Limits...
Phosphodiesterase 4B negatively regulates endotoxin-activated interleukin-1 receptor antagonist responses in macrophages

View Article: PubMed Central - PubMed

ABSTRACT

Activation of TLR4 by lipopolysaccharide (LPS) induces both pro-inflammatory and anti-inflammatory cytokine production in macrophages. Type 4 phosphodiesterases (PDE4) are key cAMP-hydrolyzing enzymes, and PDE4 inhibitors are considered as immunosuppressors to various inflammatory responses. We demonstrate here that PDE4 inhibitors enhance the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) secretion in LPS-activated mouse peritoneal macrophages, and this response was regulated at the transcriptional level rather than an increased IL-1Ra mRNA stability. Studies with PDE4-deficient macrophages revealed that the IL-1Ra upregulation elicited by LPS alone is PKA-independent, whereas the rolipram-enhanced response was mediated by inhibition of only PDE4B, one of the three PDE4 isoforms expressed in macrophages, and it requires PKA but not Epac activity. However, both pathways activate CREB to induce IL-1Ra expression. PDE4B ablation also promoted STAT3 phosphorylation (Tyr705) to LPS stimulation, but this STAT3 activation is not entirely responsible for the IL-1Ra upregulation in PDE4B-deficient macrophages. In a model of LPS-induced sepsis, only PDE4B-deficient mice displayed an increased circulating IL-1Ra, suggesting a protective role of PDE4B inactivation in vivo. These findings demonstrate that PDE4B negatively modulates anti-inflammatory cytokine expression in innate immune cells, and selectively targeting PDE4B should retain the therapeutic benefits of nonselective PDE4 inhibitors.

No MeSH data available.


Related in: MedlinePlus

STAT3 phosphorylation enhanced by rolipram or PDE4B ablation does not contribute to the increased IL-1Ra production in LPS-stimulated macrophages.Peritoneal macrophages from PDE4B+/+ mice were incubated with LPS (100 ng/ml) in the presence or absence of 10 μM rolipram for indicated times (A), or the cells from both PDE4B+/+ and PDE4B−/− mice were treated with LPS (100 ng/ml) for 2 h (C). The STAT3 phosphorylation (Tyr705) was detected by immunoblotting as described in Methods (A and C). Representative Western blots are shown. The blots were cropped for improving clarity and full-length blots are presented in Supplementary Figs 4 and 5. The level of phosphorylation relative to total STAT3 protein was quantified and expressed as fold induction to unstimulated control (B) or to the PDE4B+/+ group (D). *P < 0.05, compared with the corresponding groups treated with LPS alone (n = 4 in B); **P < 0.005, compared with the PDE4B+/+ group (n = 3 in D). (E) Bone marrow-derived macrophages prepared from STAT3+/+ and STAT3−/− mice were incubated with increasing concentrations of rolipram for 20 min prior to LPS (100 ng/ml) stimulation for 8 h. IL-1Ra accumulation in the medium was measured by ELISA. Data are the mean ± SEM (n = 7–8).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5382768&req=5

f7: STAT3 phosphorylation enhanced by rolipram or PDE4B ablation does not contribute to the increased IL-1Ra production in LPS-stimulated macrophages.Peritoneal macrophages from PDE4B+/+ mice were incubated with LPS (100 ng/ml) in the presence or absence of 10 μM rolipram for indicated times (A), or the cells from both PDE4B+/+ and PDE4B−/− mice were treated with LPS (100 ng/ml) for 2 h (C). The STAT3 phosphorylation (Tyr705) was detected by immunoblotting as described in Methods (A and C). Representative Western blots are shown. The blots were cropped for improving clarity and full-length blots are presented in Supplementary Figs 4 and 5. The level of phosphorylation relative to total STAT3 protein was quantified and expressed as fold induction to unstimulated control (B) or to the PDE4B+/+ group (D). *P < 0.05, compared with the corresponding groups treated with LPS alone (n = 4 in B); **P < 0.005, compared with the PDE4B+/+ group (n = 3 in D). (E) Bone marrow-derived macrophages prepared from STAT3+/+ and STAT3−/− mice were incubated with increasing concentrations of rolipram for 20 min prior to LPS (100 ng/ml) stimulation for 8 h. IL-1Ra accumulation in the medium was measured by ELISA. Data are the mean ± SEM (n = 7–8).

