- Top of page
- Material and methods
Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection (p < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.
Flavivirus are important human pathogens causing variety of diseases ranging from mild febrile illness to severe encephalitis and hemorrhagic fever. Among them, Japanese encephalitis virus (JEV), a neurotropic one commonly affects children and is a major cause of acute encephalopathy (Chen et al. 2002). JEV is active over a vast geographic area that includes India, China, Japan, and virtually all of South-East Asia. Approximately 3 billion people live in the JEV endemic area covering much of Asia with nearly 50 000 cases of Japanese encephalitis (JE) reported each year. Of these, about 10 000 cases results in fatality and a high proportion of survivors have serious neurological and psychiatric sequelae (Kaur and Vrati 2003). Therapy for JE is supportive and no clearly effective antiviral agents exist. Therefore, the search is on for compounds which is cheap, easily available and with no or tolerable side effects combined with a protective potential when administered several hours after infection.
Minocycline, a semisynthetic tetracycline, has demonstrated remarkably broad neuroprotective properties in experimental models of ischemic stroke, Huntington’s disease, amyotrophic lateral sclerosis (ALS), traumatic brain injury, multiple sclerosis, Parkinson’s disease, and diabetic retinopathy (Yrjanheikki et al. 1999; Chen et al. 2000; Sanchez Mejia et al. 2001; Popovic et al. 2002; Wu et al. 2002; Zhu et al. 2002; Krady et al. 2005). Minocycline was shown to possess antiapoptotic (neuroprotective) as well as antiviral and anti-inflammatory properties against human immunodeficiency virus-induced encephalitis (Zink et al. 2005). A very recent in vitro study indicates that minocycline inhibits West Nile virus (WNV) replication and apoptosis in human neuronal cells (Michaelis et al. 2007). The mechanism of minocycline-mediated neuroprotection have been demonstrated to result, at least in part, from inhibiting release of cytochrome c from the mitochondria in a transgenic mouse model of ALS (Zhu et al. 2002). Additional effects have been ascribed to minocycline that include inhibition of caspase 1, 3, and inducible nitric oxide transcriptional up-regulation and activation, reactive microgliosis, and activation of p38 mitogen-activated protein kinase (MAPK) and down-regulation of proinflammatory cytokines (Nikodemova et al. 2006). All inhibitory properties of minocycline, other than microgliosis, likely result from inhibiting downstream events after cytochrome c release. Inhibition of reactive microgliosis is a direct effect of minocycline in vitro (Tikka et al. 2001). At present it is not clear whether in vivo inhibition of reactive microgliosis is a direct effect of minocycline or a secondary event resulting from inhibition of neuronal death.
We have recently shown that proinflammatory mediators released by activated microglia induces neuronal death in JE (Ghoshal et al. 2007). In this study, we show that minocycline at relatively low doses is very effective neuroprotective drug against an experimental model of JE even when the administration is started next day following viral inoculation, indicating a clinically relevant therapeutic time window for this tetracycline derivative. Furthermore, our results also suggested that minocycline rescue 70% of animals even in animals with established infection of JEV. In addition, we report that the beneficial effect is associated with reduction of (i) proinflammatory cytokines, (ii) active caspase 3 activity, (iii) microgliosis, (iv) viral titer, and (v) neuronal death. As minocycline is well tolerated, it represents a potential new therapeutic for preventing or controlling the neurological complications of JE.
- Top of page
- Material and methods
The antibiotic minocycline has been shown to have neuroprotective properties in diverse models of neurodegeneration and CNS injury (Arvin et al. 2002; Kriz et al. 2002; Zhu et al. 2002); (Thomas et al. 2003); (Du et al. 2001); (Van Den Bosch et al. 2002); (Tikka et al. 2001);(Hirsch et al. 2003); (Krady et al. 2005); (Yrjanheikki et al. 1998); (Fan et al. 2007). However, until recently this drug had not been tested in experimental models of CNS infection. It has been previously reported that minocycline protects infected mice from neuroadapted Sindbis virus-induced spinal motor neuron death (Darman et al. 2004). Another study demonstrated that the treatment of reovirus-infected neonatal mice with minocycline delays mortality of the resulting encephalitis (Richardson-Burns and Tyler 2005). A recent study indicates that therapy with minocycline aggravates experimental rabies in mice (Jackson et al. 2007). Another finding suggests that minocycline protects detrimental host immune responses of mice from fatal alpha virus encephalitis (Irani and Prow 2007) and finally a very recent study reported the effect of minocycline on viral titer and neuronal apoptosis in WNV (Michaelis et al. 2007).The major finding in this study is that treatment with minocycline provides a complete protection against experimental JE. Minocycline’s neuroprotective action is associated with marked decreases in (i) neuronal apoptosis, (ii) the level of active caspase, (iii) microgliosis, (iv) viral titer, and (v) the level of proinflammatory mediators. Furthermore, treatment with minocycline also improves the behavioral outcome following JE.
We showed previously that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis and it is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to death (Ghoshal et al. 2007). Inhibition of chronic neuroinflammation, particularly of microglial activation, has been suggested to be a practical strategy in the treatment of neurodegenerative diseases. We show here that the treatment with minocycline following JE reduces the number of activated microglia as well as the level of proinflammatory cytokines IL-6, tumor necrosis factor-α, interferon-γ, IL-12p70, and chemokine monocytes chemoattractant protein-1. These data further support that microglial activation and subsequent inflammation is critical in determining the outcome of viral pathogenesis observed in JE.
