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Inhibition of PDE4B suppresses inflammation by increasing expression of the deubiquitinase CYLD.

Komatsu K, Lee JY, Miyata M, Hyang Lim J, Jono H, Koga T, Xu H, Yan C, Kai H, Li JD - Nat Commun (2013)

Bottom Line: Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response.Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4.These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

View Article: PubMed Central - PubMed

Affiliation: Center for Inflammation, Immunity & Infection and Department of Biology, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303, USA.

ABSTRACT
The deubiquitinase CYLD acts as a key negative regulator to tightly control overactive inflammation. Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response. Here, we show that inhibition of phosphodiesterase 4B (PDE4B) markedly enhances upregulation of CYLD expression in response to bacteria, thereby suggesting that PDE4B acts as a negative regulator for CYLD. Interestingly, in Cyld-deficient mice, inhibition of PDE4B no longer suppresses inflammation. Moreover, PDE4B negatively regulates CYLD via specific activation of JNK2 but not JNK1. Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4. These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

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PDE4B has a crucial role in mediating NTHi-induced inflammation by negatively regulating CYLD expression.(a) PDE4 mRNA expression was measured in A549 cells stimulated with NTHi for 1.5 h. (b) Cells were treated with NTHi, and cell lysates were analysed by immunoblotting with the indicated antibodies. PDE4 mRNA expression was measured in (c) lung and (d) middle ear tissues from C57BL/6J mice inoculated with NTHi for 4 h. Mouse tissues from (e) lung and (f) middle ear were stained against PDE4B after NTHi inoculation (magnification × 200 in e; × 400 in f, respectively). Scale bars, 20 μm. (g) Lysates from cells treated with NTHi were immunoprecipitated with anti-PDE4B antibody, and analysed by PDE activity assay. (h) Cells transfected with siCON or siPDE4B were pretreated with Rolipram (10 μM) for 1 h, and IL-8 mRNA expression was measured post NTHi treatment. (i) A549 cells transfected with siCON or siPDE4B were pretreated with Rolipram for 1 h, and CYLD mRNA expression was measured post NTHi treatment. Data in a, c, d and g–i are mean±s.d. (n=3). *P<0.05. Statistical analysis was performed using Student’s t-test. CON, control. Data are representative of three or more independent experiments. NS, nonsignificant.
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f4: PDE4B has a crucial role in mediating NTHi-induced inflammation by negatively regulating CYLD expression.(a) PDE4 mRNA expression was measured in A549 cells stimulated with NTHi for 1.5 h. (b) Cells were treated with NTHi, and cell lysates were analysed by immunoblotting with the indicated antibodies. PDE4 mRNA expression was measured in (c) lung and (d) middle ear tissues from C57BL/6J mice inoculated with NTHi for 4 h. Mouse tissues from (e) lung and (f) middle ear were stained against PDE4B after NTHi inoculation (magnification × 200 in e; × 400 in f, respectively). Scale bars, 20 μm. (g) Lysates from cells treated with NTHi were immunoprecipitated with anti-PDE4B antibody, and analysed by PDE activity assay. (h) Cells transfected with siCON or siPDE4B were pretreated with Rolipram (10 μM) for 1 h, and IL-8 mRNA expression was measured post NTHi treatment. (i) A549 cells transfected with siCON or siPDE4B were pretreated with Rolipram for 1 h, and CYLD mRNA expression was measured post NTHi treatment. Data in a, c, d and g–i are mean±s.d. (n=3). *P<0.05. Statistical analysis was performed using Student’s t-test. CON, control. Data are representative of three or more independent experiments. NS, nonsignificant.

Mentions: We next sought to determine which PDE4 subfamily member is specifically involved in mediating NTHi-induced inflammation by negatively regulating CYLD. PDE4 consists of four subfamily genes, PDE4A to D, encoding Rolipram-sensitive PDEs27. PDE4 exerts its cellular functions by catalysing and degrading cyclic AMP (cAMP), one of the important second messengers in regulating numerous pathological processes in response to stimulants, for example, bacterial pathogens22. Previous studies indicate that upregulation of PDE4 has an important role in modulating pathological responses283132. Thus, we first determined whether PDE4 is upregulated by NTHi. As shown in Fig. 4a and Supplementary Fig. S5a,b, PDE4B is selectively and markedly upregulated by NTHi at mRNA and protein levels in HMEEC, A549 and primary NHBE cells, in vitro. Similar to the in vitro findings, NTHi-induced upregulation of PDE4B at both mRNA and protein levels was also confirmed in the mouse lung and middle ear in vivo as assessed by performing Q–PCR and IF analysis (Fig. 4c–f). Consistent with these results, PDE4B enzymatic activity was also upregulated by NTHi (Fig. 4g). Together, these data suggest that PDE4B may have an important role in mediating NTHi-induced inflammation via negative regulation of CYLD.


