Melatonin decreases brain apoptosis,oxidative stress,and CD200 expression and increased survival rate in mice infected by Venezuelan equine encephalitis virus

Abstract

Background

Pro-inflammatory and oxidative events during brain Venezuelan equine encephalitis virus infection could lead to apoptosis and induce anti-inflammatory responses (increased expression of CD200). The aim of this study was to determine the effect of melatonin on brain apoptosis, oxidative stress, and CD200 molecule in mice and neuroblastoma cultures infected by Venezuelan equine encephalitis virus.

Methods

Mice were infected with 10 median lethal doses (LD₅₀) of Venezuelan equine encephalitis virus, treated with melatonin (500 µg/kg bw; three days before infection and during all experimental time) and sacrificed on days 1, 3, and 5 postinfection. Brain samples were obtained at those periods of time. In addition, infected neuroblastoma cell cultures (multiplicity of infection [MOI]: 1) were treated with 0, 0.1, 0.5, and 1 mM of melatonin and analyzed at 2, 4, and 6 h. CD200 and apoptosis expressions were analyzed by immunohistochemistry and TUNEL assay, respectively. Nitrites and malondialdehyde were determined by appropriate biochemical methods.

Results

Increased brain expression of apoptosis, nitrite, and malondialdehyde productions and CD200 of infected mice were found. Melatonin diminished those expressions. Similarly, high apoptosis expression and nitrite and malondialdehyde productions on infected neuroblastoma cultures were diminished by melatonin. Melatonin increased the survival rate (25%) in Venezuelan equine encephalitis virus-infected animals compared with untreated infected mice (0%).

Conclusions

Neurological damage during brain Venezuelan equine encephalitis virus infection could be mediated by apoptosis and oxidative stress and CD200 molecule could be an important anti-inflammatory response. Melatonin could be beneficial reducing apoptosis and oxidative stress.

Introduction

Venezuelan equine encephalitis (VEE) is a viral disease transmitted by mosquitoes mainly to human and equines. The inflammation induced by the VEE virus (VEEV) is associated with a high mortality rate in mice.^1,2 Melatonin (MLT), an indole, has been used to ameliorate diverse events during experimental VEE. In this regard, increased survival, increased production of interleukin-1β (IL-1 β), and decreased production of tumor necrosis factor-alpha (TNF-α) and brain viral replication in VEEV-infected mice after MLT treatment have been reported.^3–7 In addition, decreased inducible nitric oxide synthase (iNOS) expression, NO production, and lipid peroxidation were also observed in VEEV-infected mice treated with MLT.^6,8–10 These data suggest a beneficial effect of MLT in experimental VEE. On the other hand, increased production of pro-inflammatory cytokines and oxidative stress in experimental VEE could be associated with apoptosis.^11–13 VEE courses with inflammatory events, in which, anti-inflammatory response can be expected in order to limit the inflammation. In this regard, the immunosuppressive molecule CD200 expressed in the brain^14,15 could have a role during VEE. The interaction of CD200 with its receptor (CD200R) decreases the inflammatory response. Therefore, increased expression of CD200 could represent an anti-inflammatory response against the inflammation induced by VEEV, regulating the possible damage of viral infection. Thus, the expressions of apoptosis and CD200 in the brain of VEEV-infected mice may represent both harmful and beneficial effects during this infection. The modulatory effects of MLT on those expressions remain to be studied in experimental VEE. Therefore, the aim of this study was to determine the expression of apoptosis, the oxidative stress (as apoptosis inducer), CD200 molecule, and the survival rate in animals infected with VEEV and evaluates the effect of MLT on those events.

Materials and methods

Animals

Adult male NMRI mice weighing 25–30 g (Central Animal Facility, Instituto de Investigaciones Científicas, IVIC, Venezuela) were used. Animals were housed at room temperature (24℃) with water and food (Ratarina, Proteinal, Valencia, Venezuela) ad libitum and a natural day–night light cycle.

