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Contrasting effect of the latency-reversing agents bryostatin-1 and JQ1 on astrocyte-mediated neuroinflammation and brain neutrophil invasion.

ABSTRACT: Despite effectiveness of the combined antiretroviral therapy, HIV-1 persists in long-lived latently infected cells. Consequently, new therapeutic approaches aimed at eliminating this latent reservoir are currently being developed. A "shock and kill" strategy using latency-reversing agents (LRA) to reactivate HIV-1 has been proposed. However, the impact of LRA on the central nervous system (CNS) remains elusive.We used human fetal astrocytes and investigated the effects of several LRA on their functional and secretory activities. Astrocytes were infected with VSV-G-pseudotyped HIV-1 before treatment with various blood-brain barrier (BBB)-permeable LRA at subcytotoxic doses, which allow HIV-1 reactivation based on previous in vitro and clinical studies. Cells and supernatants were then used to evaluate effects of infection and LRA on (i) viability and metabolic activity of astrocytes using a colorimetric MTS assay; (ii) chemokines and proinflammatory cytokines secretion and gene expression by astrocytes using ELISA and RT-qPCR, respectively; (iii) expression of complement component 3 (C3), a proxy for astrogliosis, by RT-qPCR; (iv) glutamate uptake capacity by a fluorometric assay; and (v) modulation of neutrophil transmigration across an in vitro BBB model.We demonstrate that bryostatin-1 induces secretion of chemokines CCL2 and IL-8 and proinflammatory cytokines IL-6 and GM-CSF, whereas their production is repressed by JQ1. Bryostatin-1 also increases expression of complement component 3 and perturbs astrocyte glutamate homeostasis. Lastly, bryostatin-1 enhances transmigration of neutrophils across an in vitro blood-brain barrier model and induces formation of neutrophil extracellular traps.These observations highlight the need to carefully assess the potential harmful effect to the CNS when selecting LRA for HIV-1 reactivation strategies.

SUBMITTER: Proust A

PROVIDER: S-EPMC5725742 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Contrasting effect of the latency-reversing agents bryostatin-1 and JQ1 on astrocyte-mediated neuroinflammation and brain neutrophil invasion.

Proust Alizé A Barat Corinne C Leboeuf Mathieu M Drouin Jean J Tremblay Michel J MJ

Journal of neuroinflammation 20171211 1

<h4>Background</h4>Despite effectiveness of the combined antiretroviral therapy, HIV-1 persists in long-lived latently infected cells. Consequently, new therapeutic approaches aimed at eliminating this latent reservoir are currently being developed. A "shock and kill" strategy using latency-reversing agents (LRA) to reactivate HIV-1 has been proposed. However, the impact of LRA on the central nervous system (CNS) remains elusive.<h4>Methods</h4>We used human fetal astrocytes and investigated the ...[more]

PMID: 29228979

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Project description:The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

Project description:Eradicating HIV-1 infection is difficult because of the reservoir of latently infected cells that gets established soon after infection, remains hidden from antiretroviral drugs and host immune responses, and retains the capacity to reignite infection following the cessation of treatment. Drugs called latency-reversing agents (LRAs) are being developed to reactivate latently infected cells and render them susceptible to viral cytopathicity or immune killing. Whereas individual LRAs have failed to induce adequate reactivation, pairs of LRAs have been identified recently that act synergistically and hugely increase reactivation levels compared to individual LRAs. The maximum synergy achievable with LRA pairs is of clinical importance, as it would allow latency-reversal with minimal drug exposure. Here, we employed stochastic simulations of HIV-1 transcription and translation in latently infected cells to estimate this maximum synergy. We incorporated the predominant mechanisms of action of the two most promising classes of LRAs, namely, protein kinase C agonists and histone deacetylase inhibitors, and quantified the activity of individual LRAs in the two classes by mapping our simulations to corresponding in vitro experiments. Without any adjustable parameters, our simulations then quantitatively captured experimental observations of latency-reversal when the LRAs were used in pairs. Performing simulations representing a wide range of drug concentrations, we estimated the maximum synergy achievable with these LRA pairs. Importantly, we found with all the LRA pairs we considered that concentrations yielding the maximum synergy did not yield the maximum latency-reversal. Increasing concentrations to increase latency-reversal compromised synergy, unravelling a trade-off between synergy and efficacy in LRA combinations. The maximum synergy realizable with LRA pairs would thus be restricted by the desired level of latency-reversal, a constrained optimum we elucidated with our simulations. We expect this trade-off to be important in defining optimal LRA combinations that would maximize synergy while ensuring adequate latency-reversal.

Dataset Information

Contrasting effect of the latency-reversing agents bryostatin-1 and JQ1 on astrocyte-mediated neuroinflammation and brain neutrophil invasion.

Publications

Contrasting effect of the latency-reversing agents bryostatin-1 and JQ1 on astrocyte-mediated neuroinflammation and brain neutrophil invasion.

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