Chintan Chhatbar

Upon Intranasal Vesicular Stomatitis Virus Infection, Astrocytes in the Olfactory Bulb Are Important Interferon Beta Producers That Protect from Lethal Encephalitis

ABSTRACT Previously we found that following intranasal (i.n.) infection with neurotropic vesicular stomatitis virus (VSV) type I interferon receptor (IFNAR) triggering of neuroectodermal cells was critically required to constrain intracerebral virus spread. To address whether locally active IFN-β was induced proximally, we studied spatiotemporal conditions of VSV-mediated IFN-β induction. To this end, we performed infection studies with IFN-β reporter mice. One day after intravenous (i.v.) VSV infection, luciferase induction was detected in lymph nodes. Upon i.n. infection, luciferase induction was discovered at similar sites with delayed kinetics, whereas on days 3 and 4 postinfection enhanced luciferase expression additionally was detected in the foreheads of reporter mice. A detailed analysis of cell type-specific IFN-β reporter mice revealed that within the olfactory bulb IFN-β was expressed by neuroectodermal cells, primarily by astrocytes and to a lesser extent by neurons. Importantly, locally induced type I IFN triggered distal parts of the brain as indicated by the analysis of ISRE-eGFP mice which after i.n. VSV infection showed enhanced green fluorescent protein (eGFP) expression throughout the brain. Compared to wild-type mice, IFN-β−/− mice showed increased mortality to i.n. VSV infection, whereas upon i.v. infection no such differences were detected highlighting the biological significance of intracerebrally expressed IFN-β. In conclusion, upon i.n. VSV instillation, IFN-β responses mounted by astrocytes within the olfactory bulb critically contribute to the antiviral defense by stimulating distal IFN-β-negative brain areas and thus arresting virus spread. IMPORTANCE The central nervous system has long been considered an immune privileged site. More recently, it became evident that specialized immune mechanisms are active within the brain to control pathogens. Previously, we showed that virus, which entered the brain via the olfactory route, was arrested within the olfactory bulb by a type I IFN-dependent mechanism. Since peripheral type I IFN would not readily cross the blood-brain barrier and within the brain thus far no abundant type I IFN responses have been detected, here we addressed from where locally active IFN originated from. We found that upon intranasal VSV instillation, primarily astrocytes, and to a lesser extent neurons, were stimulated within the olfactory bulb to mount IFN-β responses that also activated and protected distal brain areas. Our results are surprising because in other infection models astrocytes have not yet been identified as major type I IFN producers.

Macrophage depletion by liposome-encapsulated clodronate suppresses seizures but not hippocampal damage after acute viral encephalitis

Viral encephalitis is a major risk factor for the development of seizures and epilepsy, but the underlying mechanisms are only poorly understood. Mouse models such as viral encephalitis induced by intracerebral infection with Theilers virus in C57BL/6 (B6) mice allow advancing our understanding of the immunological and virological aspects of infection-induced seizures and their treatment. Previous studies using the Theilers virus model in B6 mice have indicated that brain-infiltrating inflammatory macrophages and the cytokines released by these cells are key to the development of acute seizures and hippocampal damage in this model. However, approaches used to prevent or reduce macrophage infiltration were not specific, so contribution of other mechanisms could not be excluded. In the present study, we used a more selective and widely used approach for macrophage depletion, i.e., systemic administration of clodronate liposomes, to study the contribution of macrophage infiltration to development of seizures and hippocampal damage. By this approach, almost complete depletion of monocytic cells was achieved in spleen and blood of Theilers virus infected B6 mice, which was associated with a 70% decrease in the number of brain infiltrating macrophages as assessed by flow cytometry. Significantly less clodronate liposome-treated mice exhibited seizures than liposome controls (P<0.01), but the development of hippocampal damage was not prevented or reduced. Clodronate liposome treatment did not reduce the increased Iba1 and Mac3 labeling in the hippocampus of infected mice, indicating that activated microglia may contribute to hippocampal damage. The unexpected mismatch between occurrence of seizures and hippocampal damage is thought-provoking and suggests that the mechanisms involved in degeneration of specific populations of hippocampal neurons in encephalitis-induced epilepsy are more complex than previously thought.

The antiviral drug ganciclovir does not inhibit microglial proliferation and activation

Ganciclovir is effective in the treatment of human infections with viruses of the Herpesviridae family. Beside antiviral properties, recently ganciclovir was described to inhibit microglial proliferation and disease severity of experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. Microglial activation and proliferation are main characteristics of neuroinflammatory CNS diseases and inhibition of microglial functions might be beneficial in autoimmune diseases, or detrimental in infectious diseases. The objective of this study was to determine potential inhibitory effects of ganciclovir in three different murine animal models of CNS neuroinflammation in which microglia play an important role: Theiler´s murine encephalomyelitis, the cuprizone model of de- and remyelination, and the vesicular stomatitis virus encephalitis model. In addition, in vitro experiments with microglial cultures were performed to test the hypothesis that ganciclovir inhibits microglial proliferation. In all three animal models, neither microglial proliferation or recruitment nor disease activity was changed by ganciclovir. In vitro experiments confirmed that microglial proliferation was not affected by ganciclovir. In conclusion, our results show that the antiviral drug ganciclovir does not inhibit microglial activation and proliferation in the murine CNS.

Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.

Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis

BACKGROUND & AIM Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation. METHODS Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-β induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1+/gfp mice revealed the origin of reconstituted KC. Finally, mixed bone marrow chimeric mice were studied to specifically analyze the effect of IFNAR triggering on liver infiltrating monocytes. RESULTS VACV infection induced local IFN-β responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution. CONCLUSION Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis. LAY SUMMARY Viral infection can cause fulminant hepatitis, which in turn is a major cause of acute liver failure. Herein, we aimed to study the role of type 1 interferon responses in acute viral hepatitis. We identified that during infection with DNA-encoded viruses, type 1 interferon receptor triggering of blood monocytes delays the development of monocyte-derived Kupffer cells. This points to new therapeutic strategies for acute viral hepatitis.

Chemokine receptors CCR2 and CX3CR1 regulate viral encephalitis-induced hippocampal damage but not seizures

Significance Viral encephalitis is a frequent medical emergency, often resulting in acute seizures and brain damage, which reduce quality of life, promote the development of epilepsy, and can cause death. The relative roles of activation of microglia, the brain-resident innate immune cells, versus invasion of blood-borne immune cells such as monocytes in the acute and chronic consequences of viral encephalitis are only incompletely understood. Here we show that lack of the chemokine receptors CCR2 or CX3CR1, which regulate the responses of myeloid cells such as monocytes and microglia, prevents hippocampal damage but not seizures in a mouse model of viral encephalitis. Treatment strategies aimed at inhibiting peripheral immune cells from entering the brain during encephalitis could reduce brain damage. Viral encephalitis is a major risk factor for the development of seizures, epilepsy, and hippocampal damage with associated cognitive impairment, markedly reducing quality of life in survivors. The mechanisms underlying seizures and hippocampal neurodegeneration developing during and after viral encephalitis are only incompletely understood, hampering the development of preventive treatments. Recent findings suggest that brain invasion of blood-born monocytes may be critically involved in both seizures and brain damage in response to encephalitis, whereas the relative role of microglia, the brain’s resident immune cells, in these processes is not clear. CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation. We used Ccr2-KO and Cx3cr1-KO mice to understand the role of these receptors in viral encephalitis-associated seizures and neurodegeneration, using the Theiler’s virus model of encephalitis in C57BL/6 mice. Our results show that CCR2 as well as CX3CR1 plays a key role in the accumulation of myeloid cells in the CNS and activation of hippocampal myeloid cells upon infection. Furthermore, by using Cx3cr1-creER+/−tdTomatoSt/Wt reporter mice, we show that, with regard to CD45 and CD11b expression, some microglia become indistinguishable from monocytes during CNS infection. Interestingly, the lack of CCR2 or CX3CR1 receptors was associated with almost complete prevention of hippocampal damage but did not prevent seizure development after viral CNS infection. These data are compatible with the hypothesis that CNS inflammatory mechanism(s) other than the infiltrating myeloid cells trigger the development of seizures during viral encephalitis.

The olfactory epithelium as a port of entry in neonatal neurolisteriosis.

Bacterial infections of the central nervous system (CNS) remain a major cause of mortality in the neonatal population. Commonly used parenteral infection models, however, do not reflect the early course of the disease leaving this critical step of the pathogenesis largely unexplored. Here, we analyzed nasal exposure of 1-day-old newborn mice to Listeria monocytogenes (Lm). We found that nasal, but not intragastric administration, led to early CNS infection in neonate mice. In particular, upon bacterial invasion of the olfactory epithelium, Lm subsequently spread along the sensory neurons entering the brain tissue at the cribriform plate and causing a significant influx of monocytes and neutrophils. CNS infection required listeriolysin for penetration of the olfactory epithelium and ActA, a mediator of intracellular mobility, for translocation into the brain tissue. Taken together, we propose an alternative port of entry and route of infection for neonatal neurolisteriosis and present a novel infection model to mimic the clinical features of late-onset disease in human neonates.Listeria monocytogenes causes meningitis in newborns. Here, Pägelow et al. present a mouse model of neonatal cerebral listeriosis, and show that nasal inoculation, but not intragastric administration, leads to early brain infection in the absence of bacteraemia during the neonatal period.

Microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage

Abstract In the central nervous system (CNS), innate immune surveillance is mainly coordinated by microglia. These CNS resident myeloid cells are assumed to help orchestrate the immune response against infections of the brain. However, their specific role in this process and their interactions with CNS infiltrating immune cells, such as blood-borne monocytes and T cells are only incompletely understood. The recent development of PLX5622, a specific inhibitor of colony-stimulating factor 1 receptor that depletes microglia, allows studying the role of microglia in conditions of brain injury such as viral encephalitis, the most common form of brain infection. Here we used this inhibitor in a model of viral infection-induced epilepsy, in which C57BL/6 mice are infected by a picornavirus (Theiler’s murine encephalomyelitis virus) and display seizures and hippocampal damage. Our results show that microglia are required early after infection to limit virus distribution and persistence, most likely by modulating T cell activation. Microglia depletion accelerated the occurrence of seizures, exacerbated hippocampal damage, and led to neurodegeneration in the spinal cord, which is normally not observed in this mouse strain. This study enhances our understanding of the role of microglia in viral encephalitis and adds to the concept of microglia-T cell crosstalk.