RESUMEN
Toll-like receptors (TLRs) are a family of molecules that function as sensors for the detection of foreign pathogens through the recognition of nonvariable microbial motifs. Although numerous studies have focused on singular TLRs, less attention has been focused on how simultaneous signaling of multiple TLRs may result in counter-regulation of the effects of each. Here, we examine the counter-regulation that occurs during simultaneous stimulation of TLR7 and TLR9 on human plasmacytoid dendritic cells (PDCs) and B cells. Interestingly, we observed that the capacity for potent IFN-alpha-induction by TLR9 ligands like CpG-C and CpG-A is markedly reduced by concurrent small molecule TLR7 stimulation. However, this inhibition is specific to particular CpG motif-containing immunostimulatory sequence (ISS) functions such as IFN-alpha induction and BDCA-2 down-regulation. Other ISS activities such as PDC expression of CD80/CD86, secretion of IL-6, and B cell proliferation are not altered by the presence of TLR7 ligands (TLR7Ls). In concordance with the ability of TLR7Ls to decrease IFN-alpha secretion induced by ISS, we also find that the expression of interferon regulatory factor-7 (IRF-7), a transcriptional factor critical for IFN-alpha expression, is reduced. Furthermore, down-regulation of TLR9 mRNA expression is accelerated after TLR7 stimulation. These data indicate that TLR7 and TLR9 costimulation do not combine synergistically for IFN-alpha induction and demonstrate that, instead, a negative feedback mechanism has evolved, possibly to prevent levels of IFN-alpha secretion potentially detrimental to the host.
Asunto(s)
Linfocitos B/efectos de los fármacos , Interferón-alfa/biosíntesis , Oligodesoxirribonucleótidos/farmacología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Linfocitos B/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Diferenciación Celular , Proliferación Celular , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Lectinas Tipo C/metabolismo , Ligandos , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , ARN Mensajero/metabolismo , Receptores Inmunológicos/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 9/genéticaRESUMEN
The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.