School of Medicine and Health Sciences Poster Presentations

Title

Targeting Sphingosine-1-phosphate reduces latent HIV infection

Document Type

Poster

Abstract Category

Basic Biomedical Sciences

Keywords

HIV, S1P, latency

Publication Date

Spring 5-1-2019

Abstract

Background: Sphingosine-1-phosphate (S1P) is an established modulator of cell cycle and chemotaxis and the therapeutic targeting of this pathway has been the subject of investigation for the treatment of multiple sclerosis and other autoimmune diseases. In the context of Human Immunodeficiency Virus 1 (HIV-1) infection, alterations in the expression of S1P receptor 1 in thymocytes as well as impaired S1P signaling in CD4+ T cells have been described. Recently agonists of the S1P receptor 1 have been shown to reverse HIV latency. Here, we sought to determine the role of the S1P in the establishment of latent HIV infection. Methods: We examined whether targeting Sphingosine Kinase (SPK) would alter the establishment of the latent reservoir in memory CD4+ T cells using a primary cell model. Briefly, we isolated naïve human CD4+ T cells from HIV-negative donors, activated and expanded them, and infected them with NL4-3 virus by spin infection. Three days later, cells were treated with either N,N-dimethyl sphingosine (D.M.S.), a SPK inhibitor; or FTY720, a S1P receptor modulator. We quantified the effects of these inhibitors on latent infection by measuring the frequency of cells harboring total and integrated HIV DNA by qPCR and the ability to reactivate virus from latency following T cell receptor stimulation by intracellular HIV Gag. Results: Treatment with D.M.S. reduced the establishment of latent HIV infection in a dose dependent manner as measured by the frequency of cells producing HIV Gag upon T cell receptor stimulation (6 M n=9, 95% reduction, p=0.0067; 600 nM n=8, 38% reduction, p=0.0236; 60 nM n=5, 23.3% reduction, p=0.2). Moreover, FTY720, which is used in the clinical setting for the treatment of multiple sclerosis, recapitulated this effect (100nM n=7, 70% reduction, p=0.0425). These inhibitors reduced latent infection during or before reverse transcription since the treatments reduced to a similar extent both total (D.M.S. 600 nM n=4, 34% reduction; FTY720 100nM n=4, 52% reduction) and integrated HIV DNA (D.M.S. 600 nM n=4, 29.5% reduction; FTY720 100nM n=4, 57% reduction). Conclusions: Our results show that targeting S1P has an effect on latent infection. Mechanistically, D.M.S. and FTY720 force CD4+ T cells into a G0 state of the cell cycle as measured by expression of Ki67. Our research suggests that the therapeutic targeting of this pathway early in infection may aid in the development of strategies to promote a functional cure by preventing the establishment of the latent reservoir.

Open Access

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Presented at Research Days 2019.

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Targeting Sphingosine-1-phosphate reduces latent HIV infection

Background: Sphingosine-1-phosphate (S1P) is an established modulator of cell cycle and chemotaxis and the therapeutic targeting of this pathway has been the subject of investigation for the treatment of multiple sclerosis and other autoimmune diseases. In the context of Human Immunodeficiency Virus 1 (HIV-1) infection, alterations in the expression of S1P receptor 1 in thymocytes as well as impaired S1P signaling in CD4+ T cells have been described. Recently agonists of the S1P receptor 1 have been shown to reverse HIV latency. Here, we sought to determine the role of the S1P in the establishment of latent HIV infection. Methods: We examined whether targeting Sphingosine Kinase (SPK) would alter the establishment of the latent reservoir in memory CD4+ T cells using a primary cell model. Briefly, we isolated naïve human CD4+ T cells from HIV-negative donors, activated and expanded them, and infected them with NL4-3 virus by spin infection. Three days later, cells were treated with either N,N-dimethyl sphingosine (D.M.S.), a SPK inhibitor; or FTY720, a S1P receptor modulator. We quantified the effects of these inhibitors on latent infection by measuring the frequency of cells harboring total and integrated HIV DNA by qPCR and the ability to reactivate virus from latency following T cell receptor stimulation by intracellular HIV Gag. Results: Treatment with D.M.S. reduced the establishment of latent HIV infection in a dose dependent manner as measured by the frequency of cells producing HIV Gag upon T cell receptor stimulation (6 M n=9, 95% reduction, p=0.0067; 600 nM n=8, 38% reduction, p=0.0236; 60 nM n=5, 23.3% reduction, p=0.2). Moreover, FTY720, which is used in the clinical setting for the treatment of multiple sclerosis, recapitulated this effect (100nM n=7, 70% reduction, p=0.0425). These inhibitors reduced latent infection during or before reverse transcription since the treatments reduced to a similar extent both total (D.M.S. 600 nM n=4, 34% reduction; FTY720 100nM n=4, 52% reduction) and integrated HIV DNA (D.M.S. 600 nM n=4, 29.5% reduction; FTY720 100nM n=4, 57% reduction). Conclusions: Our results show that targeting S1P has an effect on latent infection. Mechanistically, D.M.S. and FTY720 force CD4+ T cells into a G0 state of the cell cycle as measured by expression of Ki67. Our research suggests that the therapeutic targeting of this pathway early in infection may aid in the development of strategies to promote a functional cure by preventing the establishment of the latent reservoir.