EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

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

Morphine withdrawal contributes to Th cell differentiation by biasing cells toward the Th2 lineage

The consequences that drug withdrawal has on immune functioning has only recently been appreciated; however, given the wide variety of use and abuse of opiate analgesics, understanding the decrements to immune function that withdrawal from these drugs causes is of crucial importance. In previous work, we have demonstrated that morphine treatment contributes to immunosuppression by polarizing Th cells toward the Th2 lineage. In the current study, it was hypothesized that morphine withdrawal would result in Th2 differentiation and subsequent immune dysfunction. To address this hypothesis, mice were chronically treated with morphine for 72 h followed by a 24-h withdrawal period. It was determined that 24-h morphine withdrawal resulted in a decrease in IFN-gamma, the Th1 signature cytokine, whereas the Th2 cytokine, IL-4, was increased. In addition, Western blot and EMSA experiments revealed that morphine withdrawal-induced Th2 differentiation was mediated through the classical Th2 transcription factors Stat-6 and GATA-3. In addition, the consequence of morphine withdrawal in the presence of an immune stimulation was also examined by treating mice in vivo with LPS before morphine withdrawal. Following withdrawal, it was found that the Th1-polarizing cytokine IL-12 was significantly decreased, providing further support for the observation that withdrawal results in Th2 differentiation by possibly impacting the generation of an appropriate innate immune response which directs subsequent adaptive Th1/Th2 responses

In vivo activation of a mutant mu-opioid receptor by naltrexone produces a potent analgesic effect but no tolerance: role of mu-receptor activation and delta-receptor blockade in morphine tolerance

Opioid analgesics are the standard therapeutic agents for the treatment of pain, but their prolonged use is limited because of the development of tolerance and dependence. Recently, we reported the development of a mu-opioid receptor knock-in (KI) mouse in which the mu-opioid receptor was replaced by a mutant receptor (S196A) using a homologous recombination gene-targeting strategy. In these animals, the opioid antagonist naltrexone elicited antinociceptive effects similar to those of partial agonists acting in wild-type (WT) mice; however, development of tolerance and physical dependence were greatly reduced. In this study, we test the hypothesis that the failure of naltrexone to produce tolerance in these KI mice is attributable to its simultaneous inhibition of delta-opioid receptors and activation of mu-opioid receptors. Simultaneous implantation of a morphine pellet and continuous infusion of the delta-opioid receptor antagonist naltrindole prevented tolerance development to morphine in both WT and KI animals. Moreover, administration of SNC-80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], a delta agonist, in the naltrexone-pelleted KI animals resulted in a dose-dependent induction in tolerance development to both morphine- and naltrexone-induced analgesia. We conclude that although simultaneous activation of both mu- and delta-opioid receptors results in tolerance development, mu-opioid receptor activation in conjunction with delta-opioid receptor blockade significantly attenuates the development of tolerance

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

Morphine withdrawal inhibits IL-12 induction in a macrophage cell line through a mechanism that involves cAMP

There are very few studies that examine the effects that morphine withdrawal has on immune functioning, and of these even fewer describe the mechanisms by which withdrawal brings about these changes. Our previous work demonstrated that morphine withdrawal contributed to Th cell differentiation by biasing cells toward the Th2 lineage. A major finding from these studies was that IL-12 was decreased following withdrawal, and it was concluded that this decrease may be a mechanism by which morphine withdrawal is mediating Th2 polarization. Therefore, it was the aim of the current studies to develop an in vitro model to examine the process of morphine withdrawal and to understand the signaling mechanisms that withdrawal may use to effect IL-12 production through the use of this model. It was demonstrated and concluded that morphine withdrawal may be effecting IL-12 production by increasing cAMP levels, which activates protein kinase A. Protein kinase A activation then prevents the phosphorylation and subsequent degradation of IkappaB, which in turn prevents translocation of the NF-kappaB p65 subunit to the nucleus to transactivate the IL-12 p40 gene, ultimately resulting in decreased IL-12 production following LPS stimulation

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