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, has ability to orchestrate humoral immune system through antigen presentation to the CD+4 T-cells (Xiao, 2017). It facilitates the first line of defence systems 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 recruitment other immune cells to the inflamed site and link the innate and adaptive immune response (McElroy and Nichol, 2012).
Inflammation pays a pivotal role in eliminating infections. However, deregulated inflammation can lead 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 production of the IFNs as targeted by the NSs virulence factor. This is thought to be the mechanism in which RVFV use to 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 translational arrest of both cellular and viral mRNA (Habjan et al., 2009).
Weakened inflammatory responses from RVFV infection has been suggested to contribute to RVFV pathogenesis and fatality (Nfon et al., 2012). Contrary to these suggestions, 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. This work examined lithium as a potential drug to restore regulatory patterns of inflammation and innate immune system. In this study lithium has shown to stimulate production of the primary pro-inflammatory cytokine, TNF-α, in Raw 264.7 cells as early as 3 hrs post infected with RVFV. All three concentration of lithium were shown to significantly stimulate production of this primary pro-inflammatory cytokine.
Kleinerman et al, have observed TNF-α increment in LPS-stimulated macrophages treated with lithium as early as 10 min post stimulation with the production plateau reached within 12 hrs post stimulation. Kleinerman and colleagues hypothesised that lithium induced production of TNF-α by macrophages and the produced TNF-α subsequently stimulate production of the granulocyte-macrophage CSF (GM-CSF) from the endothelial cells. These observation can be associated with the 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 which showed that lithium did not induce significant production of the IL-6 in both stimulated and unstimulated cells (Maes at al., 1999).
Moreover, Makola et al showed inhibitory properties of 10 mM lithium on RANTES production 24 hrs post stimulation with lipopolysaccharide (LPS) in a GSK-independent manner (Makola et al., 2020). This could suggest that lithium may not possess sufficient ability to stimulate IL-6 and RANTES production. 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 reverse the elevated production of this pro-inflammatory cytokine in RVFV-infected cells. In another study, Maes et al has shown that lithium enhance 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 from 3 hrs post infection in a concentration-dependent manner (Fig 1, A). This work shows that lithium stimulates production of some pro-inflammation cytokines which is the most important phenomenon since Nfon et al link weakened inflammation with pathogenesis and lethality. A review by Nassar and Azab, support findings from this current study as they are in agreement with previous reports. However, most of the reports show the inhibitory role of lithium on this cytokine rather than stimulation (Nassar and Azab, 2014). Nfon et al have linked elevated levels of IFN-γ to survival of infected goats in animal experimental models. IFN-γ is suggested to inhibit viral replication and stimulate cytotoxic activity of the NK cells since lowered viremia has been observed in surviving infected goats (Nfon et al., 2012). In the current in vitro model system, lithium has been suggested to inhibit viral replication through induction of early apoptosis in virus-infected cells, leading to abortion of replicating viral progeny.
Elevated IFN-γ levels could be another mechanism used by lithium to lower viral replication. In addition to pro-inflammatory cytokines, lithium stimulated production of the anti-inflammatory cytokine, IL-10, and the cytokine production reached its peak as early as 12 hpi (fig 1 C). Similar findings have been reported in other studies (Maes at al 1999; Rapaport and Manji, 2001). These studies showed that lithium stimulate expression of IL-10 and IL-1R anti-inflammatory molecules. This is suggested to be a regulatory mechanism as a result of overwhelming production of inflammatory mediators known to have deleterious outcomes. Lithium is 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 suggested that lithium could be restoring the balance in production of inflammatory mediators, as pro-inflammatory molecules are later balanced by regulatory cytokines to limit over production of pro-inflammatory molecules. Lithium remains an effective and preferred treatment option for bipolar disorders despite the sparse and limited understanding of its mechanism of action (Nassar and Azab, 2014). 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). An in vitro study by Gray et al observed strong and prolonged expression of IL-6, GCSF and MCP-1 three days pi, this has led to suggestion that these molecules could be involved in endothelial leakage that lead to haemorrhagic fever (Gray et al., 2012).
The RVFV-infected lithium treated cells have shown to lower production of the reactive oxygen and nitrogen species. The lowered production of this reactive molecules has been depicted in figure 2 A and 3 A in a qualitative assay. Quantitative findings (fig 2 B and 3 B) show the same trend as in the pictures. As represented in Figure 1 B and 2B, there is a significant difference in production of these reactive species 24 hrs post infection. Previous work has shown lithium at 10 mM to reduce ROS while 5 mM being effective in reduction of NO production in LPS-stimulated Raw 264.7 cells (Makola et al., 2020). More interestingly, in the current study, lithium down-regulated expression of iNOS enzyme (fig 3 C), which correlate with lowered NO production. In addition to the inhibited iNOS, lithium stimulate 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 show 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 treatments in a concentration dependent manner (fig 4 B & C). Previous studies (Narayanan et al., 2014) have shown that RVFV stimulate NF-κB nuclear translocation, culminating in production of inflammatory mediators and resulting in oxidative stress. 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, hypothesise that RVFV prevalent liver disease emanate from oxidative stress that lead to hepatic cell demise (Narayanan et al., 2014). Thus, inflammatory deregulation and oxidative stress has been linked with a number of pathogenic outcomes. This study suggest 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 at high lithium concentration (10 mM) showed expression of 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 show that NSs selectively tempers with the type I IFN signalling while sparing the other 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 as the transcription factors involved in production of type I IFNs or perhaps targeting the transcription of ISGs, leading to silencing of antiviral molecules (Ghaemi-Bafghi and Haghparast, 2013) (fig 5). 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 elevated inflammatory mediators and oxidative stress. Thus, this work link regulatory mechanism of lithium with inhibition of the NF-κB signalling pathway as the inflammatory transcription factor that is not targeted by NSs.
The NF-κB signalling pathway is suggested to be stimulated by the glycol proteins 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 hypothesis that lithium restore balance in the inflammatory system emanates from its regulatory mechanism on the NF-κB pathway as NSs inhibit translocation of IRFs and not other transcription factors. 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). The activated NF-κB pathway continue producing these inflammatory mediators that are suggested to participate in the RVFV pathogenesis. Therefore, lithium restores production of excessive inflammatory mediators as it has been observed to limit NF-κB translocation through upregulation of the IκB molecule.