Peroxisome injury in multiple sclerosis: protective effects of 4-phenylbutyrate in CNS-associated macrophages.

OBJECTIVES: Multiple sclerosis (MS) is a progressive and inflammatory demyelinating disease of the central nervous system (CNS). Peroxisomes perform critical functions that contribute to CNS homeostasis. We investigated peroxisome injury and mitigating effects of peroxisome-restorative therapy on inflammatory demyelination in models of MS. METHODS: Human autopsied CNS tissues (male and female), human cell cultures and cuprizone-mediated demyelination mice (female) were examined by RT-PCR, western blotting and immunolabeling. The therapeutic peroxisome proliferator, 4-phenylbutyrate (4-PBA) was investigated in vitro and in vivo. RESULTS: White matter from MS patients showed reduced peroxisomal transcript and protein levels, including PMP70, compared to non-MS controls. Cultured human neural cells revealed that human microglia contained abundant peroxisomal proteins. TNF-α-exposed microglia displayed reduced immunolabeling of peroxisomal proteins, PMP70 and PEX11ß, which was prevented with 4-PBA. In human myeloid cells exposed to TNF-α or nigericin, suppression of PEX11ß and catalase protein levels were observed to be dependent on NLRP3 expression. Hindbrains from cuprizone-exposed mice showed reduced Abcd1, Cat, and Pex5l transcript levels, with concurrent increased Nlrp3 and Il1b transcript levels, which was abrogated by 4-PBA. In the central corpus callosum, Iba-1 in CNS-associated macrophages (CAMs) and peroxisomal thiolase immunostaining after cuprizone exposure was increased by 4-PBA. 4-PBA prevented decreased myelin basic protein and neurofilament heavy chain immunoreactivity caused by cuprizone exposure. Cuprizone-induced neurobehavioral deficits were improved by 4-PBA treatment. CONCLUSIONS: Peroxisome injury in CAMs, contributed to neuroinflammation and demyelination that was prevented by 4-PBA treatment. A peroxisome-targeted therapy might be valuable for treating inflammatory demyelination and neurodegeneration in MS.Significance statement:Multiple sclerosis (MS) is a common and disabling disorder of the CNS with no curative therapies for its progressive form. The present studies implicate peroxisome impairment in CNS-associated macrophages (CAMs), which include resident microglia and blood-derived macrophages, as an important contributor to inflammatory demyelination and neuroaxonal injury in MS. We also show that the inflammasome molecule NLRP3 is associated with peroxisome injury in vitro and in vivo, especially in CAMs. Treatment with the peroxisome proliferator 4-phenylbutyrate exerted protective effects with improved molecular, morphological and neurobehavioral outcomes that were associated with a neuroprotective CAM phenotype. These findings offer novel insights into the contribution of peroxisome injury in MS together with preclinical testing of a rational therapy for MS.

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

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-1ß 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.

Inflammasomes in neurological diseases: emerging pathogenic and therapeutic concepts.

Inflammasome activation in the central nervous system occurs in both health and disease. Inflammasomes are cytosolic protein complexes that sense specific infectious or host stimuli and initiate inflammatory responses through caspase activation. Assembly of inflammasomes results in caspase-1-mediated proteolytic cleavage and release of the pro-inflammatory cytokines, interleukin-1ß and interleukin-18, with initiation of pyroptosis, an inflammatory programmed cell death. Recent developments in the inflammasome field have uncovered novel molecular mechanisms that contribute to a broad range of neurological disorders including those associated with specific mutations in inflammasome genes as well as diseases modulated by inflammasome activation. This update focuses on recent developments in the field of inflammasome biology highlighting different inflammasome activators and pathways discovered in the nervous system. We also discuss targeted therapies that regulate inflammasomes and improve neurological outcomes.

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.

Reduced antiretroviral drug efficacy and concentration in HIV-infected microglia contributes to viral persistence in brain.

BACKGROUND: In patients with HIV/AIDS receiving antiretroviral therapy (ART), HIV-1 persistence in brain tissue is a vital and unanswered question. HIV-1 infects and replicates in resident microglia and trafficking macrophages within the brain although the impact of individual ART drugs on viral infection within these brain myeloid cells is unknown. Herein, the effects of contemporary ART drugs were investigated using in vitro and in vivo models of HIV-1 brain infection. RESULTS: The EC50 values for specific ART drugs in HIV-infected human microglia were significantly higher compared to bone marrow-derived macrophages and peripheral blood mononuclear cells. Intracellular ART drug concentrations in microglia were significantly lower than in human lymphocytes. In vivo brain concentrations of ART drugs in mice were 10 to 100-fold less in brain tissues compared with plasma and liver levels. In brain tissues from untreated HIV-infected BLT mice, HIV-encoded RNA, DNA and p24 were present in human leukocytes while ART eradicated viral RNA and DNA in both brain and plasma. Interruption of ART resulted in detectable viral RNA and DNA and increased human CD68 expression in brains of HIV-infected BLT mice. In aviremic HIV/AIDS patients receiving effective ART, brain tissues that were collected within hours of last ART dosing showed HIV-encoded RNA and DNA with associated neuroinflammatory responses. CONCLUSIONS: ART drugs show variable concentrations and efficacies in brain myeloid cells and tissues in drug-specific manner. Despite low drug concentrations in brain, experimental ART suppressed HIV-1 infection in brain although HIV/AIDS patients receiving effective ART had detectable HIV-1 in brain. These findings suggest that viral suppression in brain is feasible but new approaches to enhancing ART efficacy and concentrations in brain are required for sustained HIV-1 eradication from brain.

