The innate immune response plays a central role in the immune system as the first line of defence against foreign and infectious agents. Innate immunity, although non-specific, can orchestrate the humoral immune system through antigen presentation to the CD+4 T-cells (Xiao, 2017). It facilitates the first line of defence through inflammation; an ontogenetically old defence mechanism regulated by cytokines, products of the plasma enzyme systems, lipid mediators released from different cells, and vasoactive mediators released from mast cells, basophils, platelets and macrophages (Ross et al., 2002). Macrophages are antigen presenter cells (APC) that produce cytokines as inflammatory mediators, which recruit other immune cells to the inflamed site and link the innate and adaptive immune response (McElroy and Nichol, 2012).
Despite the essential role played by inflammation, under-controlled inflammation leads to tissue damage and various adverse conditions that include neurodegeneration disorders, diabetes, cancer and endothelial leakage (Koriyama et al., 2013; Jope et al., 2007; Wang et al., 2004). Viruses such as RVFV target macrophages to invade the innate immune system and use them as a vehicle to target tissues such as brain and liver (McElroy and Nichol, 2012). This virus is known to inhibit the production of the IFNs as targeted by the NSs. This is thought to be the mechanism in which RVFV circumvent the immune system in favour of viral replication (Nfon et al., 2012). In addition to inhibited IFNs, NSs is shown to induce direct degradation and inhibition of the PKR involved in the translational arrest of both cellular and viral mRNA (Habjan et al., 2009).
Weakened inflammatory responses by RVFV infection has been suggested to contribute to RVFV pathogenesis and fatality (Nfon et al., 2012). Contrary to this hypothesis, a recent body of evidence (Caroline et al., 2016; van Vuren et al., 2015; Gray et al., 2012) suggests that deregulation and prolonged inflammation correlate with viral pathogenesis and fatality. The combination of this contradicting inflammatory evidence and the IFNs inhibitory role of the NSs led to the hypothesis that unbalanced and deregulated inflammation could be central to the RVFV pathogenesis and lethality. Our work examined lithium as a potential drug to restore regulatory patterns of inflammation and innate immune system. In this study, lithium with and without the viral stimulant has shown to stimulate the production of the primary pro-inflammatory cytokine, TNF-α, in Raw 264.7 cells as early as 3 hrs pi.
Analogous with these findings, Kleinerman et al., 1989 observed elevated TNF-α production in LPS-stimulated macrophages treated with lithium as early as 10 min post-stimulation, with the plateau reached within 12 hrs post-stimulation. Other studies hypothesised that lithium induced the production of TNF-α in macrophages subsequently this stimulate the production of the granulocyte-macrophage CSF (GM-CSF) from the endothelial cells. These observation can be associated with observed lithium-induced leukocytosis and granulocytosis as a result of GM-CSF (Merendino et al., 1994; Kleinerman et al., 1989). The secondary pro-inflammatory cytokine, IL-6, and a chemokine, RANTES, were shown to be produced during the late hours of infection 12 and 24 hrs with 1.25 mM LiCl being the most effective (Fig 1 B & D). These findings coincide with observations by Maes et al., 1999 which showed that lithium did not induce significant production of the IL-6 in both stimulated and unstimulated cells.
Recent work on lithium-based inflammation studies reported inhibitory properties of lithium at 10 mM on RANTES production 24 hrs post stimulation with lipopolysaccharide (LPS) in a GSK-independent manner. This could suggest that the ability of lithium to modulate IL-6 and RANTES production is dose-dependent and that this cytokines production is NF-κB dependent, a pathway inhibited by lithium (Makola et al., 2020). In vitro studies by van Vuren showed a 10 times elevated production of IL-6 in fatal cases as compared to non-fatal patients. This observation suggests that elevated production of this cytokine could be favouring virus survival as opposed to host defence (van Vuren et al., 2015). Interestingly, lithium was shown to delay the elevated production of IL-6 pro-inflammatory cytokine in RVFV-infected cells. Another study showed that lithium enhances the production of another secondary pro-inflammatory cytokine IL-8 in both LPS and phytohemagglutinin (PHA) stimulated and unstimulated cells (Maes at al., 1999).
This current work demonstrated significant up-regulation of IFN-γ by lithium on RVFV stimulated cells as from 3 hrs post infection (Fig 1, A), however, lithium alone did not show any modulatory effects on this cytokine production. This work shows that lithium stimulates the production of some pro-inflammation cytokines which is the most important inflammatory phenomenon since Nfon et al., 2012 linked the weakened inflammation with pathogenesis and lethality. A review by Nassar and Azab, supports the findings from this current study as they are in agreement with previous reports. However, a general view from a review by Nassar and Azab showed inhibitory role of lithium on various cytokines production rather than stimulation (Nassar and Azab, 2014). Nfon et al., 2012 have linked elevated levels of IFN-γ to the survival of infected goats in animal experimental models. The IFN-γ is suggested to inhibit viral replication and stimulate the cytotoxic activity of the NK cells since lowered viremia has been observed in surviving infected goats (Nfon et al., 2012).