Mentions: Stimulation of macrophages with LPS has been shown to induce phosphorylation of signal transducer and activator of transcription (STAT)3 at Tyr705, and this STAT3 activation is enhanced by the presence of the cAMP elevator prostaglandin E2 (PGE2)30. Additionally, a previous study by Carl et al. demonstrated that the IL-10-induced IL-1Ra expression in LPS-stimulated macrophages is mediated by an increase in STAT3-Tyr705 phosphorylation and transcription14. These findings prompted us to investigate whether elevation of cAMP by PDE4 inhibitor or PDE4B ablation promotes STAT3 phosphorylation, and whether such phosphorylation mediates the enhanced IL-1Ra production in LPS-stimulated PDE4B−/− macrophages. For this purpose, we first treated wild-type peritoneal macrophages with LPS in the presence or absence of rolipram for various times. Western blotting using an antibody specific to Tyr705-phospho-STAT3 revealed that little or no STAT3 phosphorylation was detected in the untreated cells (Fig. 7A and B). The protein phosphorylation was increased with time up to 2 h and declined thereafter. The level of phosphorylation was significantly enhanced when the cells were incubated with rolipram (p < 0.05), reaching approximately 2.0- and 2.6-fold increases at 2 h and 3 h LPS stimulation, respectively (Fig. 7A and B). We further probed the effect of PDE4B on STAT3 phosphorylation and found that following 2 h LPS treatment the level of STAT3 Tyr705 phosphorylation was significantly increased in 4B−/− macrophages compared with the wild-type cells (P < 0.005) (Fig. 7C and D). These results support the notion that STAT3 activation can be induced by cAMP-elevating agents in LPS-stimulated macrophages.


Phosphodiesterase 4B negatively regulates endotoxin-activated interleukin-1 receptor antagonist responses in macrophages
STAT3 phosphorylation enhanced by rolipram or PDE4B ablation does not contribute to the increased IL-1Ra production in LPS-stimulated macrophages.Peritoneal macrophages from PDE4B+/+ mice were incubated with LPS (100 ng/ml) in the presence or absence of 10 μM rolipram for indicated times (A), or the cells from both PDE4B+/+ and PDE4B−/− mice were treated with LPS (100 ng/ml) for 2 h (C). The STAT3 phosphorylation (Tyr705) was detected by immunoblotting as described in Methods (A and C). Representative Western blots are shown. The blots were cropped for improving clarity and full-length blots are presented in Supplementary Figs 4 and 5. The level of phosphorylation relative to total STAT3 protein was quantified and expressed as fold induction to unstimulated control (B) or to the PDE4B+/+ group (D). *P < 0.05, compared with the corresponding groups treated with LPS alone (n = 4 in B); **P < 0.005, compared with the PDE4B+/+ group (n = 3 in D). (E) Bone marrow-derived macrophages prepared from STAT3+/+ and STAT3−/− mice were incubated with increasing concentrations of rolipram for 20 min prior to LPS (100 ng/ml) stimulation for 8 h. IL-1Ra accumulation in the medium was measured by ELISA. Data are the mean ± SEM (n = 7–8).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382768&req=5

