Dimethyl Fumarate, an Approved Multiple Sclerosis Treatment, Reduces Brain Oxidative Stress in SIV-Infected Rhesus Macaques: Potential Therapeutic Repurposing for HIV Neuroprotection.

Dimethyl fumarate (DMF), an antioxidant/anti-inflammatory drug approved for the treatment of multiple sclerosis, induces antioxidant enzymes, in part through transcriptional upregulation. We hypothesized that DMF administration to simian immunodeficiency virus (SIV)-infected rhesus macaques would induce antioxidant enzyme expression and reduce oxidative injury and inflammation throughout the brain. Nine SIV-infected, CD8+-T-lymphocyte-depleted rhesus macaques were studied. Five received oral DMF prior to the SIV infection and through to the necropsy day. Protein expression was analyzed in 11 brain regions, as well as the thymus, liver, and spleen, using Western blot and immunohistochemistry for antioxidant, inflammatory, and neuronal proteins. Additionally, oxidative stress was determined in brain sections using immunohistochemistry (8-OHdG, 3NT) and optical redox imaging of oxidized flavoproteins containing flavin adenine dinucleotide (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The DMF treatment was associated with no changes in virus replication; higher expressions of the antioxidant enzymes NQO1, GPX1, and HO-1 in the brain and PRDX1 and HO-2 in the spleen; lower levels of 8-OHdG and 3NT; a lower optical redox ratio. The DMF treatment was also associated with increased expressions of cell-adhesion molecules (VCAM-1, ICAM-1) and no changes in HLA-DR, CD68, GFAP, NFL, or synaptic proteins. The concordantly increased brain antioxidant enzyme expressions and reduced oxidative stress in DMF-treated SIV-infected macaques suggest that DMF could limit oxidative stress throughout the brain through effective induction of the endogenous antioxidant response. We propose that DMF could potentially induce neuroprotective brain responses in persons living with HIV.

Induction of Heme Oxygenase-1 Deficiency and Associated Glutamate-Mediated Neurotoxicity Is a Highly Conserved HIV Phenotype of Chronic Macrophage Infection That Is Resistant to Antiretroviral Therapy.

UNLABELLED: Expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) is significantly reduced in the brain prefrontal cortex of HIV-positive individuals with HIV-associated neurocognitive disorders (HAND). Furthermore, this HO-1 deficiency correlates with brain viral load, markers of macrophage activation, and type I interferon responses. In vitro, HIV replication in monocyte-derived macrophages (MDM) selectively reduces HO-1 protein and RNA expression and induces production of neurotoxic levels of glutamate; correction of this HO-1 deficiency reduces neurotoxic glutamate production without an effect on HIV replication. We now demonstrate that macrophage HO-1 deficiency, and the associated neurotoxin production, is a conserved feature of infection with macrophage-tropic HIV-1 strains that correlates closely with the extent of replication, and this feature extends to HIV-2 infection. We further demonstrate that this HO-1 deficiency does not depend specifically upon the HIV-1 accessory genes nef, vpr, or vpu but rather on HIV replication, even when markedly limited. Finally, antiretroviral therapy (ART) applied to MDM after HIV infection is established does not prevent HO-1 loss or the associated neurotoxin production. This work defines a predictable relationship between HIV replication, HO-1 loss, and neurotoxin production in MDM that likely reflects processes in place in the HIV-infected brains of individuals receiving ART. It further suggests that correcting this HO-1 deficiency in HIV-infected MDM could provide neuroprotection above that provided by current ART or proposed antiviral therapies directed at limiting Nef, Vpr, or Vpu functions. The ability of HIV-2 to reduce HO-1 expression suggests that this is a conserved phenotype among macrophage-tropic human immunodeficiency viruses that could contribute to neuropathogenesis. IMPORTANCE: The continued prevalence of HIV-associated neurocognitive disorders (HAND) underscores the need for adjunctive therapy that targets the neuropathological processes that persist in antiretroviral therapy (ART)-treated HIV-infected individuals. To this end, we previously identified one such possible process, a deficiency of the antioxidative and anti-inflammatory enzyme heme oxygenase-1 (HO-1) in the brains of individuals with HAND. In the present study, our findings suggest that the HO-1 deficiency associated with excess glutamate production and neurotoxicity in HIV-infected macrophages is a highly conserved phenotype of macrophage-tropic HIV strains and that this phenotype can persist in the macrophage compartment in the presence of ART. This suggests a plausible mechanism by which HIV infection of brain macrophages in ART-treated individuals could exacerbate oxidative stress and glutamate-induced neuronal injury, each of which is associated with neurocognitive dysfunction in infected individuals. Thus, therapies that rescue the HO-1 deficiency in HIV-infected individuals could provide additional neuroprotection to ART.

Heme oxygenase-1 deficiency accompanies neuropathogenesis of HIV-associated neurocognitive disorders.

