Maureen H. Richards

Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.

Experimental autoimmune encephalomyelitis (EAE) and Theilers Murine Encephalitis Virus-Induced Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question being asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed. In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro proliferation, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assessment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.

Virus Expanded Regulatory T Cells Control Disease Severity in the Theiler’s Virus Mouse Model of MS

Theilers murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) serves as virus-induced model of chronic progressive multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and a progressive autoimmune demyelinating disease mediated by myelin-specific T cells activated via epitope spreading. In contrast, virus is rapidly cleared by a robust CTL response in TMEV-IDD-resistant C57BL/6 mice. We investigated whether differential induction of regulatory T cells (Tregs) controls susceptibility to TMEV-IDD. Infection of disease-susceptible SJL/J, but not B6 mice, leads to rapid activation and expansion of Tregs resulting in an unfavorable CNS ratio of Treg:Teffector cells. In addition, anti-CD25-induced inactivation of Tregs in susceptible SJL/J, but not resistant B6, mice results in significantly decreased clinical disease concomitant with enhanced anti-viral CD4(+), CD8(+) and antibody responses resulting in decreased CNS viral titers. This is the first demonstration that virus-induced Treg activation regulates susceptibility to autoimmune disease differentially in susceptible and resistant strains of mice and provides a new mechanistic explanation for the etiology of infection-induced autoimmunity.

A critical role for virus-specific CD8(+) CTLs in protection from Theiler's virus-induced demyelination in disease-susceptible SJL mice.

Theilers murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is a relevant mouse model of multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and development of a chronic T cell-mediated autoimmune demyelinating disease triggered via epitope spreading to endogenous myelin epitopes. Potent CNS-infiltrating CD8(+) T cell responses to TMEV epitopes have previously been shown to be induced in both disease-susceptible SJL/J and resistant C57BL/6 mice, in which the virus is rapidly cleared. Specific tolerization of SJL CD8(+) T cells specific for the immunodominant TMEV VP3(159)(-)(166) epitope has no effect on viral load or development of clinical TMEV-IDD, but adoptive transfer of activated CD8(+) VP3(159)(-)(166)-specific T cell blasts shortly after TMEV infection to boost the early anti-viral response leads to clearance of CNS virus and protection from subsequent TMEV-IDD. These studies have important implications for vaccine strategies and treatment of chronic infections in humans.

Human FasL Gene Is a Target of β-Catenin/T-Cell Factor Pathway and Complex FasL Haplotypes Alter Promoter Functions

FasL expression on human immune cells and cancer cells plays important roles in immune homeostasis and in cancer development. Our previous study suggests that polymorphisms in the FasL promoter can significantly affect the gene expression in human cells. In addition to the functional FasL SNP -844C>T (rs763110), three other SNPs (SNP -756A>G or rs2021837, SNP -478A>T or rs41309790, and SNP -205 C>G or rs74124371) exist in the proximal FasL promoter. In the current study, we established three major FasL hyplotypes in humans. Interestingly, a transcription motif search revealed that the FasL promoter possessed two consensus T-cell factor (TCF/LEF1) binding elements (TBEs), which is either polymorphic (SNP -205C>G) or close to the functional SNP -844C>T. Subsequently, we demonstrate that both FasL TBEs formed complexes with the TCF-4 and β-catenin transcription factors in vitro and in vivo. Co-transfection of LEF-1 and β-catenin transcription factors significantly increased FasL promoter activities, suggesting that FasL is a target gene of the β-catenin/T-cell factor pathway. More importantly, we found that the rare allele (-205G) of the polymorphic FasL TBE (SNP -205C>G) failed to bind the TCF-4 transcription factor and that SNP -205 C>G significantly affected the promoter activity. Furthermore, promoter reporter assays revealed that FasL SNP haplotypes influenced promoter activities in human colon cancer cells and in human T cells. Finally, β-catenin knockdown significantly decreased the FasL expression in human SW480 colon cancer cells. Collectively, our data suggest that β-catenin may be involved in FasL gene regulation and that FasL expression is influenced by FasL SNP haplotypes, which may have significant implications in immune response and tumorigenesis.

