RESUMEN
We show that the pertussis toxin B oligomer (PTX-B), and the PTX mutant PT9K/129G, which is safely administered in vivo, inhibit both transcription and secretion of TGF-beta elicited by HIV-1 Tat in NK cells. Tat-induced TGF-beta mRNA synthesis is also blocked by the ERK1 inhibitor PD98059, suggesting that ERK1 is needed for TGF-beta production. Moreover, Tat strongly activates the c-Jun component of the multimolecular complex AP-1, whereas TGF-beta triggers c-Fos and c-Jun. Of note, treatment of NK cells with PTX-B or PT9K/129G inhibits Tat- and TGF-beta-induced activation of AP-1. TGF-beta enhances starvation-induced NK cell apoptosis, significantly reduces transcription of the antiapoptotic protein Bcl-2, and inhibits Akt phosphorylation induced by oligomerization of the triggering NK cell receptor NKG2D. All these TGF-beta-mediated effects are prevented by PTX-B or PT9K/129G through a PI3K-dependent mechanism, as demonstrated by use of the specific PI3K inhibitor, LY294002. Finally, PTX-B and PT9K/129G up-regulate Bcl-x(L), the isoform of Bcl-x that protects cells from starvation-induced apoptosis. It is of note that in NK cells from patients with early HIV-1 infection, mRNA expression of Bcl-2 and Bcl-x(L) was consistently lower than that in healthy donors; interestingly, TGF-beta and Tat were detected in the sera of these patients. Together, these data suggest that Tat-induced TGF-beta production and the consequent NK cell failure, possibly occurring during early HIV-1 infection, may be regulated by PTX-B and PT9K/129G.
Asunto(s)
Apoptosis/inmunología , Regulación hacia Abajo/inmunología , Productos del Gen tat/fisiología , Células Asesinas Naturales/inmunología , Toxina del Pertussis/genética , Toxina del Pertussis/inmunología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/biosíntesis , Adyuvantes Inmunológicos/antagonistas & inhibidores , Adyuvantes Inmunológicos/fisiología , Sustitución de Aminoácidos/genética , Apoptosis/genética , Células Cultivadas , Regulación hacia Abajo/genética , Activación Enzimática/inmunología , Productos del Gen tat/antagonistas & inhibidores , Glicina/genética , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Lisina/genética , Toxina del Pertussis/toxicidad , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Subunidades de Proteína/toxicidad , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/toxicidad , Proteína bcl-X , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin and granzymes, and IFN-gamma secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-gamma release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.