Viral titration and infection

The Guajira VEEV strain is a strain isolated in the Venezuelan Guajira, specifically in the Paez municipality (Zulia state: between meridians 71 and 73 west longitude and parallels of latitude 9 and 12 north), during the epidemic in 1968. ¹⁶ This strain was identified as IC subtype of the VEEV complex subtypes: AB, C, and F (Centers for Disease Control and Prevention, Georgia, USA) whose range has been described for Venezuela and Colombia. This strain was selected because it has been characterized and identified on epizootic diseases and/or epizoodemics occurred in the country. ¹⁷ This strain severely affects humans and equines and causes encephalitis in most cases compared to other subtypes. ¹⁸ Moreover, this particular strain has been widely described in experimental models in mice, rats, hamsters, etc. as well as in vitro models. Compared to other strains, this virus had a higher rate of involvement than other circulating strains such as the Trinidad Donkey (Sub IA), including the last epidemic described in 1995 in Venezuela, where it was again identified and characterized the Guajira strain. ¹⁹ Virus was replicated using the plaque assays in African green monkey kidney (Vero) cell cultures. Virus titers were 6.8 × 10⁷ plaque forming units per ml. Titrations were performed in male young mice using 50 µl of log₁₀ dilutions of virus (10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶). End points were determined by the method of Reed and Muench ²⁰ and expressed as median lethal doses (LD₅₀). For the experiments, mice were inoculated intraperitoneal with 10 LD₅₀ of virus (50 µl of sterile 0.4% bovine albumin in borate saline, pH 7.4. 10 PFU/mouse) ³ under different protocols as described below.

Neuroblastoma cell cultures

Murine Na₂ neuroblastoma cells were obtained from the Instituto Nacional de Higiene “Rafael Rangel” (Caracas, Venezuela). The cells were grown in Eagle’s MEM supplemented with 10% (v/v) of fetal bovine serum, 1% penicillin–streptomycin in a CO₂ incubator maintained at 5% CO₂ and 37℃. The medium was changed every three days and the cells were passed at 80% of confluence. Cells were adjusted at 1 × 10⁶ cells/ml and used for the experiments.

Experimental protocol

Experiments designed to determine the effect of MLT (Research Biochemical International, MA, USA) according to time of treatment were performed. In this regard, VEEV-infected animals (10 LD₅₀) were divided into four groups (n = 10 per group): untreated VEEV animals, MLT pretreated VEEV animals (MLT three days before viral infection), MLT treated VEEV animals during infection (MLT immediately after viral infection), and MLT posttreated VEEV animals (MLT 24 h after infection); 500 µg/kg bw of MLT was used for these experiments. MLT was dissolved in phosphate buffer, pH 7.2 using a vortex and administrated by subcutaneous injection. These experiments were performed to analyze the effect of MLT on the infected mice survival rate according to different MLT administration protocol. Mortality rates were monitored and recorded daily for 10 days and blood samples were obtained from survived animals. The viral and MLT doses were obtained from previous reports. In this regard, the mortality rate of VEEV-infected mice was reduced from 100 to 16% by increasing the dose of MLT from 0 to 1000 µg/kg bw. In addition, high titers of VEEV IgM antibodies were found seven weeks after virus inoculation. MLT also significantly reduced VEEV levels in blood and brain of infected mice. ³ Animals received these treatments daily (5–6 pm). ²¹

Other experiments were designed to determine the effect of MLT on the brain expressions of apoptosis, CD200, and oxidative stress in controls and infected mice. In this regard, mice were divided in four groups: untreated control, untreated VEEV-infected animals (10LD₅₀), MLT-treated control, and MLT-treated infected animals. Animals were treated with 500 µg/kg bw, starting three days before and continuing for five days after virus inoculation. Controls and infected mice were sacrificed on days 1, 3, and 5 postinfection and brains were obtained (n = 9 per group). Under sodium pentobarbital anesthesia (100 mg/kg bw, intraperitoneal. Nembutal, Boehringer Ingelheim, Germany), brain was perfused by intracardiac via using a 0.9% saline solution. Cerebrum was obtained for microscopy and oxidative stress studies. Experimental procedures followed the ethical guidelines of the committee of bioethical and biosecurity of FONACIT (Caracas, Venezuela) and the committee of bioethical of Medical School (Universidad del Zulia, Maracaibo, Venezuela). All experiments were carried out in a biosafety level 2 facility.

For in vitro studies, neuroblastoma cells were seeded at 1 × 10⁶cells/ml in a 24-well plate. The cells were allowed to attach and grow for two days prior to the treatment. A group of cells was cocultured with VEEV at MOI of 1, while other groups were cultured without virus. These cells were treated with several final concentrations of MLT: 0, 0.1, 0.50, and 1 mM. ²² After 2, 4, and 6 h of culture, apoptosis, nitrites, and thiobarbituric acid reactive substances (TBARS) were determined by the TUNEL assay and respective biochemical assays. Analyses were performed in triplicate and the results represent three different experiments.

