Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism.

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR).

Dysregulated brain regulatory T cells fail to control reactive gliosis following repeated antigen stimulation.

This study was undertaken to investigate the role of CD4+FoxP3+ regulatory T cells (Tregs) in regulating neuroinflammation during viral Ag-challenge and re-challenge. CD8 lymphocytes persisting within tissues are designated tissue-resident memory T cells (T), within brain: bT. Reactivation of bT with T cell epitope peptides generates rapid antiviral recall, but repeated stimulation leads to cumulative dysregulation of microglial activation, proliferation, and prolonged neurotoxic mediator production.

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism.

Unlabelled: The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses EBV and KSHV and the alpha-herpesviruses HSV-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR).

Regulatory T-Cells Suppress Cytotoxic T Lymphocyte Responses against Microglia.

Regulatory T-cells (Tregs) play pivotal roles during infection, cancer, and autoimmunity. In our previous study, we demonstrated a role for the PD-1:PD-L1 pathway in controlling cytolytic responses of CD8 T lymphocytes against microglial cells presenting viral peptides. In this study, we investigated the role of Tregs in suppressing CD8 T-cell-mediated cytotoxicity against primary microglial cells.

Differential Cytokine-Induced Responses of Polarized Microglia.

The role of select pro- and anti-inflammatory mediators in driving microglial cell polarization into classically (M1), or alternatively, (M2) activated states, as well as the subsequent differential responses of these induced phenotypes, was examined. Expression of PD-L1, MHC-II, MHC-I, arginase 1 (Arg-1), and inducible nitric oxide synthase (iNOS) was assessed using multi-color flow cytometry. We observed that both pro- and anti-inflammatory mediators induced PD-L1 expression on non-polarized microglia.

Dysregulated Microglial Cell Activation and Proliferation Following Repeated Antigen Stimulation.

Upon reactivation of quiescent neurotropic viruses antigen (Ag)-specific brain resident-memory CD8+ T-cells (bT) may respond to -produced viral Ag through the rapid release of IFN-γ, which drives subsequent interferon-stimulated gene expression in surrounding microglia. Through this mechanism, a small number of adaptive bT may amplify responses to viral reactivation leading to an organ-wide innate protective state. Over time, this brain-wide innate immune activation likely has cumulative neurotoxic and neurocognitive consequences.

Programmed death ligand-1 induction restrains the cytotoxic T lymphocyte response against microglia.

Microglial cells are the main reservoir for HIV-1 within the brain and potential exists for negative immune checkpoint blockade therapies to purge this viral reservoir. Here, we investigated cytolytic responses of CD8 T lymphocytes against microglia loaded with peptide epitopes. Initially, flow cytometric analysis demonstrated efficient killing of HIV-1 p24 AI9 or YI9 peptide-loaded splenocytes in MHC-matched recipients.

Recall Responses from Brain-Resident Memory CD8 T Cells (bT) Induce Reactive Gliosis.

HIV-associated neurocognitive disorders (HAND) persist even during effective combination antiretroviral therapy (cART). Although the cause of HAND is unknown, studies link chronic immune activation, neuroinflammation, and cerebrospinal fluid viral escape to disease progression. In this study, we tested the hypothesis that specific, recall immune responses from brain-resident memory T cells (bT) could activate glia and induce neurotoxic mediators.

Antiallodynic Effects of Cannabinoid Receptor 2 (CBR) Agonists on Retrovirus Infection-Induced Neuropathic Pain.

The most common neurological complication in patients receiving successful combination antiretroviral therapy (cART) is peripheral neuropathic pain. Data show that distal symmetric polyneuropathy (DSP) also develops along with murine acquired immunodeficiency syndrome (MAIDS) after infection with the LP-BM5 murine retrovirus mixture. Links between cannabinoid receptor 2 (CBR) and peripheral neuropathy have been established in animal models using nerve transection, chemotherapy-induced pain, and various other stimuli.

Glial Cell Expression of PD-L1.

The programmed death (PD)-1/PD-L1 pathway is a well-recognized negative immune checkpoint that results in functional inhibition of T-cells. Microglia, the brain-resident immune cells are vital for pathogen detection and initiation of neuroimmune responses. Moreover, microglial cells and astrocytes govern the activity of brain-infiltrating antiviral T-cells through upregulation of PD-L1 expression.

Brain-Resident T Cells Following Viral Infection.

Activated CD8 lymphocytes infiltrate the brain in response to many viral infections; where some remain stationed long term as memory T cells. Brain-resident memory T cells (bT) are positioned to impart immediate defense against recurrent or reactivated infection. The cytokine and chemokine milieu present within a tissue is critical for T generation and retention; and reciprocal interactions exist between brain-resident glia and bT.

Reactive glia promote development of CD103 CD69 CD8 T-cells through programmed cell death-ligand 1 (PD-L1).

Introduction: Previous work from our laboratory has demonstrated in vivo persistence of CD103 CD69 brain resident memory CD8 T-cells (bT ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these T cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-γ-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway.

