Brienne McKenzie

HIV-1 Viral Protein R Activates NLRP3 Inflammasome in Microglia: implications for HIV-1 Associated Neuroinflammation

Human Immunodeficiency virus (HIV) enters the brain soon after seroconversion and induces chronic neuroinflammation by infecting and activating brain macrophages. Inflammasomes are cytosolic protein complexes that mediate caspase-1 activation and ensuing cleavage and release of IL-1β and −18 by macrophages. Our group recently showed that HIV-1 infection of human microglia induced inflammasome activation in NLRP3-dependent manner. The HIV-1 viral protein R (Vpr) is an accessory protein that is released from HIV-infected cells, although its effects on neuroinflammation are undefined. Infection of human microglia with Vpr-deficient HIV-1 resulted in reduced caspase-1 activation and IL-1β production, compared to cells infected with a Vpr-encoding HIV-1 virus. Vpr was detected at low nanomolar concentrations in cerebrospinal fluid from HIV-infected patients and in supernatants from HIV-infected primary human microglia. Exposure of human macrophages to Vpr caused caspase-1 cleavage and IL-1β release with reduced cell viability, which was dependent on NLRP3 expression. Increased NLRP3, caspase-1, and IL-1β expression was evident in HIV-1 Vpr transgenic mice compared to wild-type littermates, following systemic immune stimulation. Treatment with the caspase-1 inhibitor, VX-765, suppressed NLRP3 expression with reduced IL-1β expression and associated neuroinflammation. Neurobehavioral deficits showed improvement in Vpr transgenic animals treated with VX-765. Thus, Vpr-induced NLRP3 inflammasome activation, which contributed to neuroinflammation and was abrogated by caspase-1 inhibition. This study provides a new therapeutic perspective for HIV-associated neuropsychiatric disease.

Insulin Treatment Prevents Neuroinflammation and Neuronal Injury with Restored Neurobehavioral Function in Models of HIV/AIDS Neurodegeneration.

HIV-1 infection of the brain causes the neurodegenerative syndrome HIV-associated neurocognitive disorders (HAND), for which there is no specific treatment. Herein, we investigated the actions of insulin using ex vivo and in vivo models of HAND. Increased neuroinflammatory gene expression was observed in brains from patients with HIV/AIDS. The insulin receptor was detected on both neurons and glia, but its expression was unaffected by HIV-1 infection. Insulin treatment of HIV-infected primary human microglia suppressed supernatant HIV-1 p24 levels, reduced CXCL10 and IL-6 transcript levels, and induced peroxisome proliferator-activated receptor gamma (PPAR-γ) expression. Insulin treatment of primary human neurons prevented HIV-1 Vpr-mediated cell process retraction and death. In feline immunodeficiency virus (FIV) infected cats, daily intranasal insulin treatment (20.0 IU/200 μl for 6 weeks) reduced CXCL10, IL-6, and FIV RNA detection in brain, although PPAR-γ in glia was increased compared with PBS-treated FIV+ control animals. These molecular changes were accompanied by diminished glial activation in cerebral cortex and white matter of insulin-treated FIV+ animals, with associated preservation of cortical neurons. Neuronal counts in parietal cortex, striatum, and hippocampus were higher in the FIV+/insulin-treated group compared with the FIV+/PBS-treated group. Moreover, intranasal insulin treatment improved neurobehavioral performance, including both memory and motor functions, in FIV+ animals. Therefore, insulin exerted ex vivo and in vivo antiviral, anti-inflammatory, and neuroprotective effects in models of HAND, representing a new therapeutic option for patients with inflammatory or infectious neurodegenerative disorders including HAND. SIGNIFICANCE STATEMENT HIV-associated neurocognitive disorders (HAND) represent a spectrum disorder of neurocognitive dysfunctions resulting from HIV-1 infection. Although the exact mechanisms causing HAND are unknown, productive HIV-1 infection in the brain with associated neuroinflammation is a potential pathogenic mechanism resulting in neuronal damage and death. We report that, in HIV-infected microglia cultures, insulin treatment led to reduced viral replication and inflammatory gene expression. In addition, intranasal insulin treatment of experimentally feline immunodeficiency virus-infected animals resulted in improved motor and memory performances. We show that insulin restored expression of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which is suppressed by HIV-1 replication. Our findings indicate a unique function for insulin in improving neurological outcomes in lentiviral infections, implicating insulin as a therapeutic intervention for HAND.

