To ascertain which individual PDE4 subtype(s) mediate the inhibitory effect on T cell function, PDE4 subtype-specific siRNAs were nucleofected into human primary CD4+ T cells. After a 24 h resting period, transfected cells were stimulated with anti-CD3/CD28. 1.5 µM non-targeting siRNA was used as control for statistical analysis (NEG_si). The functional impact of individual siRNAs on cytokine release (Figure 37, Figure 38, and Figure 39) and on cell proliferation (Figure 40) is reported in % of the RP73401 effect shown in Figure 35 (second bar
of experimental read-out parameters), which can be assumed to represent the maximal effect and was set therefore to 100%. Because additional application of the PDE3 inhibitor motapizone can enlarge the effects of PDE4 inhibition in an overadditive manner (see Figure 35, last column of experimental read-out parameters), all experiments were performed both in the absence and in the presence of 10 µM motapizone. The functional impact of individual siRNAs on cytokine release and on cell proliferation in the presence of motapizone is reported in % of the combined RP73401 and motapizone effect, which was set to 100% in these experiments.
The application of non-targeting siRNA (NEG_si in Figures) had no effect on any of the investigated functional read-out parameters. For better clarity, the individual effects of PDE4 subtype-specific siRNAs on T cell function will not only be reported in the following chapters, but will also be summarized in Table 10 (chapter 3.8.5, reported as mean ± SD).
3.8.1 Functional impact of PDE4 subtype-specific siRNAs on IL-2 release in CD4+ T cells Regarding IL-2 synthesis 24 h after stimulation, siRNA targeting PDE4A showed no significant inhibitory effect, however, knockdown of either PDE4B or PDE4D significantly suppressed anti-CD3/CD28 induced IL-2 release to an extent close to that observed with RP73401 (Figure 37A). Remarkably, the combined application of siRNAs targeting PDE4A, PDE4B, and PDE4D (PDE4_all_si), caused the most pronounced inhibitory effect on IL-2 release 24 h after stimulation. The additional application of motapizone qualitatively confirmed the effects of PDE4 subtype-specific siRNAs, however, the overall efficacy of the siRNAs to suppress IL-2 release 24 h after stimulation quantitatively decreased in the presence of motapizone when compared to the pronounced effects of the combined application of RP73401/motapizone (Figure 37B).
To study the functional effects of PDE4 subtype-specific siRNAs on IL-2 release at a later time point, cells were stimulated for 48 h. At this time point, the efficacies of PDE4 subtype-specific siRNAs to suppress anti-CD3/CD28 induced IL-2 release were overall less pronounced than the effects observed with 1 µM RP73401. However, whereas siRNA targeting PDE4A showed no significant inhibitory effect on IL-2 synthesis, knockdown of either PDE4B or PDE4D significantly suppressed anti-CD3/CD28 induced IL-2 release, with the combined application of all siRNAs being most effective (Figure 37C). Likewise to the experiments performed with the 24 h stimulation period, the additional application of motapizone qualitatively confirmed the individual effects of the applied PDE4-subtype specific siRNAs on IL-2 secretion 48 h after stimulation (Figure 37D).
Figure 37. Suppression of anti-CD3/CD28 induced IL-2 synthesis by PDE4 subtype-specific siRNAs. 1.5 µM non-targeting siRNA (NEG_si) or PDE4 subtype-specific siRNAs were nucleofected into human primary CD4+ T cells. Cells were left in culture for 24 h and were then stimulated with 0.3 µg anti-CD3/well in combination with 0.3 µg anti-CD28/ml. The siRNA-mediated suppression of IL-2 secretion after 24 h in the absence (A) or presence (B) of 10 µM motapizone and the siRNA-mediated suppression of IL-2 secretion after 48 h in the absence (C) or presence (D) of 10 µM motapizone is reported in % of the RP73401 effect. NEG_si, non-targeting control siRNA; PDE4_all_si, combined application of PDE4A_si, PDE4B_si, and PDE4D_si. Results are expressed as mean ± SD of 4 - 6 donors. Significance of differences is indicated: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to 1.5 µM NEG_si.