The dose of minocycline that was used in these animals (45 mg/kg, twice daily) is within in the tolerated range of humans. It can be difficult to compare effective or toxic doses from one species to another. Two double-blind, randomized, placebo-controlled feasibility trials of minocycline in patients with ALS have been reported few years back (Gordon et al. 2004). In the first, 19 patients were treated with 200 mg/day of minocycline for 6 months with no difference in adverse events compared with those in the placebo group. In second, 23 patients received up to 400 mg/day in an 8-month crossover trial. The mean tolerated dose in this study was 387 mg/day. These findings suggest that minocycline at the dose that suppressed CNS inflammation, neuronal apoptosis, and virus replication in animal may be well tolerated in JEV-infected individuals.
We have recently reported that JEV infection is also accompanied by profound neuronal apoptosis (Mishra et al. 2007b). In addition to its direct actions on microglia, minocycline also has been shown to exert antiapoptotic effects by inhibiting caspases 1 and 3, and inhibiting the release of cytochrome c from mitochondria (Chen et al. 2000); (Sanchez Mejia et al. 2001); (Wang et al. 2003); (Zhu et al. 2002). These actions likely contributed to our in vivo finding of reduced caspase 3 activity as well as our in vitro findings with N2a cell line showing reduced apoptosis. These data suggest that minocycline is also working as an antiapoptotic molecule in this model of infection. The modulation of apoptosis by minocycline could be because of its effects on apoptogenic molecules such as caspase in concert with its promotion of Bcl-2. In vivo microglial activation could be a response to neuronal damage with the subsequent inflammation resulting in negative consequences. Henceforth, early inhibition of neuronal apoptosis compound with a decrease in the subsequent release of proinflammatory mediators by activated microglia would attenuate the severity of disease observed in JE. Because minocycline’s both anti-inflammatory and antiapoptotic effects will be beneficial in reducing the severity of diseases induced by JEV, our finding provides compelling evidence to support the administration of minocycline for treating JE patients. Furthermore, our results also suggested that minocycline rescue 70% of animals even in animals with established infection of JEV. JE is often accompanied by several neurological sequeale including severe movement disorders. To determine whether the preservation of brain tissue correlated with a preservation of neurological function, we used wire-hang test as a measure of motor neuron function. We observed significant improvement in the behavior of JEV infected versus JEV infected but minocycline treated mice in this test (data not shown).
We have found that JEV infections down-regulate the level of PKC-α, and treatment with minocycline reverses it in a significant extent. The decrease in the activity of PKC suggests that intracellular cAMP levels may be down-regulated, and it is also consistent with the level of apoptosis. In an interesting study it was reported earlier that cAMP protects against Staurosporine and Wortmannin induced neuronal apoptosis (Goswami et al. 1998). Staurosponne was initially described as an inhibitor of PKC but was then discovered to activate a specific 60-kDa serine/threonine kinase and has since been used to induce apoptosis in a range of cells from chondrocytes and oligodendrocytes to embryonic neurons (Wiesner and Dawson 1996a) and neuroblastoma cell lines (Wiesner and Dawson 1996b). Staurosporine may directly activate the caspases that cause degradation of poly (ADP-ribose) polymerase and lamins and ultimately cell death or may work through inhibition of the antiapoptotic kinase, PKC, which is believed to phosphorylate Bad and prevent it from inactivating the protective proteins of the Bcl-2 family (Ito et al. 1997). It is highly possible that JE virus may be exploiting the same pathway to induce neuronal death and minocycline inhibiting this cell death cascade by up-regulating PKC, the antiapoptotic kinase. However more research is necessary to derive any firm conclusion.
Perhaps, the most unexpected result of these studies was the ability of minocycline to substantially inhibit replication of JEV. A very recent study describing minocycline inhibition of WNV in human neuronal cells was recently reported (Michaelis et al. 2007). Compared with anti-inflammatory or antiapoptotic property, minocycline’s antiviral effect is relatively new phenomena. Another very recent studies indicate that minocycline has antiviral property against human immunodeficiency virus (Zink et al. 2005). Moreover, WNV and JEV belong to same family of viruses. These two recent studies prompted us to evaluate the antiviral role of minocycline in JE. Our study clearly indicates the antiviral role of minocycline against JEV. Therefore, individual suffering from severe encephalitis induced by JEV may benefit from minocycline by antiviral effects and by neuroprotective and anti-inflammatory effects that are independent of antiviral activity. It seems unlikely that minocycline has classic antiviral activity, as do reverse transcriptase and protease inhibitors because the antibiotic was not engineered to target a specific viral protein.
We propose that rather than exerting direct antiviral activity, minocycline modifies the intracellular or extracellular environment making it non-permissive for JEV replication. The ability of minocycline to modify environments differentially in primary macrophages and T lymphocytes (as evidenced by the differential effect of minocycline on p38 activation) raises the possibility that each cell type has a unique mechanism of suppression (Cohen et al. 1997; Shapiro et al. 1997; Darcissac et al. 2000). An important potential therapeutic advantage of this differential effect is that if the virus develops mutations that confer resistance to minocycline in one target cell type, that resistance might not confer a replicative advantage in the other cell type.
Minocycline is a safe drug commonly used for prolonged treatment of infections, rheumatoid arthritis, and acne vulgaris (Chopra 2001). Our data support, minocycline, which is in clinical trials for both Parkinson’s disease and Huntington’s disease, may be a an ideal candidate for considering in a human trial for JE. It is an attractive candidate for clinical assessment because it is profoundly effective even when given after the inoculation of virus, lacks obvious significant toxic side effects, can be delivered systemically with relatively good CNS penetration, and is reasonably inexpensive.