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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

Rolipram does not alter sIL-1Ra mRNA stability in LPS-stimulated macrophages.Mouse peritoneal macrophages were treated with LPS (10 ng/ml) in the presence or absence of 10 μM rolipram for 3 h, followed by the addition of actinomycin D (10 μg/ml) to inhibit RNA transcription. The levels of sIL-1Ra mRNA were measured at 0, 0.5, 1, 2, 4 and 6 h after actinomycin D addition by real-time PCR. The amounts of sIL-1Ra mRNA were normalized to GAPDH mRNA levels. Values are expressed as percent of total sIL-1Ra mRNA at the time of actinomycin D addition. Data are the mean ± SEM (n = 4–6).
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f3: Rolipram does not alter sIL-1Ra mRNA stability in LPS-stimulated macrophages.Mouse peritoneal macrophages were treated with LPS (10 ng/ml) in the presence or absence of 10 μM rolipram for 3 h, followed by the addition of actinomycin D (10 μg/ml) to inhibit RNA transcription. The levels of sIL-1Ra mRNA were measured at 0, 0.5, 1, 2, 4 and 6 h after actinomycin D addition by real-time PCR. The amounts of sIL-1Ra mRNA were normalized to GAPDH mRNA levels. Values are expressed as percent of total sIL-1Ra mRNA at the time of actinomycin D addition. Data are the mean ± SEM (n = 4–6).

Mentions: To investigate whether the steady-state level of sIL-1Ra mRNA induced by PDE4 inhibitors in macrophages is associated with an increase in transcription or mRNA stability, the RNA synthesis inhibitor actinomycin D was used to monitor the IL-1Ra mRNA expression profile in mouse peritoneal macrophages. The cells were stimulated with LPS in the presence or absence of rolipram for 3 h, followed by incubation with actinomycin D for different times. Real-time PCR analysis showed that the cells with or without rolipram treatment displayed a similar pattern of decrease in sIL-1Ra mRNA level up to at least 6 h (Fig. 3), indicating that the turnover rate or stability of this RNA was not affected by rolipram. Therefore, the rolipram-enhanced sIL-1Ra mRNA expression and protein secretion was mainly attributed to the increase in RNA transcription.


Phosphodiesterase 4B negatively regulates endotoxin-activated interleukin-1 receptor antagonist responses in macrophages
Rolipram does not alter sIL-1Ra mRNA stability in LPS-stimulated macrophages.Mouse peritoneal macrophages were treated with LPS (10 ng/ml) in the presence or absence of 10 μM rolipram for 3 h, followed by the addition of actinomycin D (10 μg/ml) to inhibit RNA transcription. The levels of sIL-1Ra mRNA were measured at 0, 0.5, 1, 2, 4 and 6 h after actinomycin D addition by real-time PCR. The amounts of sIL-1Ra mRNA were normalized to GAPDH mRNA levels. Values are expressed as percent of total sIL-1Ra mRNA at the time of actinomycin D addition. Data are the mean ± SEM (n = 4–6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Rolipram does not alter sIL-1Ra mRNA stability in LPS-stimulated macrophages.Mouse peritoneal macrophages were treated with LPS (10 ng/ml) in the presence or absence of 10 μM rolipram for 3 h, followed by the addition of actinomycin D (10 μg/ml) to inhibit RNA transcription. The levels of sIL-1Ra mRNA were measured at 0, 0.5, 1, 2, 4 and 6 h after actinomycin D addition by real-time PCR. The amounts of sIL-1Ra mRNA were normalized to GAPDH mRNA levels. Values are expressed as percent of total sIL-1Ra mRNA at the time of actinomycin D addition. Data are the mean ± SEM (n = 4–6).
Mentions: To investigate whether the steady-state level of sIL-1Ra mRNA induced by PDE4 inhibitors in macrophages is associated with an increase in transcription or mRNA stability, the RNA synthesis inhibitor actinomycin D was used to monitor the IL-1Ra mRNA expression profile in mouse peritoneal macrophages. The cells were stimulated with LPS in the presence or absence of rolipram for 3 h, followed by incubation with actinomycin D for different times. Real-time PCR analysis showed that the cells with or without rolipram treatment displayed a similar pattern of decrease in sIL-1Ra mRNA level up to at least 6 h (Fig. 3), indicating that the turnover rate or stability of this RNA was not affected by rolipram. Therefore, the rolipram-enhanced sIL-1Ra mRNA expression and protein secretion was mainly attributed to the increase in RNA transcription.

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