3.8.2 Functional impact of PDE4 subtype-specific siRNAs on IFN-γ release in CD4+ T cells Because substantial amounts of IFN-γ were only measured at later time points, the functional impact of PDE4 subtype-specific siRNAs on IFN-γ was investigated 48 h and 72 h after anti-CD3/CD28 stimulation. After stimulation for 48 h, siRNA targeting PDE4A and PDE4B had no significant inhibitory effect on IFN-γ release. In contrast, siRNA targeting PDE4D had a similar suppressive effect on IFN-γ synthesis than RP73401 (Figure 38A). However, the inhibitory effect of PDE4D-siRNA was increased by the combined application of all siRNAs. Although the overall efficacy of the PDE4 subtype-specific siRNAs quantitatively decreased by the additional application of motapizone, the predominant inhibitory effect of the siRNA targeting PDE4D was qualitatively confirmed (Figure 38B).
To ascertain whether the functional effects of PDE4 subtype-specific siRNAs on anti-CD3/CD28 induced IFN-γ generation were maintained for a longer time period, the experiments were repeated and cells were stimulated for 72 h. At this time point, similar results were observed than reported for the 48 h stimulation period. Whilst PDE4A-siRNA had again no significant inhibitory effect on IFN-γ release, PDE4B-siRNA had a slightly higher inhibitory
effect, and PDE4D-siRNA showed the most pronounced inhibitory effect on IFN-γ synthesis similar to RP73401 (Figure 38C). The combined application of all siRNAs was most effective in inhibiting IFN-γ release 72 h after stimulation. Considering the additional application of motapizone (Figure 38D), the results qualitatively confirmed the individual suppressive effects of the applied PDE4-subtype specific siRNAs on anti-CD3/CD28 induced IFN-γ synthesis observed in the absence of motapizone. However, the overall efficacies of the siRNAs decreased compared to the pronounced effect of RP73401 in combination with motapizone.
Figure 38. Suppression of anti-CD3/CD28 induced IFN-γ synthesis by PDE4 subtype-specific siRNAs. Experiments were performed as detailed in the legend of Figure 37, however, read-out parameters were the siRNA-mediated suppression of anti-CD3/CD28 induced IFN-γ secretion after 48 h in the absence (A) or presence (B) of 10 µM motapizone and after 72 h in the absence (C) or presence (D) of 10 µM motapizone. Results are expressed as mean ± SD of 5 - 6 donors.
Significance of differences is indicated: ns, not significant; *, p < 0.05; ***, p < 0.001, compared to 1.5 µM NEG_si.
3.8.3 Functional impact of PDE4 subtype-specific siRNAs on IL-5 release in CD4+ T cells Although substantial amounts of IL-5 were measured in the supernatants of anti-CD3/CD28 stimulated human primary CD4+ T cells at 48 h and 72 h (see Figure 34), the reported effects of the nucleofection procedure alone (see chapter 3.6.3) resulted in IL-5 levels that were at the detection limit at 48 h. Thus, the functional impact of PDE4 subtype-specific siRNAs on IL-5 release was only investigated at 72 h. At this time point, IL-5 synthesis was not significantly affected by the application of siRNAs targeting PDE4A and PDE4B, but was significantly inhibited by knockdown of PDE4D, to an extent close to that obtained with RP73401 (Figure 39A). The combined application of siRNAs targeting PDE4A, PDE4B, and PDE4D caused the most pronounced inhibitory effect on cytokine release. Whilst the combination of
RP73401 and motapizone was highly effective to suppress anti-CD3/CD28 induced IL-5 release, the combination of PDE4 subtype-specific siRNAs and motapizone was overall less effective (Figure 39B). However, the application of PDE4D-siRNA alone and the combined application of siRNAs were shown to significantly suppress IL-5 secretion in the presence of motapizone, which qualitatively confirmed the results obtained in the absence of motapizone.
Figure 39. Suppression of anti-CD3/CD28 induced IL-5 synthesis by PDE4 subtype-specific siRNAs. Experiments were performed as detailed in the legend of Figure 37, however, read-out parameters were the siRNA-mediated suppression of anti-CD3/CD28 induced IL-5 secretion after 72 h in the absence (A) or presence (B) of 10 µM motapizone. Results are expressed as mean ± SD of 4 donors. Significance of differences is indicated: ns, not significant; **, p < 0.01; ***, p < 0.001, compared to 1.5 µM NEG_si.