Rapid inflammasome activation in microglia contributes to brain disease in HIV/AIDS.

BACKGROUND: Human immunodeficiency virus type 1(HIV-1) infects and activates innate immune cells in the brain resulting in inflammation and neuronal death with accompanying neurological deficits. Induction of inflammasomes causes cleavage and release of IL-1ß and IL-18, representing pathogenic processes that underlie inflammatory diseases although their contribution HIV-associated brain disease is unknown. RESULTS: Investigation of inflammasome-associated genes revealed that IL-1ß, IL-18 and caspase-1 were induced in brains of HIV-infected persons and detected in brain microglial cells. HIV-1 infection induced pro-IL-1ß in human microglia at 4 hr post-infection with peak IL-1ß release at 24 hr, which was accompanied by intracellular ASC translocation and caspase-1 activation. HIV-dependent release of IL-1ß from a human macrophage cell line, THP-1, was inhibited by NLRP3 deficiency and high extracellular [K+]. Exposure of microglia to HIV-1 gp120 caused IL-1ß production and similarly, HIV-1 envelope pseudotyped viral particles induced IL-1ß release, unlike VSV-G pseudotyped particles. Infection of cultured feline macrophages by the related lentivirus, feline immunodeficiency virus (FIV), also resulted in the prompt induction of IL-1ß. In vivo FIV infection activated multiple inflammasome-associated genes in microglia, which was accompanied by neuronal loss in cerebral cortex and neurological deficits. Multivariate analyses of data from FIV-infected and uninfected animals disclosed that IL-1ß, NLRP3 and caspase-1 expression in cerebral cortex represented key molecular determinants of neurological deficits. CONCLUSIONS: NLRP3 inflammasome activation was an early and integral aspect of lentivirus infection of microglia, which was associated with lentivirus-induced brain disease. Inflammasome activation in the brain might represent a potential target for therapeutic interventions in HIV/AIDS.

HIV-1 and IL-1ß regulate astrocytic CD38 through mitogen-activated protein kinases and nuclear factor-κB signaling mechanisms.

BACKGROUND: Infection with human immunodeficiency virus type-1 (HIV)-1 leads to some form of HIV-1-associated neurocognitive disorders (HAND) in approximately half of the cases. The mechanisms by which astrocytes contribute to HIV-1-associated dementia (HAD), the most severe form of HAND, still remain unresolved. HIV-1-encephalitis (HIVE), a pathological correlate of HAD, affects an estimated 9-11% of the HIV-1-infected population. Our laboratory has previously demonstrated that HIVE brain tissues show significant upregulation of CD38, an enzyme involved in calcium signaling, in astrocytes. We also reported an increase in CD38 expression in interleukin (IL)-1ß-activated astrocytes. In the present investigation, we studied regulatory mechanisms of CD38 gene expression in astrocytes activated with HIV-1-relevant stimuli. We also investigated the role of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in astrocyte CD38 regulation. METHODS: Cultured human astrocytes were transfected with HIV-1(YU-2) proviral clone and levels of CD38 mRNA and protein were measured by real-time PCR gene expression assay, western blot analysis and immunostaining. Astrocyte activation by viral transfection was determined by analyzing proinflammatory chemokine levels using ELISA. To evaluate the roles of MAPKs and NF-κB in CD38 regulation, astrocytes were treated with MAPK inhibitors (SB203580, SP600125, U0126), NF-κB interfering peptide (SN50) or transfected with dominant negative IκBα mutant (IκBαM) prior to IL-1ß activation. CD38 gene expression and CD38 ADP-ribosyl cyclase activity assays were performed to analyze alterations in CD38 levels and function, respectively. RESULTS: HIV-1(YU-2)-transfection significantly increased CD38 mRNA and protein expression in astrocytes (p < 0.01) in a dose-dependent manner and induced astrocyte activation. IL-ß-activation of HIV-1(YU-2)-transfected astrocytes significantly increased HIV-1 gene expression (p < 0.001). Treatment with MAPK inhibitors or NF-κB inhibitor SN50 abrogated IL-1ß-induced CD38 expression and activity in astrocytes without altering basal CD38 levels (p < 0.001). IκBαM transfection also significantly inhibited IL-1ß-mediated increases in CD38 expression and activity in astrocytes (p < 0.001). CONCLUSION: The present findings demonstrate a direct involvement of HIV-1 and virus-induced proinflammatory stimuli in regulating astrocyte-CD38 levels. HIV-1(YU-2)-transfection effectively induced HIV-1p24 protein expression and activated astrocytes to upregulate CCL2, CXCL8 and CD38. In astrocytes, IL-1ß-induced increases in CD38 levels were regulated through the MAPK signaling pathway and by the transcription factor NF-κB. Future studies may be directed towards understanding the role of CD38 in response to infection and thus its role in HAND.

