Cytoprotective effects of lithium on endothelial integrity and immune profiles induced by rift valley fever virus on huvec and raw 264.7 cells

Abstract

Introduction: Rift Valley fever virus (RVFV) is an arthropod-born RNA zoonotic virus causing Rift Valley fever (RVF) disease. RVFV is prevalent across sub-Saharan Africa and the Arabian Peninsula with no existing effective and approved antiviral remedies for humans or animals. RVFV has developed mechanisms to hide from immune recognition and induce anti-apoptosis processes to keep the infected host cells viable in an attempt to advance their viral progeny. RVFV is a single-stranded enveloped RNA genome virus composed of 3 segments; the L, M and S segments. The S segment is known to encode a non-structural protein (NSs) identified to be the main virulence factor promoting viral replication through immune suppression. RVFV elicits a set of diverse symptoms ranging from a febrile illness to more severe symptoms that usually culminate in life-threatening haemorrhagic fever with high fatality rates. Thus, this study was designed to investigate the efficacy of lithium as a potential drug for reduction of RVFV load and amelioration of imbalanced and dysregulated inflammatory responses observed in Huvec and Raw 264.7 macrophages infected with this virus. Methods and results: The MTT and Cyquant viability assays were used to demonstrate that lithium exerts no cytotoxic effects on non-infected Raw 264.7 macrophage cells but rather promotes cell growth and proliferation. Conversely, lithium was shown to significantly induce cell death in RVFV-infected Raw 264.7 macrophages. The Annexin-V/PI apoptosis assay was employed to demonstrate that RVFV induces apoptosis as a mode of cell death on Raw 264.7 cells. RVFV-induced apoptosis was accompanied by antagonistic Bax/Bcl-2 protein expression ratios. RVFV-infected cells treated with lithium resulted in higher levels of apoptosis signals compared to untreated RVFV-infected cells. Analysis of apoptosis stages using the real-time cell analyser (RTCA) also revealed that lithium induced early forms of apoptosis in RVFV-infected cells. Interestingly, induction of early apoptosis in these cells corresponds with lower viral load, probably as a result of early inhibition of viral progeny replication, as determined using viral titration assay. Immune response profiles elicited in Raw 264.7 macrophages infected with RVFV and treated with lithium were monitored. An ELISA assay was used to determine the effect of lithium on cytokines and chemokine production in this cell model. The results obtained showed that lithium significantly stimulated production of IFN-γ as RVFV-infected lithium-treated cells produced high levels of IFN-γ compared to lithium-free RVFV-infected control cells. Furthermore, in the same setting, the secondary pro-inflammatory cytokine, IL-6, and chemokine, RANTES, were stimulated by lithium 12 hrs post-infection (pi). Lithium was shown to significantly stimulate TNF-α production as early as 3 hrs pi. In addition to TNF-α expression, the expression of the regulatory cytokine, IL-10, was significantly stimulated by lithium with the highest expression peak at 12 hrs pi. As determined using the H2DCF-DA and DAF-2 DA florigenic assays, reduced production of the ROS and RNS was observed in RVFV-infected lithium-treated cells as opposed to untreated RVFV-infected controls. This was further supported by the Western blot assay results that showed low expression of the iNOS while upregulating expression of heme oxygenase and IκB in RVFV-infected lithium-treated cells. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/ml LPS controls. This study hypothesises persistent and deregulated inflammation as the central phenomenon responsible for endothelial damage and haemorrhagic fever in RVFV pathogenesis. Supernatants were collected from RVFV-infected macrophage cells treated with lithium and their effects on the integrity of endothelial cells were evaluated. The xcelligence real-time cell analyser system (RTCA) and transwell assay that measure endothelial monolayer integrity were used to demonstrate that lithium protects endothelial cells from RVFV-induced cellular damage. Moreover, lithium was shown to upregulate expression of cytoplasmic molecules such as α and β-catenins involved in attaching the cadherin molecules to the actin cytoskeleton on the endothelial cell. Expression of α-catenins, talins, zyxins and vinculins that attach integrins to the extracellular matrix and to other cells were observed to be upregulated by supernatants from RVFV-infected Raw 264.7 macrophage cells treated with lithium. Endothelial cell monolayer exposed to supernatants from RVFV-infected lithium-treated Raw 264.7 cells displayed upregulated expression of transmembrane molecules such as E-cadherins and N-cadherins. However, expression of VE-cadherins was observed to be lower compared to those treated with supernatants from lithium-free RVFV-infected Raw 264.7 control cells. Conclusion: These findings propose that lithium limits viral replication and viral load in macrophages by inducing early apoptosis in RVFV-infected cells. Since lithium was shown to promote Raw 264.7 macrophage proliferation, it is thus suggested that the use of lithium as an RVFV antiviral drug is less likely to elicit leukocytopenia. Lithium seems to regulate excessive inflammation in RVFV-infected Raw 264.7 macrophages by modulating the NF-kB signalling pathway. The endothelial integrity observed in the permeability assays has been supported by the expression of the molecules involved in keeping the cell to cell adhesion intact. This study links endothelial integrity patterns exerted by lithium with lowered production of inflammatory mediators such as ROS and RNS as these molecules are involved in destabilisation of cell junctions. Results from this study point towards the use of lithium as a potential treatment for RVFV infections by limiting viral replication, restricting viral spread and restoring the inflammation-regulating machinery. Key words. Lithium, Rift Valley fever virus, NF-kB, endothelial integrity, inflammation and apoptosis