Amber M. Paul

Neuroinflammation and Endoplasmic Reticulum Stress Are Coregulated by Crocin To Prevent Demyelination and Neurodegeneration

Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is used by cells to adapt to intercellular and intracellular changes. Moreover, ER stress is closely linked to inflammatory pathways. We hypothesized that ER stress is an integral component of neuroinflammation and contributes to the development of neurological diseases. In autopsied brain specimens from multiple sclerosis (MS) and non-MS patients, XBP-1 spliced variant (XBP-1/s) was increased in MS brains (p < 0.05) and was correlated with the expression of the human endogenous retrovirus-W envelope transcript, which encodes the glycoprotein, Syncytin-1 (p < 0.05). In primary human fetal astrocytes transfected with a Syncytin-1–expressing plasmid, XBP-1/s, BiP, and NOS2 were induced, which was suppressed by crocin treatment (p < 0.05). Crocin also protected oligodendrocytes exposed to cytotoxic supernatants derived from Syncytin-1–expressing astrocytes (p < 0.05) and NO-mediated oligodendrocytotoxicity (p < 0.05). During experimental autoimmune encephalomyelitis (EAE), the transcript levels of the ER stress genes XBP-1/s, BiP, PERK, and CHOP were increased in diseased spinal cords compared with healthy littermates (p < 0.05), although CHOP expression was not involved in the EAE disease phenotype. Daily treatment with crocin starting on day 7 post-EAE induction suppressed ER stress and inflammatory gene expression in spinal cords (p < 0.05), which was accompanied by preserved myelination and axonal density, together with reduced T cell infiltration and macrophage activation. EAE-associated neurobehavioral deficits were also ameliorated by crocin treatment (p < 0.05). These findings underscored the convergent roles of pathogenic ER stress and immune pathways in neuroinflammatory disease and point to potential therapeutic applications for crocin.

Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro.

Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP-siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP-siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.

Mouse embryonic stem cells are deficient in type I interferon expression in response to viral infections and double-stranded RNA.

Background: The antiviral mechanisms are not known in mESCs. Results: mESCs are susceptible to viral infections and dsRNA-inhibited cell proliferation but do not express type I interferons. Conclusion: mESCs have underdeveloped mechanisms for type I interferon expression. Significance: The findings are important for understanding the development of antiviral mechanisms in ESCs and stem cell physiology. Embryonic stem cells (ESCs) are considered to be a promising cell source for regenerative medicine because of their unlimited capacity for self-renewal and differentiation. However, little is known about the innate immunity in ESCs and ESC-derived cells. We investigated the responses of mouse (m)ESCs to three types of live viruses as follows: La Crosse virus, West Nile virus, and Sendai virus. Our results demonstrated mESCs were susceptible to viral infection, but they were unable to express type I interferons (IFNα and IFNβ, IFNα/β), which differ from fibroblasts (10T1/2 cells) that robustly express IFNα/β upon viral infections. The failure of mESCs to express IFNα/β was further demonstrated by treatment with polyIC, a synthetic viral dsRNA analog that strongly induced IFNα/β in 10T1/2 cells. Although polyIC transiently inhibited the transcription of pluripotency markers, the stem cell morphology was not significantly affected. However, polyIC can induce dsRNA-activated protein kinase in mESCs, and this activation resulted in a strong inhibition of cell proliferation. We conclude that the cytosolic receptor dsRNA-activated protein kinase is functional, but the mechanisms that mediate type I IFN expression are deficient in mESCs. This conclusion is further supported by the findings that the major viral RNA receptors are either expressed at very low levels (TLR3 and MDA5) or may not be active (retinoic acid-inducible gene I) in mESCs.