Suppressed oligodendrocyte steroidogenesis in multiple sclerosis: Implications for regulation of neuroinflammation

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Neurosteroids are reported to exert anti‐inflammatory effects in several neurological disorders. We investigated the expression and actions of the neurosteroid, dehydroepiandrosterone (DHEA), and its more stable 3β‐sulphated ester, DHEA‐S, in MS and associated experimental models. CNS tissues from patients with MS and animals with experimental autoimmune encephalomyelitis (EAE) displayed reduced DHEA concentrations, accompanied by diminished expression of the DHEA‐synthesizing enzyme CYP17A1 in oligodendrocytes (ODCs), in association with increased expression of inflammatory genes including interferon (IFN)‐γ and interleukin (IL)‐1β. CYP17A1 was expressed variably in different human neural cell types but IFN‐γ exposure selectively reduced CYP17A1 detection in ODCs. DHEA‐S treatment reduced IL‐1β and −6 release from activated human myeloid cells with minimal effect on lymphocyte viability. Animals with EAE receiving DHEA‐S treatment showed reduced Il1b and Ifng transcript levels in spinal cord compared to vehicle‐treated animals with EAE. DHEA‐S treatment also preserved myelin basic protein immunoreactivity and reduced axonal loss in animals with EAE, relative to vehicle‐treated EAE animals. Neurobehavioral deficits were reduced in DHEA‐S‐treated EAE animals compared with vehicle‐treated animals with EAE. Thus, CYP17A1 expression in ODCs and its product DHEA were downregulated in the CNS during inflammatory demyelination while DHEA‐S provision suppressed neuroinflammation, demyelination, and axonal injury that was evident as improved neurobehavioral performance. These findings indicate that DHEA production is an immunoregulatory pathway within the CNS and its restoration represents a novel treatment approach for neuroinflammatory diseases.

Caspase-1 inhibition prevents glial inflammasome activation and pyroptosis in models of multiple sclerosis

Significance The pore-forming protein gasdermin D (GSDMD) was recently identified as the principal executioner of pyroptosis (“fiery death”), a type of proinflammatory programmed cell death driven by inflammasomes. Caspase-1 cleaves GSDMD, but whether this process contributes to neuroinflammation is unknown. Here, we report evidence of GSDMD-mediated pyroptosis as a primary mechanism of inflammatory demyelination in the central nervous system during multiple sclerosis (MS), a debilitating and incurable demyelinating disease that causes profound loss of myelin-forming oligodendrocytes. By identifying GSDMD induction and pyroptosis in oligodendrocytes and microglia, we discovered a previously unrecognized mechanism driving neuroinflammation and demyelination. Pharmacologically inhibiting caspase-1 prevented pyroptosis in experimental models of MS, reducing demyelination and neurodegeneration. These findings highlight therapeutic approaches for understanding and treating inflammatory demyelination. Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS of unknown cause that remains incurable. Inflammasome-associated caspases mediate the maturation and release of the proinflammatory cytokines IL-1β and IL-18 and activate the pore-forming protein gasdermin D (GSDMD). Inflammatory programmed cell death, pyroptosis, was recently shown to be mediated by GSDMD. Here, we report molecular evidence for GSDMD-mediated inflammasome activation and pyroptosis in both myeloid cells (macrophages/microglia) and, unexpectedly, in myelin-forming oligodendrocytes (ODCs) in the CNS of patients with MS and in the MS animal model, experimental autoimmune encephalomyelitis (EAE). We observed inflammasome activation and pyroptosis in human microglia and ODCs in vitro after exposure to inflammatory stimuli and demonstrate caspase-1 inhibition by the small-molecule inhibitor VX-765 in both cell types. GSDMD inhibition by siRNA transduction suppressed pyroptosis in human microglia. VX-765 treatment of EAE animals reduced the expression of inflammasome- and pyroptosis-associated proteins in the CNS, prevented axonal injury, and improved neurobehavioral performance. Thus, GSDMD-mediated pyroptosis in select glia cells is a previously unrecognized mechanism of inflammatory demyelination and represents a unique therapeutic opportunity for mitigating the disease process in MS and other CNS inflammatory diseases.