Heme oxygenase-1 (HO-1) is an inducible, detoxifying enzyme that is critical for limiting oxidative stress, inflammation, and cellular injury within the CNS and other tissues. Here, we demonstrate a deficiency of HO-1 expression in the brains of HIV-infected individuals. This HO-1 deficiency correlated with cognitive dysfunction, HIV replication in the CNS, and neuroimmune activation. In vitro analysis of HO-1 expression in HIV-infected macrophages, a primary CNS HIV reservoir along with microglia, demonstrated a decrease in HO-1 as HIV replication increased. HO-1 deficiency correlated with increased culture supernatant glutamate and neurotoxicity, suggesting a link among HIV infection, macrophage HO-1 deficiency, and neurodegeneration. HO-1 siRNA knockdown and HO enzymatic inhibition in HIV-infected macrophages increased supernatant glutamate and neurotoxicity. In contrast, increasing HO-1 expression through siRNA derepression or with nonselective pharmacologic inducers, including the CNS-penetrating drug dimethyl fumarate (DMF), decreased supernatant glutamate and neurotoxicity. Furthermore, IFN-γ, which is increased in CNS HIV infection, reduced HO-1 expression in cultured human astrocytes and macrophages. These findings indicate that HO-1 is a protective host factor against HIV-mediated neurodegeneration and suggest that HO-1 deficiency contributes to this degeneration. Furthermore, these results suggest that HO-1 induction in the CNS of HIV-infected patients on antiretroviral therapy could potentially protect against neurodegeneration and associated cognitive dysfunction.

Dimethyl fumarate, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and macrophage-mediated neurotoxicity: a novel candidate for HIV neuroprotection.

Despite antiretroviral therapy (ART), HIV infection promotes cognitive dysfunction and neurodegeneration through persistent inflammation and neurotoxin release from infected and/or activated macrophages/microglia. Furthermore, inflammation and immune activation within both the CNS and periphery correlate with disease progression and morbidity in ART-treated individuals. Accordingly, drugs targeting these pathological processes in the CNS and systemic compartments are needed for effective, adjunctive therapy. Using our in vitro model of HIV-mediated neurotoxicity, in which HIV-infected monocyte-derived macrophages release excitatory neurotoxins, we show that HIV infection dysregulates the macrophage antioxidant response and reduces levels of heme oxygenase-1 (HO-1). Furthermore, restoration of HO-1 expression in HIV-infected monocyte-derived macrophages reduces neurotoxin release without altering HIV replication. Given these novel observations, we have identified dimethyl fumarate (DMF), used to treat psoriasis and showing promising results in clinical trials for multiple sclerosis, as a potential neuroprotectant and HIV disease-modifying agent. DMF, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and neurotoxin release. Two distinct mechanisms are proposed: inhibition of NF-κB nuclear translocation and signaling, which could contribute to the suppression of HIV replication, and induction of HO-1, which is associated with decreased neurotoxin release. Finally, we found that DMF attenuates CCL2-induced monocyte chemotaxis, suggesting that DMF could decrease recruitment of activated monocytes to the CNS in response to inflammatory mediators. We propose that dysregulation of the antioxidant response during HIV infection drives macrophage-mediated neurotoxicity and that DMF could serve as an adjunctive neuroprotectant and HIV disease modifier in ART-treated individuals.

A conserved dileucine motif mediates clathrin and AP-2-dependent endocytosis of the HIV-1 envelope protein.

During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxxØ motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV(mac239) Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV(HxB2) also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxxØ motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV(mac239) Env GYxxØ motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions.

Simian immunodeficiency virus (SIV)/immunoglobulin G immune complexes in SIV-infected macaques block detection of CD16 but not cytolytic activity of natural killer cells.

Natural killer cells are components of the innate immune system that play an important role in eliminating viruses and malignant cells. Using simian immunodeficiency virus (SIV) infection of macaques as a model, flow cytometry revealed a gradual loss of CD16+ NK cell numbers that was associated with disease progression. Of note, the apparent loss of NK cells was detected in whole-blood samples but not in isolated peripheral blood mononuclear cells (PBMC), suggesting that an inhibitor(s) of the antibody used to detect CD16, the low-affinity immunoglobulin G (IgG) receptor, was present in blood but was removed during PBMC isolation. (Actual decreases in CD16+ cell numbers in PBMC generally were not detected until animals became lymphopenic.) The putative decrease in CD16+ cell numbers in whole blood correlated with increasing SIV-specific antibody titers and levels of plasma virion RNA. With the addition of increasing amounts of plasma from progressor, but not nonprogressor, macaques to PBMC from an uninfected animal, the apparent percentage of CD16+ cells and the mean fluorescence intensity of antibodies binding to CD16 declined proportionately. A similar decrease was observed with the addition of monomeric IgG (mIgG) and IgG immune complexes (IgG-ICs) purified from the inhibitory plasma samples; some of the ICs contained SIV p27(gag) antigen and/or virions. Of interest, addition of purified IgG/IgG-ICs to NK cell lytic assays did not inhibit killing of K562 cells. These results indicate that during progressive SIV and, by inference, human immunodeficiency virus disease, CD16+ NK cell numbers can be underestimated, or the cells not detected at all, when one is using a whole-blood fluorescence-activated cell sorter assay and a fluorochrome-labeled antibody that can be blocked by mIgG or IgG-ICs. Although this blocking had no apparent effect on NK cell activity in vitro, the in vivo effects are unknown.