3.8.4 Functional impact of PDE4 subtype-specific siRNAs on proliferation in CD4+ T cells Whereas in the latter experiments the determination of cytokine levels was taken as functional read-out parameter (as detailed in chapters 3.8.1 - 3.8.3), the functional impact of PDE4 subtype-specific siRNAs on anti-CD3/CD28 induced T cell proliferation was ascertained in the following set of experiments. Under the conditions applied, the relatively weak inhibitory effect of RP73401 on T cell proliferation after 66 h of anti-CD3/CD28 stimulation (compare Figure 35) was mimicked by PDE4D knockdown, whereas siRNA targeting PDE4A and PDE4B were hardly effective (Figure 40A). Furthermore, the addition of the latter two siRNAs to PDE4D-siRNA did not further increase the inhibitory effect on T cell proliferation. Although the additional application of motapizone qualitatively confirmed the effects of PDE4 subtype-specific siRNAs on proliferation 66 h after stimulation (Figure 40B), the overall anti-proliferative efficacy of the siRNAs quantitatively decreased in the presence of motapizone compared to the combined effects of RP73401 and motapizone.
Figure 40. Suppression of anti-CD3/CD28 induced T cell proliferation by PDE4 subtype-specific siRNAs. Experiments were performed as detailed in the legend of Figure 37, however, read-out parameters were the siRNA-mediated suppression of anti-CD3/CD28 induced T cell proliferation ([3H]-thymidine incorporation) after 66 h in the absence (A) or presence (B) of 10 µM motapizone. Results are expressed as mean ± SD of 6 donors. Significance of differences is indicated: ns, not significant; *, p < 0.05; ***, p < 0.001, compared to 1.5 µM NEG_si.
3.8.5 Summary: Impact of PDE4 subtype-specific siRNAs on T cell function
As detailed above (chapters 3.8.1 - 3.8.4), individual PDE4 subtype-specific siRNAs were shown to affect anti-CD3/CD28 induced T cell cytokine release (IL-2, IFN-γ, and IL-5) and T cell proliferation differently. For the purpose of clarity, the functional impact of the siRNAs was reported without the respective numerical values. To get a comprehensive overview of the experimental read-outs, the functional impact of individual siRNAs on cytokine release and on cell proliferation is summarized in Table 10.
Table 10. Summary of PDE4 subtype-specific, siRNA-mediated suppression of T cell functionsa
Mota-
Mean suppression by siRNA in [%] of RP73401 effect ±±±± SD (p-value)
Parameter pizone NEG_si PDE4A_si PDE4B_si PDE4D_si PDE4_all_si n IL-2 siRNAs (PDE4A_si, PDE4B_si, or PDE4D_si, respectively). Additionally, PDE4A_si, PDE4B_si, and PDE4D_si siRNAs were applied in combination (PDE4_all_si). Cells were left in culture for 24 h and were then stimulated with 0.3 µg anti-CD3/well and 0.3 µg anti-CD28/ml. Besides, nucleofected cells (no siRNA applied) were treated with 0.1% DMSO or 1 µM RP73401 (see Figure 35). The siRNA-mediated suppression of T cell functions is reported in % of the RP73401 effect. In addition, 10 µM motapizone were included. For the latter experiments, the siRNA-effects are reported in % of the combined RP73401 and motapizone effect. Significance of differences is indicated: ns, not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared to 1.5 µM NEG_si.
4 DISCUSSION
One aim of this study was to clarify the time-dependent expression profile and regulation of the four PDE4 subtypes in human primary immune cells, such as CD4+ T cells, CD8+ T cells, monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells. Next, it was intended to validate an antisense- and a siRNA-mediated knockdown strategy to induce PDE4 subtype-specific mRNA and protein knockdown in the human lung adenocarcinoma epithelial cell line A549 and to transfer the superior strategy to human primary CD4+ T cells.
The final intension was to elucidate which PDE4 subtypes are of importance for immune cell functions such as T cell cytokine production and proliferation.