Elevated IFN-γ levels could be another mechanism used by lithium to lower viral replication. In addition to pro-inflammatory cytokines, lithium stimulated the production of anti-inflammatory cytokine, IL-10 (Fig 1 C), in both viral stimulate and virus free lithium treated cells. Similar findings have been reported in other studies (Maes at al 1999; Rapaport and Manji, 2001). These studies showed that lithium stimulates the expression of IL-10 and IL-1R anti-inflammatory molecules. This is suggested to be a regulatory mechanism as a result of the overwhelming production of inflammatory mediators known to have deleterious outcomes. Lithium has been shown to stimulate both the pro and anti-inflammatory cytokines in the current and previous studies (Nassar and Azab, 2014; Maes at al., 1999).
It is hypothesised that lithium could be restoring the balance in the production of inflammatory mediators, as pro-inflammatory molecules are later balanced by regulatory cytokines to limit over production of pro-inflammatory molecules. Besides the inflammatory properties of lithium observed in this study, lithium has been used for decades as a preferred treatment option for bipolar disorders despite the sparse and limited understanding of its mechanism of action (Nassar and Azab, 2014). Nontheless, under-regulated inflammation has been linked to pathological processes behind manic depression and bipolar disorders. Hence, studies suggest that lithium could be restoring inflammatory deregulation as the mechanism underlying its anti-depressant property (Nassar and Azab, 2014; Maes at al., 1999).
The RVFV-infected lithium treated cells have shown to lower production of the reactive oxygen and nitrogen species. The lowered production of these reactive molecules has been depicted in figure 2 A and 3 A, a qualitative assay. Quantitative findings (Fig 2 B and 3 B) show the same trend as in the qualitative assay figure 2 A and 3 A. As represented in Figure 1 B and 2B, there is adequate production of these reactive species at 24 hrs pi. Previous work has shown lithium at 10 mM to reduce ROS production while 5 mM was effective in reducing NO production in LPS-stimulated Raw 264.7 cells (Makola et al., 2020). More interestingly, in the current study, lithium downregulated the expression of NOS-2 enzyme (Fig 3 C), which correlate with low NO production. In addition to the inhibited NOS-2, lithium stimulates HO-1 expression, an antioxidant enzyme (Fig 2 C). This work aligns inflammation regulatory properties of lithium with activation of the NF-κB transcription factor.
Lithium-treated cells showed the presence of the NF-κB in both the cytoplasm and the nucleus, suggesting the reversal/ inhibition of the transcription factor from translocating to the nucleus (Fig 4 A). Molecular translocation of NF-κB into the nucleus is observed to be lowered by lithium treated cells in a concentration dependent manner (Fig 4 B & C). Previous study (Narayanan et al., 2014) has shown that RVFV stimulate NF-κB nuclear translocation, culminating in production of inflammatory mediators and resulting in oxidative stress (Narayanan et al., 2014). Oxidative stress is a condition emanating from excessive production of oxidants and free radicals, leading to imbalance between oxidants and antioxidants. Studies (Christen, 2000; Reuter et al., 2010; Narayanan et al., 2014) have shown that oxidative stress conditions elicit biomolecules deformation that lead to altered cell function and then cell demise.
Narayanan et al., 2014 hypothesised that the RVFV prevalent liver disease emanates from oxidative stress that leads to hepatic cell demise (Narayanan et al., 2014). Inflammatory deregulation and oxidative stress have been linked with several pathogenic outcomes. This study suggests that lithium could ameliorate detrimental outcomes emanating from this viral infection. Figure 4 D, show that lithium concentrations upregulate the inhibitory molecules, IκB-α. IkB-α inhibit the translocation of the NF-κB by masking its nuclear translocation domain (Garcia et al., 2009). Our previous work showed that high lithium concentration (10 mM) expressed the NF-κB inhibitors IκB-α, TRAF3, Tollip and NF-κB1/p50 to be lithium inhibition biomarkers (Makola et al., 2020). What remains profound about lithium is that in as much as it was shown to promote expression of some pro-inflammatory cytokines, it stimulates anti-inflammatory cytokines in an attempt to avoid oxidative stress and nonspecific damage to host cell biomolecules.
Previous studies showed that NSs selectively tempers with the type I IFN signalling while sparing the other signalling pathways that produces inflammation mediators such as ROS, NOS and Pro/ anti-inflammation cytokines/chemokines. On a signalling level this could mean that NSs inhibit nuclear translocation of IRF3 and 7 transcription factors since they are central to type I IFNs production, or perhaps targeting the transcription of ISGs, leading to silencing of antiviral molecules as depicted in figure 5 (Ghaemi-Bafghi and Haghparast, 2013). Since NSs selectively inhibit IFNs which are linked to IRFs transcription factors, it then implies that other inflammatory mediators expressed by other transcription factors such as AP-1 and NF-κB will continually be produced leading to elevated inflammatory mediators and then oxidative stress. Thus, this work links the regulatory mechanism of lithium with inhibition of the NF-κB signalling pathway.
The NF-κB signalling pathway is suggested to be stimulated by the glycoproteins detected by TLR-4 or ssRNA detected by TLR-7 or dsRNA detected by the RIG-I. All these PRRs are linked to the NF-κB signalling pathway in as much as others stimulate IRF signalling as well (Fig 5). The in vitro and ex vivo studies have shown cytokine and chemokines production excluding type I IFN during RVFV infection (Nfon et al., 2012; van Vuren et al., 2015). Therefore, the activated NF-κB pathway continue producing these inflammatory mediators that are suggested to participate in the RVFV pathogenesis. Therefore, lithium restores the production of excessive inflammatory mediators as it has been observed to limit NF-κB translocation through upregulation of the IκB molecule.