Functional Impact of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary Immune Cells

Abstract

As a second messenger, adenosine 3 -5 cyclic monophosphate (cAMP) plays a crucial role for intracellular signaling. In immune cells, cAMP has mainly inhibitory properties on cellular responses. Thus, type 4 phosphodiesterases (PDE4), which are capable to hydrolyze cAMP, are critical regulators of inflammatory cell signals by attenuating the negative constraint of cAMP. The PDE4 isoenzyme family consists of four distinct subtypes, each encoded by one gene. Because PDE4 subtypes are widely expressed in inflammatory cells, the PDE4 family became an attractive drug target for the treatment of chronic inflammatory diseases. In fact, PDE4 inhibitors have been shown to have anti-inflammatory and immunomodulatory effects on a broad range of inflammatory and immunocompetent cells and have proved efficacy in clinical trials. However, it is largely unknown which PDE4 subtype(s) mediate(s) the inhibitory effects in a defined immune cell.The objectives of the present study were i) to ascertain the expression patterns of PDE4 subtypes in different human primary immune cells via quantitative PCR, PDE activity assays, and immunodetection experiments; ii) to validate an antisense- and a siRNA-mediated, PDE4 subtype-specific knockdown technique in the cell line A549 and to transfer the superior knockdown strategy to human primary CD4+ T cells; and iii) to elucidate the functional impact of PDE4 subtypes in human primary CD4+ T cells.In resting human primary CD4+ T cells, low levels of PDE4A were detected, whereas PDE4B and PDE4D mRNA expression and protein activity were substantially higher, with PDE4D being the predominant subtype. After anti-CD3/CD28 stimulation, PDE4A and PDE4D mRNA as well as protein activity were upregulated within five days, whereas PDE4B mRNA and activity showed a transient upregulation. Remarkably, the PDE4B and PDE4D short forms were found to contribute largely to the time-dependent upregulation of PDE4 subtypes in human primary CD4+ T cells. In resting human primary CD8+ T cells, PDE4B and PDE4D mRNA were predominantly expressed, whereas PDE4A mRNA had low expression levels. After anti-CD3/CD28 stimulation, the induction of PDE4 subtypes in CD8+ T cells resembled, also not equal in a quantitative manner, the induction of PDE4 subtypes in CD4+ T cells. In contrast to T cells, PDE4B mRNA was the predominantly expressed PDE4 subtype in freshly isolated human primary monocytes, followed by PDE4A and very low levels of PDE4D. During differentiation of monocytes to macrophages or to dendritic cells, PDE4B mRNA expression dramatically decreased, whereas PDE4A and PDE4D mRNA levels were less regulated. LPS stimulation of macrophages or dendritic cells strongly induced PDE4B, but not PDE4A and PDE4D mRNA.With the intention to elucidate the impact of individual PDE4 subtypes on cell function and because of the lack of PDE4 subtype-specific inhibitors, two strategies were evaluated to specifically knock down PDE4 subtypes. Initially, several second generation antisense (AS2nd) constructs or pools of short interfering RNAs (siRNAs) targeting PDE4A, PDE4B, or PDE4D mRNA were transfected into the human lung adenocarcinoma epithelial cell line A549. AS2nd constructs as well as siRNAs were shown to induce substantial, PDE4 subtype-specific knockdown of mRNA and protein 24 h and 72 h after transfection. Because siRNAs were effective in lower concentrations, were well tolerated, and showed less variability in between experiments, the siRNA-mediated knockdown strategy was transferred to human primary CD4+ T cells. The transfection of individual siRNAs into CD4+ T cells with the nucleofector electroporation technique resulted in significant, specific, and well-tolerated knockdown of PDE4 subtypes on mRNA and protein level.Finally, the validated siRNA-mediated knockdown technique was used to ascertain the functional relevance of PDE4 subtypes in human primary CD4+ T cells. SiRNA-mediated knockdown of either PDE4B or PDE4D inhibited anti-CD3/CD28 induced IL-2 release 24 h after stimulation to an extent overall similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast), pointing to an overlapping function of PDE4B and PDE4D for IL-2 synthesis. At 48 h after stimulation, efficacies of PDE4B- and PDE4D-siRNAs to suppress IL-2 release were slightly diminished. Considering the anti-CD3/CD28 induced generation of IFN-g and IL-5, PDE4D-siRNA showed a predominant inhibitory effect, suggesting nonredundant roles of PDE4 subtypes for T cell cytokine production. However, the inhibition of cytokines was most effective when PDE4 subtype-specific siRNAs were applied in combination, indicating complementary functions of PDE4 subtypes. Although the effect of PDE4 inhibition on T cell proliferation was small, the PDE4D-targeting siRNA alone had similar anti-proliferative effects than RP73401, whereas PDE4A- or PDE4B-siRNAs were hardly effective.