Lithium inhibits NF-κB nuclear translocation and modulate inflammation profiles in Rift valley fever virus-infected Raw 264.7 macrophages

The RVFV AR 20368 strain was isolated in 1974 during the RVF outbreak in South Africa. The virus was propagated on Vero C1008 cells at an MOI of 0.2, followed by harvesting the monolayer after extensive cytopathic effect (CPE) was observed. The supernatant was stored at − 70°C after centrifugation at 3000 xg for 30 min [29]. The Raw 264.7 macrophage cells were obtained from Prof Lyndy McGraw (University of Pretoria, Photochemistry division, Onderstepoort campus). The cells were maintained in cell culture flasks at 37 °C, in a humidified 95% air and 5% CO₂ atmosphere. Raw 264.7 cells were propagated in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 1× penicillin-streptomycin. Vero C1008 cells were purchased from ATCC and propagated in Minimum Essential Medium (MEM), supplemented with 10% FBS, 2 mM L-glutamine and 1× penicillin-streptomycin (Lonza). Trypan blue dye and haemocytometer were used to determine cell density [14].

Lithium Chloride (LiCl) was purchased from Fluka (Chemika, Switzerland) and 500 mM stock was prepared and stored at 4 °C. Sodium Chloride was purchased from Sigma-Aldrich (USA) and the stock was prepared at 500 mM NaCl stored at 4 °C. The experiments were executed by seeding the Raw 264.7 cells at various densities depending on the experimental setting and then treated with these lithium concentrations (LiCl 2.5, 1.25 and 0.625 mM) as well as NaCl (2.5 mM).

In an attempt to measure the production of the cytokines and chemokines, Raw 264.7 cell were seeded at 2.4 × 10⁶ cells for 3 h in T25 flasks. Thereafter  cells were inoculated with 10^4.8 viral titre/mL for an hour. After one hour inoculation the supernatant was removed followed by treatment of cells with lithium concentrations (as outlined in cell treatment section) and LPS (5 mg/mL) for 24 h while the supernatant was collected in 3, 6, 12 and 24 h time intervals. The collected supernatant was frozen at − 20°C for later use. Enzyme-linked immunosorbent assay (ELISA) was executed according to the manufacturer’s protocol (Preprotech, USA). The capture antibody (for IL-10, IL-6, TNF-α, IFN-γ and RANTES) was diluted with PBS to 0.5 µg/mL and 100 µL was added to each well in a 96 well plate and incubated at room temperature (RT) overnight. The unbound excess capture antibody was aspirated and plates were washed with 300 µL wash buffer (0.05% tween-20 in 1× PBS) per well using ELx 405 auto plate washer (Bio-TEK instruments-Inc).

The standard was serially diluted 2 fold and 100µL was added in triplicates followed by addition of samples for 2 h. The added samples were aspirated and washed 4 times with wash buffer. The detection antibody was diluted in diluent to 0.5µg/mL and 100 µL was added in each well for 2 h at RT. This was then followed by aspiration of excess detection antibody and plates were washed 4 times with wash buffer. The 5.5µL avian peroxidase was then diluted 1: 2000 in 11 mL diluent, thereafter 100 µL of this diluted avian peroxidase was added to each well and incubated for 30 min at RT. This solution was aspirated and wells were washed 4 x with wash buffer. After blotting of wells, 100 µL of ABTS substrates solution was added to each well and the plates were incubated for colour development. Thereafter the colour was measured by reading the OD at 405 nm with ELx 802 universal microplate reader (Bio-TEK instruments-Inc)

The 2′,7′-Dichlorofluorescin diacetate (H₂DCF-DA) is a cell-permeable reactive oxygen species (ROS) non-fluorescent probe. This molecule is deacetylated by cellular esterases and react with ROS to form a highly fluorescent 2′,7′-dichlorofluorescein. The Raw 264.7 cells were seeded at 4 × 10⁵ cells/well in a 6 well plate for 3 h and inoculation with RVFV at 1 × 10^4.8 viral titre/mL for 1 h. This was followed by discarding excess virus and cell treatment for 24 h with lithium concentrations (as outlined in cell treatment) and LPS (5 mg/mL) followed. After 24 h of inoculation and lithium treatment, cells were stained with permeant H₂DCF-DA at RT for 30 min in the dark and fixed with 3.7% paraformaldehyde for an hour. The fluorescent images were captured at 480 and 535 nm (Ex/Em) with EVOS FL Colour imaging system (Life technologies, USA). Moreover, the ROS production was measure quantitatively by seeding Raw 264.7 cells at 5 × 10⁵ cells/well in a 96 well plate for 3 h. This was followed by inoculation of cells with RVFV at 1 × 10^3.8 viral titre/100 µL for 1 h. After 1 h of cell inoculation excess virus was removed and treatment of cells with lithium was executed as  in cell treatment section above for 12 and 24 h. After the 12 and 24 h incubation time H₂DCF-DA was added for 30 min and then fluorescence intensity was measured at ex/em 480/535 nm using a Fluoroskan Ascent FL (Thermo Fisher Scientific, USA).

