Sulagna Das

Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis.

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (Cox‐2), IL‐6, IL‐1β, TNF‐α, and MCP‐1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection.

Inflammation: A new candidate in modulating adult neurogenesis

Any pathological perturbation to the brain provokes a cascade of molecular and cellular events, which manifests in the form of microglial activation and release of various proinflammatory molecules. This eventually culminates in a profound neuroinflammatory reaction that characterizes the brains response to stress, injury, or infection. The inflammatory cascade is an attempt by the system to eliminate the challenge imposed on the brain, clear the system of the dead and damaged neurons, and rescue the normal functioning of this vital organ. However, during the process of microglial activation, the proinflammatory mediators released exert certain detrimental effects, and neural stem cells and progenitor cells are likely to be affected. Here we review how the proliferation, maturation, and migration of the neural stem cells are modulated under such an inflammatory condition. The fate of the noncommitted neural stem cells and its differentiation potency are often under strict regulation, and these proinflammatory mediators seem to disrupt this critical balance and finely tune the neurogenesis pattern in the two niches of neurogenesis, the subventricular zone and the subgranular zone of the hippocampus. Moreover, the migration ability of these stem cells, which is important for localization to the proper site, is also affected in a major way by the chemokines released following inflammation.

Critical role of lipid rafts in virus entry and activation of phosphoinositide 3′ kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells

J. Neurochem. (2010) 115, 537–549.

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)‐associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR‐1. Using mouse (Neuro2a) and human (SK‐N‐SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR‐1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria‐mediated neuronal apoptosis. As TRADD‐knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small‐interfering RNA (siRNA) and have found that TRADD is required for TNFR‐1‐initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV‐infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR‐1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.

Japanese Encephalitis Virus infection induces IL-18 and IL-1β in microglia and astrocytes: Correlation with in vitro cytokine responsiveness of glial cells and subsequent neuronal death

IL-1beta and IL-18 are members of the IL-1 family of ligands, and their receptors are members of the IL-1 receptor family. Although several biological properties overlap for these cytokines, differences exist. In order to assess functional importance of these two cytokines in viral encephalitis, we have exploited an experimental model of Japanese Encephalitis (JE) and subsequent in vitro cell culture system. We report for the first time that in Japanese Encephalitis, microglia and astrocytes both produce IL-18 and IL-1beta. In vitro, these two cytokines differentially activate microglia and astrocyte, and also alter the by stander neuronal survival following treatment with these two cytokines.

Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.

Induction of IP-10 (CXCL10) in astrocytes following Japanese encephalitis

Chemokines and their receptors are important elements for the selective attraction and activation of various subsets of leukocytes. Interferon-gamma inducible protein (IP-10 or CXCL-10) is a potent chemoattractant and has been suggested to enhance the severity of virus infection and neuronal injury. In order to assess functional importance of this chemokine in viral encephalitis, we have exploited an experimental model of Japanese encephalitis. We report for the first time that in Japanese encephalitis, astrocytes are the predominant source of IP-10. A progressive increase in IP-10 induction following viral infection is concomitant with the increase in IFN-gamma a known inducer of IP-10. However, this increase in IFN-gamma level is not sufficient to confer protection as animals eventually succumb to the infection.

Abrogated Inflammatory Response Promotes Neurogenesis in a Murine Model of Japanese Encephalitis

Background Japanese encephalitis virus (JEV) induces neuroinflammation with typical features of viral encephalitis, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. The detrimental effects of inflammation on neurogenesis have been reported in various models of acute and chronic inflammation. We investigated whether JEV-induced inflammation has similar adverse effects on neurogenesis and whether those effects can be reversed using an anti-inflammatory compound minocycline. Methodology/Principal Findings Here, using in vitro studies and mouse models, we observed that an acute inflammatory milieu is created in the subventricular neurogenic niche following Japanese encephalitis (JE) and a resultant impairment in neurogenesis occurs, which can be reversed with minocycline treatment. Immunohistological studies showed that proliferating cells were replenished and the population of migrating neuroblasts was restored in the niche following minocycline treatment. In vitro, we checked for the efficacy of minocycline as an anti-inflammatory compound and cytokine bead array showed that production of cyto/chemokines decreased in JEV-activated BV2 cells. Furthermore, mouse neurospheres grown in the conditioned media from JEV-activated microglia exhibit arrest in both proliferation and differentiation of the spheres compared to conditioned media from control microglia. These effects were completely reversed when conditioned media from JEV-activated and minocycline treated microglia was used. Conclusion/Significance This study provides conclusive evidence that JEV-activated microglia and the resultant inflammatory molecules are anti-proliferative and anti-neurogenic for NSPCs growth and development, and therefore contribute to the viral neuropathogenesis. The role of minocycline in restoring neurogenesis may implicate enhanced neuronal repair and attenuation of the neuropsychiatric sequelae in JE survivors.

Tumor necrosis factor receptor-associated death domain mediated neuronal death contributes to the glial activation and subsequent neuroinflammation in Japanese encephalitis

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and in disease state, little is known regarding how the neuronal death initiates the cascades of secondary neuroinflammation. We have exploited an experimental model of Japanese encephalitis to better understand how neuronal death following viral infection initiates microglial activation following Japanese encephalitis virus infection. We have earlier shown that the altered expression of tumor necrosis factor receptor-1 (TNFR-1) and TNFR associated death domain (TRADD) following Japanese encephalitis virus infection regulates the downstream apoptotic cascades. Here we have reported that silencing TRADD expression with small-interfering RNA reduced neuronal apoptosis and subsequent microglial and astroglial activation and release of various pro-inflammatory mediators. Our findings suggest that the engagement of TNFR-1 and TRADD following Japanese encephalitis virus infection plays a crucial role in glial activation also and influences the outcome of viral pathogenesis.

Tobacco carcinogen induces microglial activation and subsequent neuronal damage

4‐Methylnitrosamino‐1‐(3‐pyridyl)‐1‐butanone (NNK) is a tobacco‐specific procarcinogen. We have investigated whether NNK causes inflammatory upheaval in the brain by activation of resident microglia and astrocyte and result in bystander neuronal damage. We have carried out the work in both in vitro and in vivo models. We have found that treatment with NNK causes significant activation of mouse microglial (BV2) cell line as evident by increase in reactive oxygen species and nitric oxide level. Western blot analysis has showed increase in proinflammatory signaling proteins, proinflammatory effector proteins, and other stress‐related proteins. Interestingly, increased levels of proinflammatory cytokines like interleukin (IL)‐6, tumor necrosis factor‐α, monocyte chemoattractant protein 1 (MCP1), and IL‐12p70 are also detected. Work from our in vivo studies has demonstrated similar increase in proinflammatory signaling and effector molecules along with the proinflammatory cytokine levels, following NNK treatment. Immunohistochemical staining of the brain sections of NNK‐treated mice reveals massive microglial and astrocyte activation along with distinct foci of neuronal damage. Both in vitro and in vivo results provide strong indication that NNK causes significant upheaval of the inflammatory condition of brain and inflicts subsequent neuronal damage.