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-1ß 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-1ß 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-1ß release with reduced cell viability, which was dependent on NLRP3 expression. Increased NLRP3, caspase-1, and IL-1ß 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-1ß 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.

CXCL8 as a Potential Therapeutic Target for HIV-Associated Neurocognitive Disorders.

Chemokine CXCL8 is a low molecular weight neutrophil chemoattractant implicated in various neurodegenerative disorders including Alzheimer's disease and stroke. Increased expression of CXCL8 has been reported in serum, plasma and brain of human immunodeficiency virus (HIV)-1 infected individuals with neurocognitive impairment, indicating its role in neuroinflammation associated with HIV-1 infection of the brain. Since chemokines are critical in eliciting immune responses in the central nervous system (CNS), CXCL8 is of particular importance for being one of the first chemokines described in the brain. Activation of astrocytes and microglia by HIV-1 and virus associated proteins results in production of this chemokine in the brain microenvironment. Consequently, CXCL8 exerts its effect on target cells via Gprotein coupled receptors CXCR1 and CXCR2. Neutrophils are the main target cells for CXCL8; however, microglia and neurons also express CXCR1/CXCR2 and therefore are important targets for CXCL8-mediated crosstalk. The objective of this review is to focus on CXCL8 production, signaling and regulation in neuronal and glial cells in response to HIV-1 infection. We highlight the role of HIV-1 secreted proteins such as trans-activator of transcription, envelope glycoprotein, negative regulatory factor and viral protein r in the regulation of CXCL8. We discuss dual role of CXCL8 in neurodegeneration as well as neuroprotection in the CNS. Thus, targeting CXCL8 through the development of CXCR1/CXCR2-based therapeutic strategies to either selectively agonize or antagonize receptors may be able to selectively promote neuroprotective and anti-inflammatory outcomes, leading to significant clinical applications in many neuroinflammatory CNS diseases, including HIV-associated neurocognitive disorders.

Chemokine CXCL8 promotes HIV-1 replication in human monocyte-derived macrophages and primary microglia via nuclear factor-κB pathway.

BACKGROUND: Chemokine CXCL8 is an important neutrophil chemoattractant implicated in various neurodegenerative disorders. Cytokine/chemokine imbalance, with an increase in proinflammatory cytokines like interleukin-1ß and tumor necrosis factor-α within the central nervous system, is a hallmark of human immunodeficiency virus (HIV)-1 infection. We previously reported that HIV-1 infection is linked to upregulation of CXCL8 in brain tissues and human astrocytes. Chemokines play crucial roles in trafficking of leukocytes and trafficking of HIV-1-infected across the blood-brain barrier play an important role in HIV-1 central nervous system disease. In the post-antiretroviral therapy era, low level of productive replication of HIV-1 in brain is a critical component of neuropathogenesis regulation. The present study investigated the effect of CXCL8 on productive infection of HIV-1 in human monocytes-derived macrophages (MDM) and primary human microglia. RESULTS: Human MDM and microglia were infected with the blood or brain derived HIV-1 isolates, HIV-1ADA or HIV-1JRFL. Treatment with CXCL8 significantly upregulated HIV-1p24 levels in supernatants of both HIV-1-infected MDM as well as microglia. In addition, the formation of 2-long terminal repeat (LTR) circles, a measure of viral genome integration, was significantly higher in CXCL8-treated, HIV-1-infected MDM and microglia. Transient transfection of U937 cells with HIV-1 LTR luciferase reporter construct resulted in increased promoter activity when treated with CXCL8. Moreover, increased nuclear translocation of nuclear factor-κB was seen in HIV-1-infected MDM following CXCL8 treatment. Blocking CXCL8 receptors CXCR1 and CXCR2 abrogated the CXCL8-mediated enhanced HIV-1 replication. CONCLUSION: Our results show that CXCL8 mediates productive infection of HIV-1 in MDM and microglia via receptors CXCR1 and CXCR2. These results demonstrate that CXCL8 exerts its downstream effects by increasing translocation of nuclear factor-κB into the nucleus, thereby promoting HIV-1 LTR activity.

Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and p38 regulate the expression of chemokine CXCL8 in human astrocytes.

CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1ß activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1ß stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.