Activation of multiple inflammasomes within the central nervous system during experimental autoimmune encephalomyelitis and multiple sclerosis

There is a growing pool of evidence to support the hypothesis that events in the periphery, particularly of an inflammatory nature, can have a profound effect on the central nervous system (CNS). Specifically, inflammatory cytokines have been shown to be important mediators of neuropsychiatric disorders and variations in their expression can affect the severity of both depression and disease score, if presenting with specific comorbidities. The mechanisms driving the relationship between the CNS and the periphery remain to be fully understood. To investigate this, we have used a well characterised model of psoriasis-like skin inflammation as a tool to examine the consequential effect of localised peripheral inflammation on the brain. Aldara cream, which contains 5% Imiquimod as the active component, has been shown to induce psoriasis-like skin inflammation when applied repeatedly to the dorsal skin of mice. We have already shown that this model leads to the transcriptional upregulation of a number of classic interferon-stimulated genes (ISGs) in the brain, a response that we do not see mimicked in the peripheral blood leukocytes (PBL) at the same time point. We have since identified the transcriptional upregulation of a number of chemokines in the CNS which may be important for instigating immune cell infiltration. Furthermore, we have found that Aldara treatment causes a reduction in the level of doublecortin staining in the dentate gyrus of the hippocampus, indicating a negative effect on neurogenesis. Finally, a basic behavioural model of anhedonia has allowed us to determine a phenotypic consequence of Aldaratreatment, whereby the burrowing activity of the treated group is significantly reduced compared with the control group.

HIV-1 Vpr activates the NLRP3 inflammasome in primary human microglia

responses during autoimmunity, we investigated the role of Nur77 in modulation of human and murine T cell responses in vitro and characterised autoreactive T cell responses in vivo making use of the animal model of Multiple Sclerosis (MS), i.e. experimental autoimmune encephalomyelitis (EAE). We observed that Nur77 was expressed within hours after T cell receptor triggering. Upon T cell receptormediated activation, Nur77-deficient T cells proliferated stronger than wildtype T cells and exhibited increased potential to differentiate into pathogenic TH1 and TH17 cells — both, in an antigen-independent setting and upon antigen-specific activation. Also in vivo, antigenspecific T cell activation by Nur77-competent dendritic cells resulted in enhanced proliferation and cytokine production when T cells lacked Nur77. After induction of EAE, Nur77-deficient animals exhibited an earlier onset of disease and a significantly aggravated clinical score, which was accompanied by enhanced MOG35–55-specific TH1 and TH17 cell responses both in the periphery and within the CNS. Importantly, also the transfer of MOG35–55-specific Nur77-deficient T cells into healthy wildtype recipients induced a more aggravated EAE disease course than transfer of MOG35–55-specific wildtype T cells, which further demonstrated the importance of Nur77 for restriction of pathogenic T cell responses. In human T cells we observed dysregulated Nur77 expression after TCR triggering when T cells were derived from MS patients, thus indicating that Nur77 could also be important for control of human autoreactive T cell responses. Taken together, Nur77 limits T cell responses in the context of CNS autoimmunity by restricting T cell proliferation and differentiation into TH1 and TH17 effector cells. Hence, Nur77 dysregulation might contribute to enhanced T cell activation in T cell-mediated autoimmune diseases such as MS.