Neurosteroid-mediated regulation of brain innate immunity in HIV/AIDS: DHEA-S suppresses neurovirulence

Neurosteroids are cholesterol‐derived molecules synthesized within the brain, which exert trophic and protective actions. Infection by human and feline immunodeficiency viruses (HIV and FIV, respectively) causes neuroinflammation and neurodegeneration, leading to neurological deficits. Secretion of neuroinflammatory host and viral factors by glia and infiltrating leukocytes mediates the principal neuropathogenic mechanisms during lentivirus infections, although the effect of neurosteroids on these processes is unknown. We investigated the interactions between neurosteroid‐mediated effects and lentivirus infection outcomes. Analyses of HIV‐infected (HIV+) and uninfected human brains disclosed a reduction in neurosteroid synthesis enzyme expression. Human neurons exposed to supernatants from HIV+ macrophages exhibited suppressed enzyme expression without reduced cellular viability. HIV+ human macrophages treated with sulfated dehydroepiandrosterone (DHEA‐S) showed suppression of inflammatory gene (IL‐1β, IL‐6, TNF‐α) expression. FIV‐infected (FIV+) animals treated daily with 15 mg/kg body weight. DHEA‐S treatment reduced inflammatory gene transcripts (IL‐1β, TNF‐α, CD3ε, GFAP) in brain compared to vehicle‐(β‐cyclodextrin)‐treated FIV+ animals similar to levels found in vehicle‐treated FIV– animals. DHEA‐S treatment also increased CD4+ T‐cell levels and prevented neurobehavioral deficits and neuronal loss among FIV+ animals, compared to vehicle‐treated FIV+ animals. Reduced neuronal neurosteroid synthesis was evident in lentivirus infections, but treatment with DHEA‐S limited neuroinflammation and prevented neurobehavioral deficits. Neurosteroid‐derived therapies could be effective in the treatment of virus‐ or inflammation‐mediated neurodegeneration.—Maingat, F. G., Polyak, M. J., Paul, A. M., Vivithanaporn, P., Noorbakhsh, F., Ahboucha S., Baker, G. B., Pearson, K., Power, C. Neurosteroid‐mediated regulation of brain innate immunity in HIV/AIDS: DHEA‐S suppresses neurovirulence. FASEB J. 27, 725–737 (2013). www.fasebj.org

Antiviral Responses in Mouse Embryonic Stem Cells DIFFERENTIAL DEVELOPMENT OF CELLULAR MECHANISMS IN TYPE I INTERFERON PRODUCTION AND RESPONSE

Background: mESCs are deficient in type I IFN expression. Results: mESCs can respond to type I IFNs and express interferon-stimulated genes. Conclusion: mESCs are unable to express type I IFNs but can respond to type I IFNs. Significance: The findings are important for understanding the antiviral mechanisms and innate immunity in ESCs. We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFNs) in response to viral infection and synthetic viral RNA analogs (Wang, R., Wang, J., Paul, A. M., Acharya, D., Bai, F., Huang, F., and Guo, Y. L. (2013) J. Biol. Chem. 288, 15926–15936). Here, we report that mESCs are able to respond to type I IFNs, express IFN-stimulated genes, and mediate the antiviral effect of type I IFNs against La Crosse virus and chikungunya virus. The major signaling components in the IFN pathway are expressed in mESCs. Therefore, the basic molecular mechanisms that mediate the effects of type I IFNs are functional in mESCs; however, these mechanisms may not yet be fully developed as mESCs express lower levels of IFN-stimulated genes and display weaker antiviral activity in response to type I IFNs when compared with fibroblasts. Further analysis demonstrated that type I IFNs do not affect the stem cell state of mESCs. We conclude that mESCs are deficient in type I IFN expression, but they can respond to and mediate the cellular effects of type I IFNs. These findings represent unique and uncharacterized properties of mESCs and are important for understanding innate immunity development and ESC physiology.

HIV-1 Nef expression in microglia disrupts dopaminergic and immune functions with associated mania-like behaviors.