TUNEL assay

The method for nick end labeling of apoptotic cells was adapted from that of Gavrieli et al. ²³ with a commercial kit (Apop Tag Peroxidase In Situ; Chemicon International, USA). Adhered neuroblastoma cells and brain sections were treated according to the protocol of manufacture’s kit. Positive apoptotic nuclei were assessed by the peroxidase reaction using an inverted microscope and Zeiss/Nomarski differential interference equipment (Carl Zeiss Zeiss 872E, Wetzlar, Germany). The results were expressed as number of apoptotic cells per mm ² from 20 randomly selected fields of brain or as percentage of apoptotic cells in neuroblastoma cultures.

Determinations of nitrite and TBARS

Nitric oxide formation was detected by nitrite accumulation using the Griess reaction with sodium nitrite as standard. ²⁴ Briefly, proteins were precipitated with 2 N HCl and centrifuged at 2000g for 10 min. A volume of the sample was incubated with the same volume of 1% sulfanilamide, 0.1% N-1-naphthylethylenediamide dihydrochloride in 25% H₃PO₄ at room temperature for 5 min. Optical density was measured at 540 nm and the results expressed as µmol/mg of protein in brain samples and µM in supernatant cultures. TBARS content was assessed by the thiobarbituric acid assay. ²⁵ A volume of sample was added to a solution containing 8.1% SDS, 20% acetic acid, and 0.8% thiobarbituric acid, pH 3.5. The complete mixture was heated to 95℃ for 60 min; thereafter, the chromogenic substrate was extracted into the organic phase with butanol/pyridine (15:1). Absorbance of the organic layer was measured at 532 nm. As external standard the malondialdehyde (MDA) bis-dimethyl acetal was used and the results were expressed as µmol/mg of protein in brain samples and nM in culture supernatants.

Immunohistochemistry of CD200

Tissues from from controls and infected mice were fixed in 10% buffered formalin, embedded in paraffin and sectioned (4 µm). For immunohistochemistry, the sections were deparaffinized and blocked by treatment with 3% hydrogen peroxide solution (15 min). The sections were then treated overnight with a rat monoclonal antibody anti-mouse CD200 (Serotec Inc., NC, USA). Thereafter, sections were incubated with a horseradish peroxidase-labeled rabbit anti-rat IgG (Serotec Inc., NC, USA). The sections were then exposed to the substrate diaminobenzidine-tetrahydrochloride and counterstained with methyl green. The results were expressed as number of CD200 positive cells per mm² from 20 randomly selected fields of brain.

Determination of anti-VEEV antibodies

Survived infected mice were tested for the levels of serum antibodies anti-VEEV. The presence of specific IgM against the VEEV was determined by immunoenzymatic assay (SIGMA Chemical Co, MO, USA).

Statistical analysis

Data were expressed as mean ± standard deviation and analyzed by Chi squared, by unpaired t test or by nonparametric ANOVA (Kruskal–Wallis test) followed by Dunn’s multiple comparisons test. Log-rank test was used to compare survival curves. Significance was assumed to be at two tailed p < 0.05.

Results

Analysis of mortality on VEEV-infected animals

MLT was capable of decreasing the mortality rate of mice infected by VEEV (10 LD₅₀). Analysis of survival curves showed 0% of survival rate in untreated infected mice compared to 100% of survival rate in MLT-treated infected animals at day 6. Thereafter, MLT pretreatment (500 µg/kg bw, starting three days before) and MLT treatment during infection (500 µg/kgbw, at the moment of viral infection) had 25% of survival rate at day 10. However, infected animals treated 24 h after infection with MLT had 0% of survival rate at day 7. Log-rank test analysis showed significant differences when untreated animal curves were compared with treated animal curves (VEE versus VEE + MLT previous: p = 0.01; VEE versus VEE + MLT immediately: p = 0.01) (Figure 1). Very high titers of anti-VEEV IgM antibodies (dilution range: 1:2560 to 1:5120 at week 3 postinfection and 1:640 to 1:1280 at week 7 postinfection) in surviving animals were found.

Figure 1.