The 5,6-Diaminofluorescein diacetate (DAF-2 Da) is a cell permeant NO indicator that is deacetylated by intracellular esterase to DAF-2 that react with NO to yield a highly fluorescent triazolofluorescein (DAF-2T). The Raw 264.7 cells were seeded at 4 × 10⁵ cells/well in a 6 well plate for 3 h and inoculation with RVFV at 1 × 10^4.8 viral titer/mL for 1 h. The excess viral inoculum was replaced with lithium treatment (as outlined in cell treatment) and LPS (5 mg/mL) for 24 h. After 24 h of inoculation, cells were stained with DAF-2 DA at RT for 30 min in the dark then cell were fixed with 3.7% paraformaldehyde for an hour. The pictures were captured with EVOS FL Colour imaging system (Life technologies, USA) at 480 and 535 nm (Ex/Em). For quantitative measure of NO, Raw 264.7 cells were seeded at 5 × 10⁵ cells/well in a 96 well plate for 3 h and then inoculated with RVFV at 1 × 10^3.8 viral titre/100µL for 1 h. And then excess virus was removed and treatment of cells with lithium was executed as above for 12 and 24 h. After the incubation time, DAF-2 Da was added for 30 min and then fluorescence intensity was measured at ex/em 480/535 nm using a Fluoroskan Ascent FL (Thermo Fisher Scientific, USA).

Raw 264.7 macrophage cells were cultured in 6 well plates on the slides at 4 × 10⁵ cells/well for 3 h. This was followed by RVFV inoculation at 1 × 10^4.8 titre/mL for 1 h, and the excess virus was discarded. Cell treatment with lithium was executed as outlined in cell treatment and some wells were stimulated with LPS (5 mg/mL) for 24 h. The media was then aspirated after 24 h incubation and cells were fixated with 4% paraformaldehyde for 1 h. Thereafter, cells were permeabilised with 0.1% Triton X-100, 1%BSA for 60 min and the nonspecific binding sites were blocked by adding 1% BSA for 1 h, followed by 2× wash with wash buffer. Cells were incubated for 60 min with rabbit anti-p65 antibody (1:500) (Santa Cruiz, USA) followed by 3× wash with wash buffer. The cells were incubated with FITC-labelled goat anti-rabbit secondary anti-body for 60 min. After 5 min, the nuclear staining was done with DAPI, and then cells were mounted on slides using 50% glycerol and analysed using the fluorescent inverted Nikon Ti-E microscope at 20× magnification.

In order to examine NF-κB related protein expression, the Raw 264.7 cells were seeded in T25 flasks for 3 h at a density of 1 × 10⁶ cell/mL and inoculation at 1 × 10^4.8 titre/mL for 1 h, and the excess virus was discarded. Cell treatment with lithium was executed as outlined in cell treatment section and stimulated with LPS (5 mg/mL) for 24 h. After 24 h treatment cells were washed once with 1x PBS, thereafter, cell lysis was accomplished with 500µL lysis buffer (10 mM Tris-HCl, pH 6.8, 1% SDS, 100 mM sodium chloride, 1 mM EDTA, 1% NP 40, protease inhibitor), the cells were vortexed for 10 seconds and incubated on ice for 30 mins. The supernatant was collected by centrifugation at 15,000 ×g for 20 min at 4 °C and then the protein concentration was determined using BCA protein assay at 562 nm. For SDS PAGE 50µg proteins ware mixed with the sample buffer (1 mM Tris buffer pH 6.8, 20% SDS, 20% glycerol, 0.05% β-mercaptoethanol, 0.002% bromophenol blue) separated on 12% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane using a semi-dry blotting system (Bio-Rad).

The membranes were blocked with Tris buffered saline (TBS) [150 mM NaCl, 50 mM Tris, 0.1% Tween, pH 7.5] containing 3% fat-free dried milk. The membranes were washed with wash buffer [0.05% TBS- Tween] and then incubated each time with 1:500 dilutions of anti- NF-κB-p65, IκB, HO-1, NOS-2 and β-actin primary antibodies for 1 h at RT. After incubation, the membranes were washed 3 x with wash buffer and incubated with corresponding peroxidase-conjugated secondary antibodies at 1:10 000 dilutions for 1 h at RT. The membranes were washed with wash buffer and the immune-reactive proteins were detected using the super-signal west pico chemiluminescent substrate (Thermo Scientific, Rockford, USA). The protein bands were visualised and photographed using the ChemiDoc XRS+ (Bio-RAD, USA).

All assays were performed 3 times in duplicates or triplicates and the error bars represent the degree of variance. Graph-Pad Prism 6 software was used to plot the graphs and statistical analysis was performed using GraphPad InStat 3 software. Duncan's multiple comparison t-test was used to determine significant differences between the means of treated and untreated groups. Differences were considered significant at *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001.

Most viruses have developed mechanisms to evade the immune surveillance system which favours viral replication and increased viral progeny. RVFV is not an exception to this type of virus-induced immune invasion mechanism. RVFV is known to inhibit the innate immune system, particularly the type I IFN cytokine production as its mechanism of invasion [19]. Other studies [2, 24] outlined prolonged immune response as the primary detrimental factor in patients who suffer from this viral infection. This study examined the effects of lithium on cytokine and chemokines production after RVFV inoculation. This study has demonstrated that lithium stimulates INF-γ production in viral stimulated Raw 264.7 macrophage model system.