Loss of galectin-3 decreases the number of immune cells in the subventricular zone and restores proliferation in a viral model of multiple sclerosis.

Multiple sclerosis (MS) frequently starts near the lateral ventricles, which are lined by subventricular zone (SVZ) progenitor cells that can migrate to lesions and contribute to repair. Because MS‐induced inflammation may decrease SVZ proliferation and thus limit repair, we studied the role of galectin‐3 (Gal‐3), a proinflammatory protein. Gal‐3 expression was increased in periventricular regions of human MS in post‐mortem brain samples and was also upregulated in periventricular regions in a murine MS model, Theilers murine encephalomyelitis virus (TMEV) infection. Whereas TMEV increased SVZ chemokine (CCL2, CCL5, CCL, and CXCL10) expression in wild type (WT) mice, this was inhibited in Gal‐3−/− mice. Though numerous CD45+ immune cells entered the SVZ of WT mice after TMEV infection, their numbers were significantly diminished in Gal‐3−/− mice. TMEV also reduced neuroblast and proliferative SVZ cell numbers in WT mice but this was restored in Gal‐3−/− mice and was correlated with increased numbers of doublecortin+ neuroblasts in the corpus callosum. In summary, our data showed that loss of Gal‐3 blocked chemokine increases after TMEV, reduced immune cell migration into the SVZ, reestablished SVZ proliferation and increased the number of progenitors in the corpus callosum. These results suggest Gal‐3 plays a central role in modulating the SVZ neurogenic niches response to this model of MS. GLIA 2016;64:105–121

Dynamic interaction between astrocytes and infiltrating PBMCs in context of neuroAIDS

HIV‐mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells [peripheral blood mononuclear cells (PBMCs)]. We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary‐derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either β‐catenin‐dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10‐fold increase in HLA‐DR and a 5‐fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2‐fold, which was mediated by IFNγ in a Stat‐3‐dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte‐secreted Wnts exert an anti‐HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation. GLIA 2015;63:441–451

Porcupine Is Not Required for the Production of the Majority of Wnts from Primary Human Astrocytes and CD8+ T Cells

Wnts are small secreted glycoproteins that are highly conserved among species. To date, 19 Wnts have been described, which initiate a signal transduction cascade that is either β-catenin dependent or independent, culminating in the regulation of hundreds of target genes. Extracellular release of Wnts is dependent on lipidation of Wnts by porcupine, a membrane-bound-O-acyltransferase protein in the endoplasmic reticulum. Studies demonstrating the requirement of porcupine for Wnts production are based on cell line and non-human primary cells. We evaluated the requirement for porcupine for Wnts production in human primary astrocytes and CD8+ T cells. Using IWP-2, an inhibitor of porcupine, or siRNA targeting porcupine, we demonstrate that porcupine is not required for the release of Wnt 1, 3, 5b, 6,7a, 10b, and 16a. While IWP had no effect on Wnt 2b release, knockdown of porcupine by siRNA reduced Wnt 2b release by 60%. These data indicate that porcupine-mediated production of Wnts is context dependent and is not required for all Wnts production, suggesting that alternative mechanisms exist for Wnts production.

β-Catenin/TCF-4 Signaling Regulates Susceptibility of Macrophages and Resistance of Monocytes to HIV-1 Productive Infection

Cells of the monocyte/macrophage lineage are an important target for HIV-1 infection. They are often at anatomical sites linked to HIV-1 transmission and are an important vehicle for disseminating HIV-1 throughout the body, including the central nervous system. Monocytes do not support extensive productive HIV-1 replication, but they become more susceptible to HIV-1infection as they differentiate into macrophages. The mechanisms guiding susceptibility of HIV-1 replication in monocytes versus macrophages are not entirely clear. We determined whether endogenous activity of β-catenin signaling impacts differential susceptibility of monocytes and monocyte-derived macrophages (MDMs) to productive HIV-1 replication. We show that monocytes have an approximately 4-fold higher activity of β-catenin signaling than MDMs. Inducing β-catenin in MDMs suppressed HIV-1 replication by 5-fold while inhibiting endogenous β-catenin signaling in monocytes by transfecting with a dominant negative mutant for the downstream effector of β- catenin (TCF-4) promoted productive HIV-1 replication by 6-fold. These findings indicate that β-catenin/TCF-4 is an important pathway for restricted HIV-1 replication in monocytes and plays a significant role in potentiating HIV-1 replication as monocytes differentiate into macrophages. Targeting this pathway may provide a novel strategy to purge the latent reservoir from monocytes/macrophages, especially in sanctuary sites for HIV-1 such as the central nervous system.

