Saguna Verma

Selenoprotein K Knockout Mice Exhibit Deficient Calcium Flux in Immune Cells and Impaired Immune Responses

Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K−/− mice were generated and found to be similar to wild-type controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K−/− mice. Receptor-mediated Ca2+ flux was decreased in T cells, neutrophils, and macrophages from Sel K−/− mice compared with controls. Ca2+-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K−/− mice compared with that in cells from controls. West Nile virus infections were performed, and Sel K−/− mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, West Nile virus-infected Sel K−/− mice demonstrated significantly lower survival (2 of 23; 8.7%) compared with that of wild-type controls (10 of 26; 38.5%). These results establish Sel K as an endoplasmic reticulum-membrane protein important for promoting effective Ca2+ flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.

Reversal of West Nile virus-induced blood-brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor.

Though compromised blood-brain barrier (BBB) is a pathological hallmark of WNV-associated neurological sequelae, underlying mechanisms are unclear. We characterized the expression of matrix metalloproteinases (MMP) in WNV-infected human brain microvascular endothelial cells (HBMVE) and human brain cortical astrocytes (HBCA), components of BBB and their role in BBB disruption. Expression of multiple MMPs was significantly induced in WNV-infected HBCA cells. Naïve HBMVE cells incubated with the supernatant from WNV-infected HBCA cells demonstrated loss of tight junction proteins, which were rescued in the presence of MMP inhibitor, GM6001. Further, supernatant from WNV-infected HBCA cells compromised the in vitro BBB model integrity. Our data suggest astrocytes as one of the sources of MMP in the brain, which mediates BBB disruption allowing unrestricted entry of immune cells into the brain, thereby contributing to WNV neuropathogenesis. Because of the unavailability of WNV antivirals and vaccines, use of MMP inhibitors as an adjunct therapy to ameliorate WNV disease progression is warranted.

Inflammasome Adaptor Protein Apoptosis-Associated Speck-Like Protein Containing CARD (ASC) Is Critical for the Immune Response and Survival in West Nile Virus Encephalitis

ABSTRACT West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans. The WNV-induced innate immune response, including production of antiviral cytokines, is critical for controlling virus infection. The adaptor protein ASC mediates a critical step in innate immune signaling by bridging the interaction between the pathogen recognition receptors and caspase 1 in inflammasome complexes, but its role in WNV immunopathogenesis is not defined. Here, we demonstrate that ASC is essential for interleukin-1β (IL-1β) production and development of effective host immunity against WNV. ASC-deficient mice exhibited increased susceptibility to WNV infection, and reduced survival was associated with enhanced virus replication in the peripheral tissues and central nervous system (CNS). Infection of cultured bone marrow-derived dendritic cells showed that ASC was essential for the activation of caspase 1, a key component of inflammasome assembly. ASC−/− mice exhibited attenuated levels of proinflammatory cytokines in the serum. Intriguingly, infected ASC−/− mice also displayed reduced levels of alpha interferon (IFN-α) and IgM in the serum, indicating the overall protective role of ASC in restricting WNV infection. However, brains from ASC−/− mice displayed unrestrained inflammation, including elevated levels of proinflammatory cytokines and chemokines, such as IFN-γ, CCL2, and CCL5, which correlated with more pronounced activation of the astrocytes, enhanced infiltration of peripheral immune cells in the CNS, and increased neuronal cell death. Collectively, our data provide new insights into the role of ASC as an essential modulator of inflammasome-dependent and -independent immune responses to effectively control WNV infection.

Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death

BackgroundWNV-associated encephalitis (WNVE) is characterized by increased production of pro-inflammatory mediators, glial cells activation and eventual loss of neurons. WNV infection of neurons is rapidly progressive and destructive whereas infection of non-neuronal brain cells is limited. However, the role of neurons and pathological consequences of pro-inflammatory cytokines released as a result of WNV infection is unclear. Therefore, the objective of this study was to examine the role of key cytokines secreted by WNV-infected neurons in mediating neuroinflammatory markers and neuronal death.MethodsA transformed human neuroblastoma cell line, SK-N-SH, was infected with WNV at multiplicity of infection (MOI)-1 and -5, and WNV replication kinetics and expression profile of key pro-inflammatory cytokines were analyzed by plaque assay, qRT-PCR, and ELISA. Cell death was measured in SK-N-SH cell line in the presence and absence of neutralizing antibodies against key pro-inflammatory cytokines using cell viability assay, TUNEL and flow cytometry. Further, naïve primary astrocytes were treated with UV-inactivated supernatant from mock- and WNV-infected SK-N-SH cell line and the activation of astrocytes was measured using flow cytometry and ELISA.ResultsWNV-infected SK-N-SH cells induced the expression of IL-1β, -6, -8, and TNF-α in a dose- and time-dependent manner, which coincided with increase in virus-induced cell death. Treatment of cells with anti-IL-1β or -TNF-α resulted in significant reduction of the neurotoxic effects of WNV. Furthermore treatment of naïve astrocytes with UV-inactivated supernatant from WNV-infected SK-N-SH cell line increased expression of glial fibrillary acidic protein and key inflammatory cytokines.ConclusionOur results for the first time suggest that neurons are one of the potential sources of pro-inflammatory cytokines in WNV-infected brain and these neuron-derived cytokines contribute to WNV-induced neurotoxicity. Moreover, cytokines released from neurons also mediate the activation of astrocytes. Our data define specific role(s) of WNV-induced pro-inflammatory cytokines and provide a framework for the development of anti-inflammatory drugs as much-needed therapeutic interventions to limit symptoms associated with WNVE.

West Nile Virus-Induced Cell Adhesion Molecules on Human Brain Microvascular Endothelial Cells Regulate Leukocyte Adhesion and Modulate Permeability of the In Vitro Blood-Brain Barrier Model

Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via ‘Trojan horse’ route, and improve WNV disease outcome.

Interferon-α and -β Restrict Polyomavirus JC Replication in Primary Human Fetal Glial Cells: Implications for Progressive Multifocal Leukoencephalopathy Therapy

One of the major limitations of highly active antiretroviral therapy is its inability to inhibit the replication of polyomavirus JC (JCV), the etiologic agent of progressive multifocal leukoencephalopathy (PML), an acquired immunodeficiency syndrome-defining illness. We previously demonstrated the induction of interferon (IFN)-stimulated genes (ISGs) by JCV. In the present study, we characterize the specific viral events required to induce ISGs and the potential antiviral effects of type I IFN on JCV replication in human fetal glial cells in the presence and absence of type I IFNs. Productive JCV replication was essential for the induction of the antiviral host response. JCV replication at all steps was significantly inhibited in the presence of IFN, and neutralizing anti-IFN antibody rescued the inhibitory effect of IFN. These results support the use of IFN as an adjunct therapy for patients with PML. Because IFN cannot cross the blood-brain barrier to achieve its direct antiviral effect, intrathecal administration of IFN is warranted.

In vitro effects of selenium deficiency on West Nile virus replication and cytopathogenicity

BackgroundSelenium (Se) deficiency plays an important role in viral pathogenesis. To understand the effects of Se deficiency on West Nile virus (WNV) infection, we analyzed cytopathogenicity, apoptosis and viral replication kinetics, using a newly developed Se-deficient cell culture system.ResultsBoth Vero and SK-N-SH cells grown in Se-deficient media exhibited a gradual loss of glutathione peroxidase (GPx1) activity without any significant effect on cell growth and viability. In SK-N-SH cells, Se deficiency had no effect on the expression of key antioxidant enzymes, including manganese- and copper-zinc superoxide dismutase (MnSOD and CuZnSOD), catalase and inducible nitric oxide synthase, whereas Vero cells demonstrated a significant increase in the expression of MnSOD and an overall increase in oxidative stress (OS) at day 7 post-induction of Se deficiency. At 2 days after infection with WNV, CPE and cell death were significantly higher in WNV-infected Se-deficient Vero cells, compared to WNV-infected control cells. Furthermore, WNV-induced apoptosis was significantly heightened in Se-deficient cells and was contributed by loss of mitochondrial membrane potential and increased caspase activity. However, no significant difference was found in WNV copy numbers between control, Se-adequate and Se-deficient cell cultures.ConclusionOverall results demonstrate that the in vitro Se-deficient model can be used to study responses of WNV to this essential nutrient. Although Se deficiency has no in vitro effect on WNV replication kinetics, adequate Se is presumably critical to protect WNV-infected cells against virus-induced cell death.

