Prevention and treatment of HIV infection and cognitive disease in mice by innate immune responses

HIV associated neurocognitive impairment afflicts roughly half of infected individuals on antiretroviral therapy. This disease currently has no treatment. We have previously shown that type I interferon is induced by and partially controls infection and neuropathogenesis in mice infected by chimeric HIV, EcoHIV. Here we investigate the intentional ligation of the pattern recognition receptor Toll-like receptor 3 (TLR3) by polyinosinic-polycytidylic acid (poly I:C) for its ability to prevent or control infection and associated cognitive disease in EcoHIV infected mice. We tested topical, injection, and intranasal application of poly I:C in mice during primary infection through injection or sexual transmission or in established infection. We measured different forms of HIV DNA and RNA in tissues by real-time PCR and the development of HIV-associated cognitive disease by the radial arm water maze behavioral test. Our results indicate that poly I:C blocks primary EcoHIV infection of mice prior to reverse transcription and reduces established EcoHIV infection. Prevention or control of viral replication by poly I:C prevents or reverses HIV associated cognitive disease in mice. These findings indicate that poly I:C or other innate immune agonists may be useful in control of HIV cognitive disease

Image_1_Buprenorphine reverses neurocognitive impairment in EcoHIV infected mice: A potential therapy for HIV-NCI.jpeg

Thirty-eight million people worldwide are living with HIV, PWH, a major public health problem. Antiretroviral therapy (ART) revolutionized HIV treatment and significantly increased the lifespan of PWH. However, approximately 15-50% of PWH develop HIV associated neurocognitive disorders (HIV-NCI), a spectrum of cognitive deficits, that negatively impact quality of life. Many PWH also have opioid use disorder (OUD), and studies in animal models of HIV infection as well as in PWH suggest that OUD can contribute to HIV-NCI. The synthetic opioid agonist, buprenorphine, treats OUD but its effects on HIV-NCI are unclear. We reported that human mature inflammatory monocytes express the opioid receptors MOR and KOR, and that buprenorphine reduces important steps in monocyte transmigration. Monocytes also serve as HIV reservoirs despite effective ART, enter the brain, and contribute to HIV brain disease. Using EcoHIV infected mice, an established model of HIV infection and HIV-NCI, we previously showed that pretreatment of mice prior to EcoHIV infection reduces mouse monocyte entry into the brain and prevents NCI. Here we show that buprenorphine treatment of EcoHIV infected mice with already established chronic NCI completely reverses the disease. Disease reversal was associated with a significant reduction in brain inflammatory monocytes and reversal of dendritic injury in the cortex and hippocampus. These results suggest that HIV-NCI persistence may require a continuing influx of inflammatory monocytes into the brain. Thus, we recommend buprenorphine as a potential therapy for mitigation of HIV brain disease in PWH with or without OUD.</p

Infected mice lacking T cells develop cognitive impairment and have elevated number of monocytes/macrophages in the brain.

<p><b>A.</b> Control and EcoHIV-infected nude mice with or without ART were tested 24 h after fear conditioning training for contextual fear response and 24 h later for cued fear response. *P<0.05, **P<0.01, EcoHIV vs. EcoHIV+ART; #P<0.05, EcoHIV vs. PBS. <b>B.-D.</b> Flow cytometry analysis of macrophage or monocytes in brains of nude mice with EcoHIV, EcoHIV with cART or PBS showing representative dot plots. Cell populations were gated based on isotype control antibodies. <b>B.</b> Staining for CD45 and CD11b and negative for Ly-6G/C. <b>C.</b> Staining for CD45, CD11b and Ly-6C and negative for Ly-6G. <b>D.</b> As determined by flow cytometry using 5 mice per group, the number of cells in each population per total mouse brain are shown. <b>E.</b> Total and integrated vDNA was measured by QPCR and nested QPCR, respectively, in infiltrating leukocytes isolated from the brain of EcoHIV-infected mice with and without cART. *P<0.05, **P<0.01, ***P<0.001.</p

LASER ART (L-ART) fails to reverse HIV-NCI in chronically-infected mice.

<p><b>A.</b> Two days prior to EcoHIV infection, mice were injected once with L-ART and their virus burdens in spleen and PC were evaluated seven days after infection. <b>B.</b> Design of the experiment. L-ART was administered twice, at 28 days and 56 days p.i. <b>C.</b> Results of RAWM conducted on chronically EcoHIV-infected L-ART-treated and untreated mice starting 82 days p.i.; L-ART treated, uninfected mice served as controls; left panel: errors; middle panel: latency; right panel: visible platform control. T represents the learning trial and RT the retention trial (*Mock vs. EcoHIV, <i>p</i><0.002; #Mock vs. EcoHIV+L-ART, <i>p</i><0.03). <b>D.</b> HIV burdens of treated and untreated EcoHIV-infected mice tested in spleen cells, peritoneal macrophages, and brain 94 days p.i.</p

Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment.

<p><b>A.</b> Design of the experiment. <b>B.</b> Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. <b>C.</b> Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. <b>D.</b> Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 EcoHIV vs. EcoHIV+cART; ^#<i>P</i><0.05, ^##P<0.01, ^###<i>P</i><0.001 EcoHIV vs. MLV.</p

Features of EcoHIV-infected mice compared to HIV-infected people.

<p>Features of EcoHIV-infected mice compared to HIV-infected people.</p

EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice.

<p><b>A.-B.</b> Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in <b>A.</b> spleen and <b>B.</b> PC at the times indicated after infection. Each point represents a single mouse. <b>C.</b> EcoHIV <i>gag</i> RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. <b>D.</b> The frequency of interferon-γ expression by CD4⁺ or CD8⁺ spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. <b>E.</b> The number of CD4⁺ and CD8⁺ T cells was measured by flow cytometry at the indicated times and the CD4⁺:CD8⁺ ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. <b>F.</b> Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.</p

EcoHIV establishes latent reservoirs in resting CD4⁺ cells that are inducible by epigenetic modulators.

<p><b>A.</b> CD4⁺ T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. <b>B.</b> Six weeks after EcoHIV infection, CD4⁺ splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV <i>gag</i> RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. <b>C.-D.</b> Twelve weeks after infection, resting CD4⁺ T cells were purified from the spleen and <b>C.</b> The levels of integrated EcoHIV DNA and <b>D.</b> Genomic RNA were measured by QPCR. <b>E.</b> Eight weeks after EcoHIV infection of mice, resting CD4⁺ T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. <b>F.</b> Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4⁺ cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Mouse macrophages are susceptible to EcoHIV but not MLV.

<p><b>A.</b> Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. <b>B.</b> Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. <b>C.</b> Eight weeks after mouse infection by EcoHIV or MLV, F4/80⁺ macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80⁺ PC was measured by nested-QPCR. <b>D.</b> C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (*<i>P</i><0.05). <b>E.</b> At day 86 after EcoHIV infection, PC were isolated from 129X1 euthymic and athymic mice and forms of the viral DNA and RNA were measured by QPCR, each symbol represents a single mouse. <b>F.</b> Flow cytometric analysis of percentage of F4/80⁺ PM expressing EcoHIV-encoded EGFP in nude and wild-type mice at 25 days after infection. <b>G.</b> Expression of EcoHIV-encoded EGFP in F4/80⁺ PM at 25 days after infection in mice was detected by confocal imaging.</p