HIV and drug abuse mediate astrocyte senescence in a β‐catenin‐dependent manner leading to neuronal toxicity

Emerging evidence suggests that cell senescence plays an important role in aging‐associated diseases including neurodegenerative diseases. HIV leads to a spectrum of neurologic diseases collectively termed HIV‐associated neurocognitive disorders (HAND). Drug abuse, particularly methamphetamine (meth), is a frequently abused psychostimulant among HIV+ individuals and its abuse exacerbates HAND. The mechanism by which HIV and meth lead to brain cell dysregulation is not entirely clear. In this study, we evaluated the impact of HIV and meth on astrocyte senescence using in vitro and several animal models. Astrocytes constitute up to 50% of brain cells and play a pivotal role in marinating brain homeostasis. We show here that HIV and meth induce significant senescence of primary human fetal astrocytes, as evaluated by induction of senescence markers (β‐galactosidase and p16INK4A), senescence‐associated morphologic changes, and cell cycle arrest. HIV‐ and meth‐mediated astrocyte senescence was also demonstrated in three small animal models (humanized mouse model of HIV/NSG‐huPBMCs, HIV‐transgenic rats, and in a meth administration rat model). Senescent astrocytes in turn mediated neuronal toxicity. Further, we show that β‐catenin, a pro‐survival/proliferation transcriptional co‐activator, is downregulated by HIV and meth in human astrocytes and this downregulation promotes astrocyte senescence while induction of β‐catenin blocks HIV‐ and meth‐mediated astrocyte senescence. These studies, for the first time, demonstrate that HIV and meth induce astrocyte senescence and implicate the β‐catenin pathway as potential therapeutic target to overcome astrocyte senescence.

Migration of CD8+ T Cells into the Central Nervous System Gives Rise to Highly Potent Anti-HIV CD4dimCD8bright T Cells in a Wnt Signaling–Dependent Manner

The role of CD8+ T cells in HIV control in the brain and the consequences of such control are unclear. Approximately 3% of peripheral CD8+ T cells dimly express CD4 on their surface. This population is known as CD4dimCD8bright T cells. We evaluated the role of CD4dimCD8bright and CD8 single positive T cells in HIV-infected brain using NOD/SCID/IL-2rcγ−/− mice reconstituted with human PBMCs (NSG-huPBMC). All three T cell populations (CD4 single positive, CD8 single positive, and CD4dimCD8bright) were found in NSG-huPBMC mouse brain within 2 wk of infection. Wnts secreted from astrocytes induced CD4dimCD8bright T cells by 2-fold in vitro. Injection of highly purified CD8 single positive T cells into mouse brain induced CD4dimCD8bright T cells by 10-fold, which were proliferative and exhibited a terminally differentiated effector memory phenotype. Brain CD4dimCD8bright T cells from HIV-infected mice exhibited anti-HIV–specific responses, as demonstrated by induction of CD107ab post exposure to HIV peptide–loaded targets. Further, higher frequency of CD4dimCD8bright T cells (R = −0.62; p ≤ 0.001), but not CD8 single positive T cells (R = −0.24; p ≤ 0.27), negatively correlated with HIV gag mRNA transcripts in HIV-infected NSG-huPBMC brain. Together, these studies indicate that single positive CD8+ T cells entering the CNS during HIV infection can give rise to CD4dimCD8bright T cells, likely through a Wnt signaling–dependent manner, and that these cells are associated with potent anti-HIV control in the CNS. Thus, CD4dimCD8bright T cells are capable of HIV control in the CNS and may offer protection against HIV-associated neurocognitive disorders.