BACKGROUND Neuropsychiatric disorders during HIV/AIDS are common although the contribution of HIV-1 infection within the brain, and in particular individual HIV-1 proteins, to the development of these brain disorders is unknown. Herein, an in vivo transgenic mouse model was generated in which the HIV-1 Nef protein was expressed in microglia cells, permitting investigation of neurobehavioral phenotypes and associated cellular and molecular properties. METHODS Transgenic (Tg) mice that expressed full length HIV-1 nef under the control of the c-fms promoter and wildtype (Wt) littermates were investigated using different measures of neurobehavioral performance including locomotory, forced swim (FST), elevated plus maze (EPM) and T-maze tests. Host gene and transgene expression were assessed by RT-PCR, immunoblotting, enzymatic activity and immunohistochemistry. Biogenic amine levels were measured by HPLC with electrochemical detection. RESULTS Tg animals exhibited Nef expression in brain microglia and cultured macrophages. Tg males displayed hyperactive behaviors including augmented locomotor activity, decreased immobility in the FST and increased open-arm EPM exploration compared to Wt littermates (p<0.05). Tg animals showed increased CCL2 expression with concurrent IFN-α suppression in striatum compared with Wt littermates (p<0.05). Dopamine levels, MAO activity and the dopamine transporter (DAT) expression were reduced in the striatum of Tg animals (p<0.05). CONCLUSIONS HIV-1 Nef expression in microglia induced CCL2 expression together with disrupting striatal dopaminergic transmission, resulting in hyperactive behaviors which are observed in mania and other psychiatric comorbidities among HIV-infected persons. These findings emphasize the selective effects of individual viral proteins in the brain and their participation in neuropathogenesis.

Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity

The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.

Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal) or C6/36 cells (mosquito) through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKVmos) has slower replication than mammalian cell-derived CHIKV (CHIKVvero), when tested in both human and murine cell lines. Consistent with this, CHIKVmos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKVmos produces a lower level of viremia and less severe footpad swelling when compared with CHIKVvero. Interestingly, CHIKVmos has impaired ability to bind to glycosaminoglycan (GAG) receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKVmos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKVvero after a single passage in mammalian cells. Furthermore, CHIKVvero and CHIKVmos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells.

An Ultrasensitive Electrogenerated Chemiluminescence-Based Immunoassay for Specific Detection of Zika Virus

Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research.

Does Astroglial Protein S100B Contribute to West Nile Neuro-Invasive Syndrome?

The clinical spectrum of West Nile Virus (WNV) infection ranges from a flu-like febrile condition to a more severe neuro-invasive disease that can cause death. The exact mechanism of neurodegeneration in neuro-invasive form of WNV infection has not been elucidated; however, a destructive role played by glial cells in promoting WNV mediated neurotoxicity has widely been speculated. The clinical studies revealed that the astroglial protein S100B is significantly elevated in the blood and CSF of patients with WNV infection, even in the absence of neuro-invasive disease. Therefore, the present study was designed to explore the potential role of S100B in the pathophysiology of WNV infection. The overarching hypothesis was that WNV primes astroglia to release S100B protein, which leads to a cascade of events that may have deleterious effects in both acute and chronic stages of WNV disease. To justify our hypothesis, we first ascertained increased levels of S100B in post-mortem tissue samples from WNV patients. Next, we looked at the effects of UV-inactivated WNV particles on astroglia using astroglial cell lines or primary cultures. Astroglial activation was measured as an increase in the expression of S100B and was analyzed by immunofluorescence and real-time PCR. Further, the in vitro effects of purified S100B protein on neutrophil migration and glutamate uptake were also determined in astroglial cell lines or primary cultures. We found that incubation of cultured astroglial cells with UV-inactivated WNV particles caused induction of S100B both at the mRNA and protein levels. Varying concentrations of S100B stimulated neutrophil migration in vitro. In addition, varying amounts of S100B caused inhibition of glutamate uptake in astroglia in a dose-dependent manner. Our data suggest that inactivated WNV particles are capable of inducing S100B synthesis in astroglia in vitro. We speculate that S100B release by activated astroglia may have multiple roles in the pathophysiology of WNV neuro-invasive disease, including induction of neutrophil migration to the sites where blood brain barrier is disrupted as well as glutamate neurotoxicity. To further elucidate the WNV-S100B neurotoxic pathway, in vivo studies using mouse models are warranted.