Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells

Abstract

Exosomes, 30-200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies.

Keywords: HIV-1; T cell immunoglobulin and mucin proteins; exosomes; nanoparticle tracking analysis; phosphatidylserine.

Figure 1

Western blot and NTA validation of exosomal samples. Notes: Western blots of (A) breast milk exosomes (60 μg/lane) [(A1) clathrin and (A2) CD9] and (B, C) plasma exosomes (25 μg/lane) [(B1) CD9, (B2) CD63], (C) TIM-4. Arrows indicate proteins of interest. (D, E) NTA-generated size and concentration plots for (D) human plasma- and (E) human breast milk-derived exosomes. Abbreviations: NTA, nanoparticle tracking analysis; TIM, T cell immunoglobulin and mucin.

Figure 2

NSC- and A549-derived exosomes significantly enhance HIV-1 entry into human immune cell lines. Notes: (A, C) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of (A) NSC-derived exosomes (0.1 μg) or (C) A549-dervied exosomes (0.1 μg). (B, D) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of (B) NSC-derived exosomes (0.1 μg) or (D) A549-derived exosomes (0.1 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA *P<0.05, ***P<0.001, ****P<0.0001. YU-2, (NL-LucR.T2A-YU2.ecto) engineered to express Renilla luciferase (LucR) and ENV from the YU-2 virus strain. Abbreviations: NSC, neural stem cell; TIM, T cell immunoglobulin and mucin; ANOVA, analysis of variance; RLU, relative luminescence unit, exo, exosomes.

Figure 3

Breast milk- and plasma-derived exosomes enhance HIV-1 entry into human immune cell lines. Notes: (A, C) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of (A) breast milk-derived exosomes (0.035 μg) or (C) plasma-derived exosomes (0.05 μg). Virus entry was evaluated in the presence of exosomes and anti-TIM-4 antibodies. (B, D) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of (B) breast milk-derived exosomes (0.035 μg) or (D) plasma-derived exosomes (0.05 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA. **P<0.01, ****P<0.0001. YU-2, (NL-LucR.T2A-YU2. ecto) engineered to express Renilla luciferase (LucR) and ENV from the YU-2 virus strain. Abbreviations: TIM, T cell immunoglobulin and mucin; ANOVA, analysis of variance; RLU, relative luminescence unit; BM, breast milk; exo, exosomes.