Methods: We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8 T-cells obtained from wild type mice to investigate the role of glial cells in the development of bT .

Nitrosative damage during retrovirus infection-induced neuropathic pain.

Background: Peripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35-50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation.

The PD-1: PD-L1 pathway promotes development of brain-resident memory T cells following acute viral encephalitis.

Background: Previous work from our laboratory has demonstrated that during acute viral brain infection, glial cells modulate antiviral T cell effector responses through the PD-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Here, we evaluated the PD-1: PD-L1 pathway in development of brain-resident memory T cells (bT) following murine cytomegalovirus (MCMV) infection.

Methods: Flow cytometric analysis of immune cells was performed at 7, 14, and 30 days post-infection (dpi) to assess the shift of brain-infiltrating CD8 T cell populations from short-lived effector cells (SLEC) to memory precursor effector cells (MPEC), as well as generation of bT.

Modulation of Microglial Cell Fcγ Receptor Expression Following Viral Brain Infection.

Fcγ receptors (FcγRs) for IgG couple innate and adaptive immunity through activation of effector cells by antigen-antibody complexes. We investigated relative levels of activating and inhibitory FcγRs on brain-resident microglia following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that activating FcγRs were expressed maximally at 5 d post-infection (dpi), while the inhibitory receptor (FcγRIIB) remained highly elevated during both acute and chronic phases of infection.

Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection.

Background: Chemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19(+) B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection.

Methods: Flow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells.

Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection.

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue.

Chronic reactive gliosis following regulatory T cell depletion during acute MCMV encephalitis.

Long-term, persistent central nervous system inflammation is commonly seen following brain infection. Using a murine model of viral encephalitis (murine cytomegalovirus, MCMV) we have previously shown that post-encephalitic brains are maintained in an inflammatory state consisting of glial cell reactivity, retention of brain-infiltrating tissue-resident memory CD8 T-cells, and long-term persistence of antibody-producing cells of the B-lineage. Here, we report that this neuroinflammation occurs concomitantly with accumulation and retention of immunosuppressive regulatory T-cells (Tregs), and is exacerbated following their ablation.

Infiltrating regulatory B cells control neuroinflammation following viral brain infection.

Previous studies have demonstrated the existence of a subset of B lymphocytes, regulatory B cells (Bregs), which modulate immune function. In this study, in vivo and in vitro experiments were undertaken to elucidate the role of these Bregs in controlling neuroinflammation following viral brain infection. We used multicolor flow cytometry to phenotype lymphocyte subpopulations infiltrating the brain, along with in vitro cocultures to assess their anti-inflammatory and immunoregulatory roles.

Activated CD8+ T lymphocytes inhibit neural stem/progenitor cell proliferation: role of interferon-gamma.

The ability of neural stem/progenitor cells (NSCs) to self-renew, migrate to damaged sites, and differentiate into neurons has renewed interest in using them in therapies for neurodegenerative disorders. Neurological diseases, including viral infections of the brain, are often accompanied by chronic inflammation, whose impact on NSC function remains unexplored. We have previously shown that chronic neuroinflammation, a hallmark of experimental herpes simplex encephalitis (HSE) in mice, is dominated by brain-infiltrating activated CD8 T-cells.

Glial cells suppress postencephalitic CD8+ T lymphocytes through PD-L1.

Engagement of the programmed death (PD)-1 receptor on activated cells by its ligand (PD-L1) is a mechanism for suppression of activated T-lymphocytes. Microglia, the resident inflammatory cells of the brain, are important for pathogen detection and initiation of innate immunity, however, a novel role for these cells as immune regulators has also emerged. PD-L1 on microglia has been shown to negatively regulate T-cell activation in models of multiple sclerosis and acute viral encephalitis.

T-cell reconstitution during murine acquired immunodeficiency syndrome (MAIDS) produces neuroinflammation and mortality in animals harboring opportunistic viral brain infection.

Background: Highly active antiretroviral therapy (HAART) restores inflammatory immune responses in AIDS patients which may unmask previous subclinical infections or paradoxically exacerbate symptoms of opportunistic infections. In resource-poor settings, 25% of patients receiving HAART may develop CNS-related immune reconstitution inflammatory syndrome (IRIS). Here we describe a reliable mouse model to study underlying immunopathological mechanisms of CNS-IRIS.

Modulation of experimental herpes encephalitis-associated neurotoxicity through sulforaphane treatment.

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1).

Persistent humoral immune responses in the CNS limit recovery of reactivated murine cytomegalovirus.

Background: Experimental infection of the mouse brain with murine CMV (MCMV) elicits neuroimmune responses that terminate acute infection while simultaneously preventing extensive bystander damage. Previous studies have determined that CD8(+) T lymphocytes are required to restrict acute, productive MCMV infection within the central nervous system (CNS). In this study, we investigated the contribution of humoral immune responses in control of MCMV brain infection.