RIG-I Mediates Innate Immune Response in Mouse Neurons Following Japanese Encephalitis Virus Infection

Neuroinflammation associated with Japanese encephalitis (JE) is mainly due to the activation of glial cells with subsequent release of proinflammatory mediators from them. The recognition of viral RNA, in part, by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been indicated to have a role in such processes. Even though neurons are also known to express this receptor, its role after JE virus (JEV) infections is yet to be elucidated.Upon infecting murine neuroblastoma cells and primary cortical neurons with JEV the expression profile of key proinflammatory cyto/chemokines were analyzed by qRT-PCR and bead array, both before and after ablation of RIG-I. Immunoblotting was performed to evaluate the levels of key molecules downstream to RIG-I leading to production of proinflammatory mediators. Changes in the intracellular viral antigen expression were confirmed by intracellular staining and immunoblotting. JEV infection induced neuronal expression of IL-6, IL-12p70, MCP-1, IP-10 and TNF-α in a time-dependent manner, which showed significant reduction upon RIG-I ablation. Molecules downstream to RIG-I showed significant changes upon JEV-infection, that were modulated following RIG-I ablation. Ablation of RIG-I in neurons also increased their susceptibility to JEV.In this study we propose that neurons are one of the potential sources of proinflammatory cyto/chemokines in JEV-infected brain that are produced via RIG-I dependent pathways. Ablation of RIG-I in neurons leads to increased viral load and reduced release of the cyto/chemokines

Antiviral and Neuroprotective Role of Octaguanidinium Dendrimer-Conjugated Morpholino Oligomers in Japanese Encephalitis

Japanese encephalitis (JE) is caused by a flavivirus that is transmitted to humans by mosquitoes belonging to the Culex sp. The threat of JE looms over a vast geographical realm, encompassing approximately 10 billion people. The disease is feared because currently there are no specific antiviral drugs available. There have been reports where other investigators have shown that agents that block viral replication can be used as effective therapeutic countermeasures. Vivo-Morpholinos (MOs) are synthetically produced analogs of DNA or RNA that can be modified to bind with specific targeted regions in a genome. In this study the authors propose that in an animal model of JE, MOs specifically designed to bind with specific region of JE virus (JEV) genome, blocks virus production in cells of living organisms. This results in reduced mortality of infected animals. As the major target of JEV is the nerve cells, analysis of brain of experimental animals, post treatment with MOs, showed neuroprotection. Studies in cultured cells were also supportive of the antiviral role of the MOs. The potent anti-sense effect in animals and lack of obvious toxicity at the effective dosage make these MOs good research reagents with future therapeutic applications in JE

A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

BACKGROUND: Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels. CONCLUSIONS/SIGNIFICANCE: Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today's perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert

STING Mediates Neuronal Innate Immune Response Following Japanese Encephalitis Virus Infection

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Japanese encephalitis virus infection modulates the expression of suppressors of cytokine signaling (SOCS) in macrophages: Implications for the hosts’ innate immune response

Viruses have evolved various mechanisms to subvert the host’s immune system and one of them is preventing the infected cells from sending out chemotactic signals to activate the adaptive immune response. Japanese encephalitis virus (JEV) is a neuropathologic flavivirus that is responsible for significant number of child mortalities in various parts of South-East Asia. In this study we show that JEV modulates suppressors of cytokine signaling (SOCS)1 and 3 expression in macrophages to bring about changes in the JAK–STAT signaling cascade, so as to inhibit proinflammatory cyto/chemokine release. Using real time PCR, immunoblotting and immunofluorescent staining, we show that the expression of type 1 interferons and intracellular expression of viral genes are also affected over time. Also, following the initial activation of SOCS1 and 3, there is production of interferon-inducible anti-viral proteins in the cells which may be responsible for inhibiting viral replication. However, even at later time points, viral genes were still detected from the macrophages, albeit at lesser quantities, than earlier time points, indicative of intracellular persistence of the virus in a latent form. On knocking down SOCS1 and SOCS3 we found a significant decrease in viral gene expression at an early time point, indicating the dysregulation of the signaling cascade leading to increased production of interferon-inducible anti-viral proteins. Taken together, our study provides an insight into the role of JEV infection in modulating the JAK–STAT pathway with the help of SOCS leading to the generation of an antiviral innate immune response

Japanese encephalitis virus-infected macrophages induce neuronal death

Inflammation in the central nervous system (CNS) in Japanese encephalitis (JE) is shown to be the result of microglial activation that leads to the release of various proinflammatory mediators. Peripheral macrophages have been reported to infiltrate into the CNS in JE, though their contribution to the inflammatory process is yet to be elucidated. In this study, using an in vitro macrophage model, we have shown that upon JE virus infection, these cells secrete various soluble factors which may significantly add to the existing inflammatory milieu and lead to apoptotic or necrotic death of neurons. However, it is difficult to quantify the extent of involvement of either the microglia or infiltrating macrophages in the inflammatory processes

Minocycline differentially modulates macrophage mediated peripheral immune response following Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a neurotropic flavivirus that is the causative agent of a major mosquito-borne encephalitis in the world. Evasion of peripheral immune system facilitates the entry of the virus into the central nervous system (CNS) where it causes extensive neuronal inflammatory damage that leads to death or severe neuropschychiatric sequel in survivors. It has been proposed that after entry into the body, the virus is carried into the CNS by peripheral immune cells that act as Trojan horses. In this study we investigate whether macrophages can be considered as such a Trojan horse. We also investigate the role of minocycline, a synthetic tetracycline, in such processes. Minocycline has been found to be broadly protective in neurological disease models featuring inflammation and cell death but there has been no report of it having any modulatory role in peripheral macrophage-mediated immune response against viral infection. Persistence of internalized virus within macrophages was visualized by immunofluorescent staining. Cytotoxicity assay revealed that there was no significant cell death after 24 h and 72 h infection with JEV. Proinflammatory cytokine levels were elevated in cells that were infected with JEV but it was abrogated following minocycline treatment. Reactive oxygen species level was also increased after JEV infection. Nitric oxide level was found to increase after 72 h post infection but remained unchanged after 24 h. The cellular levels of signaling molecules such as PI3 kinase, phophoAkt and phospho p38MAP kinase were found to be altered after JEV infection and minocycline treatment. JEV infection also affected the VEGF-MMP pathway. Increased activity of MMP-9 was detected from JEV-infected macrophage culture supernatants after 72 h; minocycline treatment resulted in reduced activity. Thus it seems that minocycline dampens peripheral immune reactions by decreasing proinflammatory cytokine release from infected macrophages and the virus survives within macrophages long enough to be carried into the CNS, even though minocycline inhibits cell survival