Lithium concentrations have shown to stimulate INF-γ production in RVFV-infected cells compared to the RVFV-infected control cells not exposed to lithium (Fig. 1a). The increase in INF-γ production also increased proportionally with an increase in incubation time, however, uninfected lithium treated cells did not stimulate INF-γ production. INF-γ production in RVFV-infected cells treated with lithium was even higher than LPS stimulated cells albeit reaching comparable levels at 24 h post inoculation (pi). Lithium at 1.25 mM induce the highest amount of INF-γ production compared to other concentrations in all the time points. Another prominent inflammatory cytokine, IL-6, was shown to be produced after 12 h pi (Fig. 1b). After 12 h (pi), 0.625 mM lithium produced IL-6 almost 3 fold compared to RVFV-infected cells compared to untreated RVFV-infected cells and 1.5 fold compared to LPS stimulated cells. The chemokine (RANTES/CCL-5) showed similar increment profiles to those observed in IL-6. Lithium induced CCL-5 production 12 h pi, the low concentrations of lithium stimulated more of CCL-5 (Fig. 1d).

Furthermore, lithium concentrations were shown to increase the production of the TNF-α compared to that of RVFV-infected control cells as from 6 h pi (Fig. 1e). The increase in TNF-α production was also shown to be time-dependent. The level of TNF-α after treatment with lithium was shown to be above that of 5 mg/mL LPS positive control. Lithium was shown to stimulate IL-10 (anti-inflammatory cytokine) production in both RVFV-infected and uninfected cells (Fig. 1c), from 3 h pi, the production of IL-10 increased with incubation time. The IL-10 was production more by 1.25 mM LiCl compared to other concentrations and control RVFV showed improvement as from 6 h pi and reached production peak after 24 h pi (Fig. 1c).

Reactive oxygen species (ROS) are generated as metabolic by-products during mitochondrial respiratory chain and excessively produced during inflammation [22]. In this study, the fluorescent probes such as 2ʹ,7ʹ–dichlorofluorescein diacetate (H₂DCF-DA) and 4,5-diamino-fluorescein diacetate (DAF-2 DA) were used to determine the level of ROS and RNS production in virally infected cells. Figures 2a and 3a represent qualitative levels of ROS and RNS production in Raw 264.7 cells after treatment with lithium and infection with RVFV. Lithium concentrations are shown to downregulate the production of reactive molecules as the green fluorescence intensity is reduced in lithium treated cells. Quantitative measurement of fluorescence intensity (Figs. 2b, 3b) show that lithium lowered the production of ROS in a concentration-dependent manner. Highest inhibition of RNS production was observed in cells treated with lithium at 2.5 mM. These findings also showed that the production of ROS/RNS molecules only reached their highest levels at 24 hpi. Moreover, lithium concentrations are shown to upregulate antioxidant enzyme Heme oxygenase-1 (HO-1) (Fig. 2c). Lithium is shown to induce HO-1 expression in a concentration dependent manner.

The determination of RNS production using DAF-2 showed that lithium inhibited RNS production in RVFV-infected Raw 264.7 cells. Treatment of cells with lithium notably reduced DAF-2 fluorescence intensity. These observations were accompanied by elevation of nitric oxide synthase-2 (NOS-2) expression as depicted in Fig. 3c. The higher expression levels of the NOS-2 were elevated in control RVFV and LPS stimulated cells. Expression of NOS-2 was shown to be lowered by lithium in a concentration-dependent manner. NOS-2 is an enzyme that stimulates the production of the nitric oxide.

The molecular location of a transcription factor NF-κB is used as a molecular measure of inflammatory response. NF-κB binds to the kappa responsive element and induce expression of inflammatory mediators. Thus, the cellular location of the NF-κB molecule is an essential phenomenon in inflammatory studies. The molecular location of the NF-κB was examined using immunocytochemistry depicted in Fig. 4a. Figure 4a shows that NF-κB is located in both the nucleus and cytoplasm in lithium-treated cells, since green fluorescence is on both inside and outside of the nucleus. However, control untreated cells showed less of the green fluorescence in the nucleus and the image overlays showed an intense blue nuclear fluorescence, displaying that the two stains are not in the same location.

The positive control LPS (5 mg/mL) treatment shows the light blue nuclear fluorescence since all the green (NF-kB staining) and the blue (nucleus staining) are all in the same location in the nucleus. The control RVFV show similar patterns as control LPS, indicative of NF-kB nuclear translocation. On the other hand, Fig. 4b and c shows that lithium inhibits translocation of NF-κB with LiCl 2.5 mM showing the most inhibitory effects, although in this assay the inhibition margin is narrow. The IκB-α expression 12 hpi (Fig. 4d) showed to be elevated in lithium-treated cells in a dose-dependent manner. IκB-α is the inhibitory molecule that keeps NF-κB in the cytoplasm and is known to regulate the activity of this transcription factor [4].