BackgroundInflammation drives COPD development in people living with HIV (PLwHIV), and the HIV virus impairs T regulatory (Treg) cell responses that deter immune-mediated lung injury. This study sought to determine how cigarette smoke exposure alters lung Treg responses to increase COPD susceptibility in PLwHIV.MethodsLung lavage levels of the Treg chemoattractant CXCL11 were quantified in a cohort of 26 HIV-infected subjects and 34 age-matched controls. To ascertain how CXCL11 modifies lung Treg responses, analyses were then conducted using a chimeric HIV (EcoHIV) infection smoke exposure mouse model and elastase treatment of Treg depleted (DEREG) mice.ResultsCXCL11 lung lavage levels increased in HIV + subjects compared to controls. However, CXCL11 levels were significantly lower in those HIV + subjects with a reduced diffusing capacity for carbon monoxide compared to HIV + subjects with normal lung function. Cigarette smoke exposure reduced CXCL11 levels in HIV + current smokers and decreased lung Cxcl11 levels and Treg frequency in control and EcoHIV-infected mice. Cigarette smoke increased lung c-Src activity in mice and the c-Src inhibitor AZD0530 restored Cxcl11 expression in smoke exposed mice and alveolar macrophages. Direct administration of CXCL11 protein to the airways of EcoHIV infected or smoke exposed mice significantly enhanced lung Treg responses. Treg deficient DEREG mice exhibited increased airway resistance at baseline and had greater lung tissue destruction post elastase treatment.ConclusionsThese findings indicate that cigarette smoke activates c-Src to suppress CXCL11 levels thereby diminishing lung Treg responses that counter airways disease and lung tissue destruction in HIV-infected individuals.

BACKGROUND:Cognitive impairment is a frequent outcome of chronic viral infections linked to premature aging, including HIV. The mechanisms underlying this decline remain poorly understood. Here, we identify pro-inflammatory glycan degradation, characterized by loss of sialic acid and galactose, both hallmarks of premature aging, as a key contributor to HIV-associated cognitive impairment (HIV-CI). METHODS:We analyzed two cohorts of people with HIV, with and without HIV-CI, and tested causality through intervention studies in two complemetary mouse models of virally mediated inflammation and cognitive deceline. FINDINGS:Degradative glycomic changes were enriched in people with HIV with cognitive impairment, particularly females, and correlated with worse cognitive performance. In both a humanized mouse model of HIV and EcoHIV model, a complementary model that enables cognitive testing, pharmacological inhibition of glycan degradation with sialidase inhibitors prevented virally induced inflammation, immune activation, accelerated aging, and memory deficits. CONCLUSIONS:These findings implicate glycan degradation as a contributor to inflammation and cognitive impairment in HIV and highlight glycan preservation as a promising strategy to mitigate inflammation, premature aging, and cognitive decline during viral infections. FUNDING:This study was funded by the National Institutes of Health (NIH).

Background. EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. Results. We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. Conclusions. In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.

Background: Inflammation drives COPD in people living with HIV, and the HIV virus impairs T regulatory (Treg) cell responses that deter immune-mediated lung injury. Aims: We determined how cigarette smoke exposure alters lung Treg responses to increase the susceptibility to COPD in people living with HIV. Methods: Lung lavage levels of the Treg chemoattractant CXCL11 were quantified in a cohort of 26 HIV-infected subjects and 42 age-matched controls. To ascertain how CXCL11 modifies lung Treg responses, analyses were conducted using a chimeric HIV (EcoHIV) infection smoke exposure mouse model and elastase treatment of Treg depleted (DEREG) mice. Results: CXCL11 lung lavage levels increased in HIV+ subjects compared to controls. However, CXCL11 levels were significantly lower in those HIV+ subjects with a reduced diffusing capacity for carbon monoxide compared to HIV+ subjects with normal lung function. Cigarette smoke exposure reduced CXCL11 levels in human subjects and decreased lung Cxcl11 levels and Treg frequency in control and EcoHIV-infected mice. Cigarette smoke increased lung c-Src activity in mice and the c-Src inhibitor AZD0530 restored Cxcl11 expression in smoke exposed mice and alveolar macrophages. Direct administration of CXCL11 protein to the airways of EcoHIV infected or smoke exposed mice significantly enhanced lung Treg responses. Treg deficient DEREG mice exhibited increased airway resistance at baseline and had greater lung tissue destruction post elastase treatment. Conclusions: Cigarette smoke activates c-Src to suppress CXCL11 levels thereby diminishing lung Treg responses that counter airways disease and lung tissue destruction in HIV+ individuals.