f7: STAT3 phosphorylation enhanced by rolipram or PDE4B ablation does not contribute to the increased IL-1Ra production in LPS-stimulated macrophages.Peritoneal macrophages from PDE4B+/+ mice were incubated with LPS (100 ng/ml) in the presence or absence of 10 μM rolipram for indicated times (A), or the cells from both PDE4B+/+ and PDE4B−/− mice were treated with LPS (100 ng/ml) for 2 h (C). The STAT3 phosphorylation (Tyr705) was detected by immunoblotting as described in Methods (A and C). Representative Western blots are shown. The blots were cropped for improving clarity and full-length blots are presented in Supplementary Figs 4 and 5. The level of phosphorylation relative to total STAT3 protein was quantified and expressed as fold induction to unstimulated control (B) or to the PDE4B+/+ group (D). *P < 0.05, compared with the corresponding groups treated with LPS alone (n = 4 in B); **P < 0.005, compared with the PDE4B+/+ group (n = 3 in D). (E) Bone marrow-derived macrophages prepared from STAT3+/+ and STAT3−/− mice were incubated with increasing concentrations of rolipram for 20 min prior to LPS (100 ng/ml) stimulation for 8 h. IL-1Ra accumulation in the medium was measured by ELISA. Data are the mean ± SEM (n = 7–8).
Mentions: Stimulation of macrophages with LPS has been shown to induce phosphorylation of signal transducer and activator of transcription (STAT)3 at Tyr705, and this STAT3 activation is enhanced by the presence of the cAMP elevator prostaglandin E2 (PGE2)30. Additionally, a previous study by Carl et al. demonstrated that the IL-10-induced IL-1Ra expression in LPS-stimulated macrophages is mediated by an increase in STAT3-Tyr705 phosphorylation and transcription14. These findings prompted us to investigate whether elevation of cAMP by PDE4 inhibitor or PDE4B ablation promotes STAT3 phosphorylation, and whether such phosphorylation mediates the enhanced IL-1Ra production in LPS-stimulated PDE4B−/− macrophages. For this purpose, we first treated wild-type peritoneal macrophages with LPS in the presence or absence of rolipram for various times. Western blotting using an antibody specific to Tyr705-phospho-STAT3 revealed that little or no STAT3 phosphorylation was detected in the untreated cells (Fig. 7A and B). The protein phosphorylation was increased with time up to 2 h and declined thereafter. The level of phosphorylation was significantly enhanced when the cells were incubated with rolipram (p < 0.05), reaching approximately 2.0- and 2.6-fold increases at 2 h and 3 h LPS stimulation, respectively (Fig. 7A and B). We further probed the effect of PDE4B on STAT3 phosphorylation and found that following 2 h LPS treatment the level of STAT3 Tyr705 phosphorylation was significantly increased in 4B−/− macrophages compared with the wild-type cells (P < 0.005) (Fig. 7C and D). These results support the notion that STAT3 activation can be induced by cAMP-elevating agents in LPS-stimulated macrophages.

View Article: PubMed Central - PubMed

ABSTRACT

Activation of TLR4 by lipopolysaccharide (LPS) induces both pro-inflammatory and anti-inflammatory cytokine production in macrophages. Type 4 phosphodiesterases (PDE4) are key cAMP-hydrolyzing enzymes, and PDE4 inhibitors are considered as immunosuppressors to various inflammatory responses. We demonstrate here that PDE4 inhibitors enhance the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) secretion in LPS-activated mouse peritoneal macrophages, and this response was regulated at the transcriptional level rather than an increased IL-1Ra mRNA stability. Studies with PDE4-deficient macrophages revealed that the IL-1Ra upregulation elicited by LPS alone is PKA-independent, whereas the rolipram-enhanced response was mediated by inhibition of only PDE4B, one of the three PDE4 isoforms expressed in macrophages, and it requires PKA but not Epac activity. However, both pathways activate CREB to induce IL-1Ra expression. PDE4B ablation also promoted STAT3 phosphorylation (Tyr705) to LPS stimulation, but this STAT3 activation is not entirely responsible for the IL-1Ra upregulation in PDE4B-deficient macrophages. In a model of LPS-induced sepsis, only PDE4B-deficient mice displayed an increased circulating IL-1Ra, suggesting a protective role of PDE4B inactivation in vivo. These findings demonstrate that PDE4B negatively modulates anti-inflammatory cytokine expression in innate immune cells, and selectively targeting PDE4B should retain the therapeutic benefits of nonselective PDE4 inhibitors.

No MeSH data available.


Related in: MedlinePlus