Inhibition of phosphodiesterase type IV suppresses human immunodeficiency virus type 1 replication and cytokine production in primary T cells: involvement of NF-kappaB and NFAT

Abstract

Rolipram, a phosphosdiesterase type IV-specific inhibitor, prevented p24 antigen release from anti-CD3-activated human immunodeficiency virus (HIV)-infected T cells and CD4(+)-cell depletion associated with viral replication in a dose-responsive manner but minimally inhibited T-cell proliferation. Moreover, rolipram reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) by HIV-infected T cells. The transcriptional ability of a luciferase reporter gene under control of the HIV long terminal repeat, induced by phorbol myristic acetate plus ionomycin or by TNF-alpha, in primary T and Jurkat cells was also inhibited by rolipram. Rolipram inhibited NF-kappaB and NFAT activation induced by T-cell activation in Jurkat and primary T cells, as measured by transient transfection of reporter genes and electrophoretic mobility shift assays. Exogenous addition of TNF-alpha in the presence of rolipram restored NF-kappaB but not NFAT activation or p24 release. Addition of dibutyryl-cyclic AMP (dBcAMP) mimicked the effects of rolipram on p24 antigen release, NF-kappaB activation, and TNF-alpha secretion, but it did not affect NFAT activation or IL-10 production. The protein kinase A inhibitor KT5720 prevented the inhibition of TNF-alpha secretion but not that of HIV type 1 (HIV-1) replication caused by rolipram. Our data indicate that blockade of phosphodiesterase type IV could be of benefit against HIV-1 disease by modulating cytokine secretion and transcriptional regulation of HIV replication, and they suggest an important role of NFAT in HIV replication in primary T cells. Some of those activities cannot be ascribed solely to its ability to increase cAMP.

FIG. 1

Effect of RP on activated HIV-1-infected T cells. Human T cells were stimulated with immobilized anti-CD3 antibody and infected with HIV-1 or mock infected. RP (100 μM) was added to the cultures as indicated. At 3, 6, and 9 days after infection, cultures were assayed for the p24 viral antigen (Ag) content in the supernatants by using an antigen capture immunoassay (A), TNF-α and IL-10 contents in the supernatants of infected cells by using specific ELISAs (B), or the number of CD4⁺ cells in infected or mock-infected cultures by direct flow cytometry (C). Data shown are the means ± standard deviations for triplicate cultures.

FIG. 2

Dose-response effect of RP on activated HIV-1-infected T cells. Human cells were stimulated with immobilized anti-CD3 antibody and infected with HIV-1. RP, at the indicated concentrations, was added to the cultures. Culture supernatants were assayed for p24 viral antigen (Ag) content 9 days after infection, using an antigen capture immunoassay. Proliferation was evaluated by [³H]thymidine incorporation during the last 16 h of culture, 3 days after infection. Cytokines in the supernatants were evaluated by ELISA 3 days postinfection. Results shown are the means ± standard deviations from three experiments with different donors, each one carried out in triplicate, standardized as percentages of control values. Unstimulated HIV-infected T cells did not produce detectable amounts of either p24 antigen or cytokines and did not proliferate (not shown).

FIG. 3

Effect of dBcAMP on activated HIV-1-infected human T cells. Human T cells were stimulated with immobilized anti-CD3 antibody and were infected with HIV-1 as described in the legend to Fig. 1. dBcAMP at the indicated concentrations was added to the cultures. The results shown (means ± standard deviations for triplicate cultures) correspond to the same experiments as in Fig. 1 standardized as percentages of control values. Ag, antigen.

FIG. 4

Effect of PK-A inhibitors on RP and dBcAMP activities. Human T cells were stimulated with immobilized anti-CD3 antibody and infected with HIV-1. TNF-α secretion and p24 antigen (Ag) release were evaluated 3 and 9 days after infection as described in Materials and Methods. dBcAMP (300 μM), RP (100 μM), and KT5720 (200 nM), alone or in combination, were added to the cultures. Results are means ± standard deviations.

FIG. 5

Effect of PDE IV inhibition on nuclear factor and HIV-1 LTR-driven transcription. Human resting T cells were transfected with the reporter plasmid HIV-LTR-luc alone or cotransfected with pCMV-Tat (A) or with reporter plasmid NF-κB-luc, NFAT-luc, AP-1-luc, or CRE-luc (B). After 14 h, the cells were stimulated or not with PMA (10 ng/ml) plus ionomycin (IONO) (1 μM) in the presence or absence of RP (100 μM), dBcAMP (300 μM), or CsA (100 ng/ml) as indicated, and 12 h later the amount of luciferase in the cells was estimated. Shown are the means ± standard deviations from three experiments using T cells from three different donors.

FIG. 6

Inhibition of NF-κB and NFAT activation by RP. Human HIV-infected T cells were stimulated with immobilized anti-CD3 antibody (αCD3) or with PMA (10 ng/ml) plus ionomycin (1 μM) (P+I) in the presence of RP (100 μM), dBcAMP (300 μM), CsA (100 ng/ml), or TNF-α (30 ng/ml) as indicated. The binding activity of NFAT (A and B) or NF-κB (C) in the nuclei of T cells was assayed 14 h later, using a κB-HIV or distal IL-2 NFAT site labeled probe as described in the text. Control specific binding was detected by using as a competitor a 50-fold excess of unlabeled oligonucleotide. uns, unstimulated.

FIG. 7

Effect of exogenous TNF-α on the inhibitory effect of RP. Human T cells were stimulated with immobilized anti-CD3 antibody and infected with HIV-1. RP (500 μM) and/or TNF-α (100 ng/ml) was added to the cultures as indicated. Shown is the mean p24 antigen (Ag) released (± standard deviation) in triplicate culture supernatants at day 9 after infection from two independent experiments.

FIG. 8

Effect of RP on TNF-α-induced LTR transcription. Jurkat cells were transfected with LTR-luc. After 14 h, the cells were stimulated with TNF-α (10 or 30 ng/ml) in the presence or absence of RP (50 or 100 μM), and 12 h later the amount of luciferase in the cells was estimated. Results are means ± standard deviations.