Survival curves of controls and Venezuelan equine encephalitis (VEE) virus-infected mice. Early treatment of infected mice with melatonin (MLT: previous and during viral infection) significantly increased the percentage of survival (25%) compared with infected nontreated animals (0%). VEE virus-infected animals (10 LD₅₀) were divided into four groups (n = 10 per group): untreated VEEV animals, MLT pretreated VEEV animals (MLT three days before viral Infection), MLT-treated VEEV animals during infection (MLT immediately after viral infection), and MLT posttreated VEEV animals (MLT 24 h after infection); 500 µg/kg bw of MLT was used for these experiments. Mortality rates were monitored and recorded daily for 10 days and blood samples were obtained from survived animals. Animals received these treatments daily (5–6 pm). Log-rank test analysis showed significant differences between untreated animal curve and treated animal curves.

Apoptosis and oxidative stress

Brain apoptosis in VEEV-infected animals was observed increased, with higher values at day 5 postinfection. No apoptotic cells at day 1 were observed. MLT treatment (500 µg/kg bw, three days before infection and during all experiment) reduced the numbers of apoptotic cells at day 5 postinfection. Untreated controls and MLT-treated controls did not show apoptotic cells (Figure 2). The apoptosis inducer effect of VEEV was also observed in neuroblastoma cell cultures at 4 and 6 h. This apoptotic effect was diminished by all MLT doses (0.1, 0.50, and 1 mM). However, a pro-apoptotic effect of MLT on uninfected neuroblastoma cell cultures was also found (Figure 3).

Figure 2.

Brain apoptosis expression in Venezuelan equine encephalitis (VEE) and controls. (a) Increased expression of apoptosis in infected animals was observed, with higher values at day 5. No apoptotic cells at day 1 were observed. Melatonin (MLT) treatment diminished apoptosis expression. Untreated and MTL-treated controls did not show apoptotic cells. (b) Microphotographs from brain sections reacted for apoptosis (TUNEL). (1) Tissue from VEE virus-infected mouse. (2) Tissue from infected mouse treated with MLT (500 µg/kg bw, three days before infection and during all experiment) (arrows show apoptotic cells); 3 and 4 represent tissues from MLT control and untreated control, respectively. In this experiments, mice were divided in four groups (n = 9 per group): untreated control, untreated VEE virus-infected animals (10LD₅₀), MLT-treated control, and MLT-treated infected animals. Animals were treated with 500 µg/kgbw, starting three days before and continuing for five days after virus inoculation. Controls and infected mice were sacrificed on days 1, 3, and 5 postinfection and brains were obtained. Immunoperoxidase. Original magnification: × 400.

Figure 3.

Apoptosis expression in Venezuelan equine encephalitis (VEE) virus-infected neuroblastoma and control cultures. (a) At 2 h of culture no significant differences were observed in infected cultures compared with controls. Increased expression of apoptosis in infected cultures was observed at 4 and 6 h. Melatonin (MLT) treatment diminished apoptosis expression. MTL-treated controls also showed increased expression of apoptotic cells compared with untreated cultures. For these studies, neuroblastoma cells were seeded at 1 × 10⁶ cells/ml in a 24-well plate. The cells were allowed to attach and grow for two days prior to the treatment. A group of cells was cocultured with VEEV at MOI of 1, while other groups were cultured without virus. These cells were treated with several final concentrations of MLT: 0, 0.1, 0.50, and 1 mM. After 2, 4, and 6 h of culture, apoptosis was determined by the TUNEL assay. Analyses were performed in triplicate and the results represent three different experiments. (b) Microphotography from a neuroblastoma culture at 6 h showing apoptotic nuclei (arrows). Zeiss/Nomarski differential interference equipment. Peroxidase-TUNEL assay. Original magnification: × 400.

Oxidative stress

Figure 4 shows increased production of nitrites in the brain of mice infected by VEEV. This increment was observed from day 1 to day 5 (Figure 4(a)) and it was accompanied by high production of MDA (Figure 4(b)). Both nitrite and TBARS (MDA) production were decreased by MLT treatment. Figure 5 also shows increased productions of nitrites and MDA in neuroblastoma cultures infected by VEEV. After MLT treatment both nitrites and MDA were observed diminished.

Figure 4.