Inhibition of PDE4B suppresses inflammation by increasing expression of the deubiquitinase CYLD.

Komatsu K, Lee JY, Miyata M, Hyang Lim J, Jono H, Koga T, Xu H, Yan C, Kai H, Li JD - Nat Commun (2013)

PDE4B has a crucial role in mediating NTHi-induced inflammation by negatively regulating CYLD expression.(a) PDE4 mRNA expression was measured in A549 cells stimulated with NTHi for 1.5 h. (b) Cells were treated with NTHi, and cell lysates were analysed by immunoblotting with the indicated antibodies. PDE4 mRNA expression was measured in (c) lung and (d) middle ear tissues from C57BL/6J mice inoculated with NTHi for 4 h. Mouse tissues from (e) lung and (f) middle ear were stained against PDE4B after NTHi inoculation (magnification × 200 in e; × 400 in f, respectively). Scale bars, 20 μm. (g) Lysates from cells treated with NTHi were immunoprecipitated with anti-PDE4B antibody, and analysed by PDE activity assay. (h) Cells transfected with siCON or siPDE4B were pretreated with Rolipram (10 μM) for 1 h, and IL-8 mRNA expression was measured post NTHi treatment. (i) A549 cells transfected with siCON or siPDE4B were pretreated with Rolipram for 1 h, and CYLD mRNA expression was measured post NTHi treatment. Data in a, c, d and g–i are mean±s.d. (n=3). *P<0.05. Statistical analysis was performed using Student’s t-test. CON, control. Data are representative of three or more independent experiments. NS, nonsignificant.
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f4: PDE4B has a crucial role in mediating NTHi-induced inflammation by negatively regulating CYLD expression.(a) PDE4 mRNA expression was measured in A549 cells stimulated with NTHi for 1.5 h. (b) Cells were treated with NTHi, and cell lysates were analysed by immunoblotting with the indicated antibodies. PDE4 mRNA expression was measured in (c) lung and (d) middle ear tissues from C57BL/6J mice inoculated with NTHi for 4 h. Mouse tissues from (e) lung and (f) middle ear were stained against PDE4B after NTHi inoculation (magnification × 200 in e; × 400 in f, respectively). Scale bars, 20 μm. (g) Lysates from cells treated with NTHi were immunoprecipitated with anti-PDE4B antibody, and analysed by PDE activity assay. (h) Cells transfected with siCON or siPDE4B were pretreated with Rolipram (10 μM) for 1 h, and IL-8 mRNA expression was measured post NTHi treatment. (i) A549 cells transfected with siCON or siPDE4B were pretreated with Rolipram for 1 h, and CYLD mRNA expression was measured post NTHi treatment. Data in a, c, d and g–i are mean±s.d. (n=3). *P<0.05. Statistical analysis was performed using Student’s t-test. CON, control. Data are representative of three or more independent experiments. NS, nonsignificant.
Mentions: We next sought to determine which PDE4 subfamily member is specifically involved in mediating NTHi-induced inflammation by negatively regulating CYLD. PDE4 consists of four subfamily genes, PDE4A to D, encoding Rolipram-sensitive PDEs27. PDE4 exerts its cellular functions by catalysing and degrading cyclic AMP (cAMP), one of the important second messengers in regulating numerous pathological processes in response to stimulants, for example, bacterial pathogens22. Previous studies indicate that upregulation of PDE4 has an important role in modulating pathological responses283132. Thus, we first determined whether PDE4 is upregulated by NTHi. As shown in Fig. 4a and Supplementary Fig. S5a,b, PDE4B is selectively and markedly upregulated by NTHi at mRNA and protein levels in HMEEC, A549 and primary NHBE cells, in vitro. Similar to the in vitro findings, NTHi-induced upregulation of PDE4B at both mRNA and protein levels was also confirmed in the mouse lung and middle ear in vivo as assessed by performing Q–PCR and IF analysis (Fig. 4c–f). Consistent with these results, PDE4B enzymatic activity was also upregulated by NTHi (Fig. 4g). Together, these data suggest that PDE4B may have an important role in mediating NTHi-induced inflammation via negative regulation of CYLD.

Bottom Line: Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response.Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4.These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

View Article: PubMed Central - PubMed

Affiliation: Center for Inflammation, Immunity & Infection and Department of Biology, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303, USA.

ABSTRACT
The deubiquitinase CYLD acts as a key negative regulator to tightly control overactive inflammation. Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response. Here, we show that inhibition of phosphodiesterase 4B (PDE4B) markedly enhances upregulation of CYLD expression in response to bacteria, thereby suggesting that PDE4B acts as a negative regulator for CYLD. Interestingly, in Cyld-deficient mice, inhibition of PDE4B no longer suppresses inflammation. Moreover, PDE4B negatively regulates CYLD via specific activation of JNK2 but not JNK1. Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4. These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

Show MeSH
Related in: MedlinePlus