Impaired Virus Clearance, Compromised Immune Response and Increased Mortality in Type 2 Diabetic Mice Infected with West Nile Virus

Clinicoepidemiological data suggest that type 2 diabetes is associated with increased risk of West Nile virus encephalitis (WNVE). However, no experimental studies have elucidated the role of diabetes in WNV neuropathogenesis. Herein, we employed the db/db mouse model to understand WNV immunopathogenesis in diabetics. Nine-week old C57BL/6 WT and db/db mice were inoculated with WNV and mortality, virus burden in the periphery and brain, and antiviral defense responses were analyzed. db/db mice were highly susceptible to WNV disease, exhibited increased tissue tropism and mortality than the wild-type mice, and were unable to clear the infection. Increased and sustained WNV replication was observed in the serum, peripheral tissues and brain of db/db mice, and heightened virus replication in the periphery was correlated with enhanced neuroinvasion and replication of WNV in the brain. WNV infection in db/db mice was associated with enhanced inflammatory response and compromised antiviral immune response characterized by delayed induction of IFN-α, and significantly reduced concentrations of WNV-specific IgM and IgG antibodies. The compromised immune response in db/db mice correlated with increased viremia. These data suggest that delayed immune response coupled with failure to clear the virus leads to increased mortality in db/db mice. In conclusion, this study provides unique mechanistic insight into the immunopathogenesis of WNVE observed in diabetics and can be used to develop therapeutics for the management of WNVE among diabetic patients.

Cyclooxygenase-2 inhibitor blocks the production of West Nile virus-induced neuroinflammatory markers in astrocytes

Inflammatory immune responses triggered initially to clear West Nile virus (WNV) infection later become detrimental and contribute to the pathological processes such as blood–brain barrier (BBB) disruption and neuronal death, thus complicating WNV-associated encephalitis (WNVE). It has been demonstrated previously that WNV infection in astrocytes results in induction of multiple matrix metalloproteinases (MMPs), which mediate BBB disruption. Cyclooxygenase (COX) enzymes and their product, prostaglandin E2 (PGE2), modulate neuroinflammation and regulate the production of multiple inflammatory molecules including MMPs. Therefore, this study determined and characterized the pathophysiological consequences of the expression of COX enzymes in human brain cortical astrocytes (HBCAs) following WNV infection. Whilst COX-1 mRNA expression did not change, WNV infection significantly induced RNA and protein expression of COX-2 in HBCAs. Similarly, PGE2 production was also enhanced significantly in infected HBCAs and was blocked in the presence of the COX-2-specific inhibitor NS-398, thus suggesting that COX-2, and not COX-1, was the source of the increased PGE2. Treatment of infected HBCAs with NS-398 attenuated the expression of MMP-1, -3 and -9 in a dose-dependent manner. Similarly, expression of interleukin-1β, -6 and -8, which were markedly elevated in infected HBCAs, exhibited a significant reduction in their levels in the presence of NS-398. These results provide direct evidence that WNV-induced COX-2/PGE2 is involved in modulating the expression of multiple neuroinflammatory mediators, thereby directly linking COX-2 with WNV disease pathogenesis. The ability of COX-2 inhibitors to modulate WNV-induced COX-2 and PGE2 signalling warrants further investigation in an animal model as a potential approach for clinical management of neuroinflammation associated with WNVE.

Triggering receptor expressed on myeloid cells-1 (TREM-1): a new player in antiviral immunity?

The triggering receptor expressed on myeloid cells (TREM) family of protein receptors is rapidly emerging as a critical regulator of a diverse array of cellular functions, including amplification of inflammation. Although the ligand(s) for TREM have not yet been fully identified, circumstantial evidence indicates that danger- and pathogen-associated molecular patterns (DAMPs and PAMPs) can induce cytokine production via TREM-1 activation. The discovery of novel functions of TREM, such as regulation of T-cell proliferation and activation of antigen-presenting cells, suggests a larger role of TREM proteins in modulation of host immune responses to microbial pathogens, such as bacteria and fungi. However, the significance of TREM signaling in innate immunity to virus infections and the underlying mechanisms remain largely unclear. The nature and intensity of innate immune responses, specifically production of type I interferon and inflammatory cytokines is a crucial event in dictating recovery vs. adverse outcomes from virus infections. In this review, we highlight the emerging roles of TREM-1, including synergy with classical pathogen recognition receptors. Based on the literature using viral PAMPs and other infectious disease models, we further discuss how TREM-1 may influence host-virus interactions and viral pathogenesis. A deeper conceptual understanding of the mechanisms associated with pathogenic and/or protective functions of TREM-1 in antiviral immunity is essential to develop novel therapeutic strategies for the control of virus infection by modulating innate immune signaling.