Platelet activation suppresses HIV-1 infection of T cells

Abstract

Background: Platelets, anucleate cell fragments abundant in human blood, can capture HIV-1 and platelet counts have been associated with viral load and disease progression. However, the impact of platelets on HIV-1 infection of T cells is unclear.

Results: We found that platelets suppress HIV-1 spread in co-cultured T cells in a concentration-dependent manner. Platelets containing granules inhibited HIV-1 spread in T cells more efficiently than degranulated platelets, indicating that the granule content might exert antiviral activity. Indeed, supernatants from activated and thus degranulated platelets suppressed HIV-1 infection. Infection was inhibited at the stage of host cell entry and inhibition was independent of the viral strain or coreceptor tropism. In contrast, blockade of HIV-2 and SIV entry was less efficient. The chemokine CXCL4, a major component of platelet granules, blocked HIV-1 entry and neutralization of CXCL4 in platelet supernatants largely abrogated their anti-HIV-1 activity.

Conclusions: Release of CXCL4 by activated platelets inhibits HIV-1 infection of adjacent T cells at the stage of virus entry. The inhibitory activity of platelet-derived CXCL4 suggests a role of platelets in the defense against infection by HIV-1 and potentially other pathogens.

Figure 1

Analysis of CD62P and C-type lectin expression on platelets. (A) CD62P expression on activated and resting platelets. Platelets maintained resting by treatment with PGE₁ (R-PLT), activated by exposure to TRAP (A-PLT) or platelets left untreated (PLT) were analyzed by flow cytometry for surface expression of CD62P. The geometric mean channel fluorescence was measured. The results present the average of three experiments with samples from two different donors. Error bars indicate standard deviation (SD). (B) Lectin expression on platelets. Untreated platelets were stained with anti-DC-SIGN, -MR and -LSECtin antibodies (grey histograms) or isotype-matched control antibodies (black lines) and analyzed by flow cytometry. Unstained platelets are depicted in black filled histograms. The results are representative of three independent experiments.

Figure 2

Platelets inhibit HIV-1 spread in T cells and release an anti-HIV-1 activity upon activation. (A) PHA stimulated PBMCs were infected with 10 pg of HIV-1 NL4-3 in the presence of untreated platelets (PLT, 1 × 10⁸/mL) or medium alone (control) and p24-antigen content in the supernatants was measured on day one and six post infection. The p24-antigen levels at day one post infection were below detection range. The results of a single representative experiment performed in duplicates are shown, error bars indicate SD. The results were confirmed in an independent experiment. (B) Platelets inhibit HIV-1 spread in adjacent T cells in a concentration- and activation status- dependent manner. The indicated amounts of non-resting platelets (PLT, platelets left untreated) or activated platelets (A-PLT, platelets treated with TRAP) were added to C8166-SEAP T cells, the cultures infected with 10 pg of HIV-1 NL4-3 and SEAP-activity measured at day five post infection. The average ± SEM of three experiments performed in triplicates is shown, SEAP-activity measured in the absence of platelets was set as 100%. (C) Activation of platelets induces the release of one or more HIV-1 inhibitory factors. The indicator cell line TZM-bl was incubated with supernatants from resting (R-PLT Sup) and activated platelets (A-PLT Sup) or incubated with PBS or PBS containing 10 μM PGE₁ or 100 μM TRAP. Subsequently, the cells were infected with HIV-1 NL4-3 and luciferase activities in the lysates of infected cells were measured. The results ± SD of a single experiment performed in triplicates is shown. Similar results were obtained in a separate experiment.

Figure 3

Supernatants from activated platelets inhibit HIV-1 but not HIV-2 and SIV entry, independent of viral strain and coreceptor tropism. (A) Supernatants from activated platelets efficiently inhibit cellular entry of HIV-1 but not HIV-2 and SIV. TZM-bl indicator cells were preincubated with the supernatants from TRAP-activated platelets (A-PLT Sup) or TRAP containing PBS (TRAP) or PBS. Subsequently, the cells were infected with pseudotypes bearing the indicated Env proteins or with replication competent HIV-2 Rod and SIVmac 239 and β-galactosidase activity in cell lysates was measured. The results ± SD of a representative experiment performed in triplicates are shown. Similar results were obtained in a separate experiment. (B) Inhibition of HIV-1 entry by supernatants from activated platelets is independent of the viral coreceptor tropism. The experiment was performed as described for (A) but HIV-1 NL4-3 chimeras harboring the V3 loops of the indicated primary HIV-1 isolates were used for infection of TZM-bl cells and luciferase activities in cell lysates were quantified. The results of a single representative experiment performed in triplicates are shown; error bars indicate SD. Similar results were obtained in a separate experiment. (C) Different molecular clones of HIV-1 are inhibited by the supernatants from activated platelets. The experiment was performed as in (B), but the indicated HIV-1 infectious clones were used. The average of two to four independent experiments performed in triplicates is shown, error bars indicate SEM.

Figure 4

CXCL4 in supernatants from activated platelets inhibits HIV-1. (A) The anti-HIV-1 factor(s) released from activated platelets are heat labile. Supernatants from activated platelets were left at RT or treated at 95°C for 15 minutes and used to inhibit infection of TZM-bl cells by HIV-1 NL4-3. The results of a representative experiment performed in triplicates are shown and were confirmed in 5 independent experiments, error bars indicate SD. (B) Recombinant CXCL4 inhibits HIV-1- and MLV-Env-driven host cell entry. Pseudotypes bearing the indicated Env proteins and normalized for comparable infectivity were used for infection of TZM-bl indicator cells in the presence and absence of recombinant CXCL4. β-galactosidase activities in cell lysates were quantified at 72 h post infection. The results of a representative experiment performed in triplicates are shown; error bars indicate SD. Comparable results were obtained in two independent experiments. (C) CXCL4 accounts for the anti-HIV-1 activity of platelet supernatants. Infectivity normalized pseudotypes were incubated with PBS, platelet supernatants or platelet supernatants preincubated with CXCL4-neutralizing antibody and infection of TZM-bl indicator cells was assessed. The results of a representative experiment are shown and were confirmed in two separate experiments. Infection by PBS treated viruses was set as 100%; error bars indicate SD.