Nitrites and malondialdehyde (MDA) productions in the brain of Venezuelan equine encephalitis (VEE) virus-infected mice and controls. (a) Increased production of nitrites was observed in infected mice from day 1 to day 3. Melatonin (MLT) decreased nitrite production at days 2 and 3. (b) Increased production of nitrite was accompanied by increased production of MDA in the brain of infected mice that was reduced by MLT. Mice were divided in three groups (n = 9 per group): untreated control, untreated VEE virus-infected animals (10LD₅₀), and MLT-treated infected animals. Animals were treated with 500 µg/kgbw, starting three days before and continuing for five days after virus inoculation. Controls and infected mice were sacrificed on days 1, 3, and 5 postinfection and brains were obtained.

Figure 5.

Nitrites and malondialdehyde (MDA) productions in Venezuelan equine encephalitis (VEE) virus-infected neuroblastoma and control cultures. (a) At 6 h of culture increased nitrite concentration was observed in VEEV-infected cultures. Melatonin (MLT) decreased nitrite concentration at different doses. (b) Increased production of nitrite was accompanied by increased production of MDA in VEE virus-infected cultures that was reduced by MLT. For these studies, neuroblastoma cells were seeded at 1 × 10⁶ cells/ml in a 24-well plate. The cells were allowed to attach and grow for two days prior to the treatment. A group of cells was cocultured with VEEV at MOI of 1, while other groups were cultured without virus. These cells were treated with several final concentrations of MLT: 0, 0.1, 0.50, and 1 mM. After 2, 4, and 6 h of culture, oxidative stress was determined by biochemical methods. Analyses were performed in triplicate and the results represent three different experiments.

Brain CD200 expression

CD200 molecule was observed increased in all studied postinfection times. MLT treatment (500 µg/kg bw, three days before infection and during all experiment) reduced the number of brain CD200 positive cells. Basal expression of CD200 in uninfected controls was also reduced by MLT treatment (Figure 6).

Figure 6.

Brain CD200 expression in Venezuelan equine encephalitis (VEE) and controls. (A) Increased expression of CD200 in infected animals was observed in all experimental days. Melatonin (MLT) treatment diminished CD200 expression. CD200 values from untreated control were observed diminished after MTL treatment. Mice were divided in four groups (n = 9 per group): untreated control, untreated VEE virus-infected animals (10LD₅₀), MLT-treated control and MLT-treated infected animals. Animals were treated with 500 µg/kgbw, starting three days before and continuing for five days after virus inoculation. Controls and infected mice were sacrificed on days 1, 3, and 5 postinfection and brains were obtained. (B) Microphotographs from brain sections reacted for CD200. (a) Tissue from VEE virus-infected mouse. In upper right square: detail of CD200 positive cell. (b) Tissue from infected mouse treated with MLT (arrows show CD200 positive cells); (c) and (d) represent tissues from MLT control and untreated control, respectively. Immunoperoxidase. Original magnification: × 400.

Discussion

Previous studies have shown the beneficial effects of MLT in experimental VEE. In VEEV-infected mice, MLT postpones the onset of the disease and death and reduces the mortality rate. ³ This protective effect seems to be due to several mechanisms such as, by increasing production of IL-1 β,^4–6 decreasing production of TNF-α, ⁴ decreasing NO production,^8,9 or by decreasing iNOS expression and lipid peroxidation;^6,9 protective effects probably mediated by MLT receptors. ¹⁰ In addition, we had previously demonstrated that MLT is capable of decreasing viral replication in brain and neuroblastoma cell cultures infected by VEEV. ⁷ The oxidative stress and inflammatory events as a result of brain VEEV infection may induce apoptosis. In this study, increased number of apoptotic cells in the brain of VEEV-infected mice was observed. MLT treatment reduced the number of apoptotic cells and increased the survival rate in VEEV-infected animals, suggesting that apoptosis is a death type during VEE and the antiapoptotic effect of MLT. This antiapoptotic effect of MLT can be related to its capacity to reduce the oxidative stress and TNF-α content in experimental VEE.^6,9 A similar effect of MLT in brain neurodegeneration has been documented. ²⁶ This antiapoptotic effect of MLT was also observed in VEEV-infected neuroblastoma cell cultures. In this study increased production of nitrites (NO) and MDA suggesting lipid peroxidation was associated with increased apoptosis in neuroblastoma cultures. MLT was capable of reducing apoptosis, nitrites, and MDA, suggesting that MLT antiapoptosis effect involves its antioxidant capacity. However, a pro-apoptotic effect on uninfected MLT controls was observed. This effect of MLT has been previously reported in several neoplastic cell lines^27–29 and since neuroblastoma cells also represent a neoplastic cell line this effect is expected.

