molecules, enhancing their attractiveness and adhesion to leukocytes in vitro. Furthermore, LAMB3 and LAMC2 are upregulated in human carotid plaques and show a positive correlation with TNF expression. In summary, TNF stimulates the expression of LN332-encoding genes in human endothelial cells and LN332 promotes an endothelial phenotype characterized by compromised junctional integrity and increased leukocyte interaction. These findings highlight the importance of basement membrane proteins for endothelial integrity and the potential role of LN332 in atherosclerosis.

Background and Aims: Cells within atherosclerotic lesions have a higher glutamine demand than cells in healthy vessel although glutaminase, the enzyme converting glutamine to glutamate, is significantly downregulated in human carotid lesions. This may suggest rewiring of glutamine metabolic pathways in atherosclerotic lesions, caused by infiltrating immune cells and or their cytokines. Here we aimed at exploring the enzymes and transporters involved in glutamine metabolism in human carotid atherosclerotic tissues and aortic smooth muscle cells (hAoSMCs) exposed to interferon gamma.

Methods: Protein and mRNA from interferon gamma-treated hAoSMCs were subjected to Western blot or qRT-PCR for quantification of enzymes and transporters involved in glutamine metabolism. H2DCFDA probe was utilized for detection of intracellular reactive oxygen species (ROS) using flow cytometry. The expression of these enzymes and transporters was also evaluated in human carotid lesions (GEO accession: GSE43292).

Results: Interferon-treated hAoSMCs display a significantly lower expression of glutaminase followed by an increase in the expression of glutamine transporters, glutamine synthetase and glutamine-fructose-6-phosphate transaminase-1 (GFPT1). The level of ROS and the expression of enzymes involved in de novo synthesis of glutathione are elevated in interferon-treated cells. A similar expression pattern for these genes, expect for GFPT1, is also evident in human carotid lesions where glutaminase mRNA shows a strong positive correlation with SMC markers and a strong negative correlation with macrophage markers.

Conclusions: Glutamine metabolism is disrupted in human carotid lesions and interferon gamma alters glutamine metabolism in hAoSMCs, which may favor the production of UDP-GlcNAc and reactive oxygen species.

BACKGROUND AND AIMS: Laminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs).

METHODS: HUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively.

RESULTS: Stimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3.

CONCLUSIONS: We showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

Background and Aims: During atherosclerosis, activated vascular cells secrete chemokines to recruit leukocytes to the subendothelial space. Activated leukocytes, in particular activated T lymphocytes express IFN-γ, which affects the expression of numerous genes, including the antiviral protein viperin, in vascular cells. Viperin is expressed in endothelial cells of human carotid plaques but its relevance for vascular physiology and/or pathophysiology has not been established (Olofsson et al., 2005). We aimed at studying the function of viperin in human AoSMCs.

Methods: Immunostaining of human carotid plaques was performed to determine the expression and localization of viperin in carotid plaques. To study the role of viperin in regulation of vascular inflammation, viperin was silenced using siRNA in cultured human AoSMCs, prior to stimulation with IFN-γ. Release of inflammatory mediators was analyzed using OLINK proteomics and ELISA, and the gene expression was analyzed by qRT-PCR.

Results: Viperin is expressed in endothelial cells, macrophages and smooth muscle cells in human carotid plaques. OLINK data analysis revealed reduction in release of CCL3, CXCL9, CXCL10 and CXCL11 by AoSMCs that were targeted with anti-viperin siRNA prior to stimulation with IFN-γ. Using ELISA and qRT-PCR, we could confirm the reduction of IFN-γ-induced CXCL10 and CXCL11 in viperin knockdown AoSMCs.

Conclusions: Viperin is expressed in human carotid plaques and seem to be involved in regulating the expression of CXCL10 and CXCL11 in AoSMCs. Thus, it may play a role in recruitment of T lymphocytes to the subendothelial space in atherosclerotic plaques.