HIV enters the brain within days of infection causing neurocognitive impairment (NCI) in up to half of infected people despite suppressive antiretroviral therapy. The virus is believed to enter the brain in infected monocytes through chemotaxis to the major monocyte chemokine, CCL2, but the roles of CCL2 in established NCI are not fully defined. We addressed this question during infection of conventional and CCL2 knockout mice with EcoHIV in which NCI can be verified in behavioral tests. EcoHIV enters mouse brain within 5 days of infection, but NCI develops gradually with established cognitive disease starting 25 days after infection. CCL2 knockout mice infected by intraperitoneal injection of virus failed to develop brain infection and NCI. However, when EcoHIV was directly injected into the brain, CCL2 knockout mice developed NCI. Knockout of CCL2 or its principal receptor, CCR2, slightly reduced macrophage infection in culture. Treatment of mice prior to and during EcoHIV infection with the CCL2 transcriptional inhibitor, bindarit, prevented brain infection and NCI and reduced macrophage infection. In contrast, bindarit treatment of mice 4 weeks after infection affected neither brain virus burden nor NCI. Based on these findings we propose that HIV enters the brain mainly through infected monocytes but that resident brain cells are sufficient to maintain NCI. These findings suggest that NCI therapy must act within the brain.

Approximately 15-40% of people living with HIV develop HIV-associated neurocognitive disorders, HAND, despite successful antiretroviral therapy. There are no therapies to treat these disorders. HIV enters the CNS early after infection, in part by transmigration of infected monocytes. Currently, there is a major opioid epidemic in the United States. Opioid use disorder in the context of HIV infection is important because studies show that opioids exacerbate HIV-mediated neuroinflammation that may contribute to more severe cognitive deficits. Buprenorphine is an opioid derivate commonly prescribed for opiate agonist treatment. We used the EcoHIV mouse model to study the effects of buprenorphine on cognitive impairment and to correlate these with monocyte migration into the CNS. We show that buprenorphine treatment prior to mouse EcoHIV infection prevents the development of cognitive impairment, in part, by decreased accumulation of monocytes in the brain. We propose that buprenorphine has a novel therapeutic benefit of limiting the development of neurocognitive impairment in HIV-infected opioid abusers as well as in nonabusers, in addition to decreasing the use of harmful opioids. Buprenorphine may also be used in combination with HIV prevention strategies such as pre-exposure prophylaxis because of its safety profile.

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A correction to this paper has been published: https://doi.org/10.1007/s11481-021-09992-5

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.

Cigarette smoke usage is prevalent in human immunodeficiency virus (HIV)-positive patients, and, despite highly active antiretroviral therapy, these individuals develop an accelerated form of chronic obstructive pulmonary disease (COPD). Studies investigating the mechanisms of COPD development in HIV have been limited by the lack of suitable mouse models. Here we describe a model of HIV-induced COPD in wild-type mice using EcoHIV, a chimeric HIV capable of establishing chronic infection in immunocompetent mice. A/J mice were infected with EcoHIV and subjected to whole body cigarette smoke exposure. EcoHIV was detected in alveolar macrophages of mice. Compared with uninfected mice, concomitant EcoHIV infection significantly reduced forced expiratory flow 50%/forced vital capacity and enhanced distal airspace enlargement following cigarette smoke exposure. Lung IL-6, granulocyte-macrophage colony-stimulating factor, neutrophil elastase, cathepsin G, and matrix metalloproteinase-9 expression was significantly enhanced in smoke-exposed EcoHIV-infected mice. These changes coincided with enhanced IκBα, ERK1/2, p38, and STAT3 phosphorylation and lung cell apoptosis. Thus, the EcoHIV smoke exposure mouse model reproduces several of the pathophysiological features of HIV-related COPD in humans, indicating that this murine model can be used to determine key parameters of HIV-related COPD and to test future therapies for this disorder.

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

The roles of Type I interferon (IFN) in human immunodeficiency virus Type 1 (HIV-1) neuropathogenesis are poorly understood; both protective and deleterious effects of IFN signaling have been described. We used genetically modified mice deficient in the Type I IFN receptor (IFNRKO) to analyze the progress of HIV-1 brain infection and neuropathogenesis in the absence of IFN signaling. IFNRKO and wild-type (WT) mice on the 129xSv/Ev or C57BL/6 strain backgrounds were infected systemically with EcoHIV, a chimeric HIV-1 that productively infects mice. IFNRKO mice showed higher HIV-1 expression in spleen and peritoneal macrophages and greater virus infiltration into the brain compared to WT mice. Neuropathogenesis was studied by histopathological, immunohistochemical, immunofluorescence, and polymerase chain reaction analyses of brain tissues after the virus was inoculated into the brain by stereotaxic intracerebral injection. Both IFNRKO and WT mice showed readily detectable HIV-1 and brain lesions, including microglial activation, astrocytosis, and increased expression of genes coding for inflammatory cytokines and chemokines typical of human HIV-1 brain disease. Parameters of HIV-1 neuropathogenesis, including HIV-1 expression in microglia/macrophages, were significantly greater in IFNRKO than in WT mice. Our results show unequivocally that Type I IFN signaling and responses limit HIV-1 infection and pathogenesis in the brains of mice.

Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1-7 nights. At 1-6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70-100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2',3'-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.