Increased expression of brain CD200 protein in the VEEV-infected mice was observed in this study. CD200 represents an immunosuppressive molecule when it interacts with its receptor (CD200R), decreasing the inflammatory response. ³⁰ CD200 is expressed on neurons, ¹⁴ oligodendrocytes, and reactive astrocytes ¹⁵ but not on normal microglia; ³¹ however, microglia can express CD200 during inflammatory responses. ³² The expression of CD200R is mainly restricted to the myeloid lineage cells and microglia,^14,15 but not on neurons, ³² suggesting a neuronal inhibitory effect on inflammatory microglial activation. The upregulation of CD200 in the infected animals could represent an anti-inflammatory response against the brain inflammation induced by VEEV, maintaining the microglia and macrophages in a quiescent state, and therefore, protecting the brain from deleterious effects of inflammation. MLT treatment diminished the CD200 expression in both infected animals and controls, suggesting a potential pro-inflammatory effect of MLT, leading neurons unprotected from activated microglia or macrophages during VEEV infection. These data represent the first report of brain CD200 upregulation during VEE and the modulation of this molecule by MLT.

Apoptosis can be induced during inflammatory events by different mechanisms.^11,13 In this report, increased apoptosis and CD200 expression were observed in VEEV-infected animals. It is expected that increased expression of CD200 should be related to decreased expression of apoptosis; since decreased expression of this molecule is associated with increased production of NO and oxidative stress (apoptosis inducers). ³³ However, there was no relationship between brain CD200 expression and apoptosis. These data suggest that probably CD200 is not involved in the apoptosis events.

In this study increased apoptosis was observed in both brain of infected mice and in infected neuroblastoma cultures, associated with increased production of NO and lipid peroxidation. Since oxidative stress has been associated to apoptosis ³⁴ this could be a mechanism of apoptosis during VEEV infection. In previous study, it was demonstrated that increased VEEV replication was associated to increased NO and MDA in infected mice and neuroblastoma cultures, parameters diminished by MLT ⁷ ; therefore, it is expected that the alteration of the sequence: virus replication, oxidative stress, and apoptosis by MLT represent a possible mechanism of MLT during VEEV infection. In this regard, antiapoptotic effect of MLT has been previously reported. ³⁵

The attenuation of studied factors in VEE could be a direct effect of MLT; however, the attenuation of viral infection by MLT^3,7 could indirectly downregulate other host factors stimulated by virus infection and induce survival or prolong the time to death. In this regard, reduced VEEV proliferation by different antiviral treatments was accompanied by increased survival and reduction of several factors such as TNF-α, ³⁶ IL-12, ³⁷ and nitric oxide ⁷ among others, which could be involved in deleterious effect when they are upregulated in VEE virus-infected individuals.

The role of brain inflammation on the mortality rate during VEEV infection has been discussed. Previous experiments have shown that immunocompetent mice infected with VEEV survived an average of seven days before succumbing to fulminant encephalitis. In contrast, immunodeficient mice infected with VEEV showed a persistent replication of virus throughout all organs and survived an average of nine days. ¹ These data suggest that inflammation represents a lethal factor during VEE in the mouse model. In this study, MLT diminished the expression of brain CD200, an effect that could lead to increment of inflammatory events with increased brain damage. However, the antiapoptotic and reducing effect on oxidative stress effects of MLT observed in this study and the capacity of this hormone to diminish VEEV replication ⁷ could balance the final events during VEE infection.

The doses and time of MLT administration were evaluated on survival rate of VEEV-infected mice. Survival rates were higher when early administration of MLT was used, suggesting a beneficial preventive effect of this hormone on VEE infection. The protective effect of MLT could also be mediated by the immune humoral response during the disease. Infected survived animals had high titers of serum anti-VEEV antibodies, suggesting an effective systemic immune response, in spite of the possible immunosuppressive activity of MLT in the brain.

In conclusion, MLT has both an antiapoptotic and potential pro-inflammatory effects during experimental VEE that could reduce the number of dead brain cells, but could also lead to inflammatory events. Since MLT is used as a nutraceutical, it could certainly be used in VEEV-infected patients. The data from the manuscript suggest that early treatment is likely the most successful approach to treatment benefit.