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Inhibition of Viral Infection-Induced Inflammatory Responses by Targeting the CLOCK Regulator Casein Kinase 1 Delta/Epsilon
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A7271 - Inhibition of Viral Infection-Induced Inflammatory Responses by Targeting the CLOCK Regulator Casein Kinase 1 Delta/Epsilon
Author Block: M. Li, S. Y. Langenbach, C. Keenan, Y. C. Xia, T. Harris, A. Stewart; Pharmacology and Therapeutics, University of Melbourne, Melbourne, Australia.
Rationale: The key CLOCK component CK1delta/epsilon (CK1δ/ε) is implicated in TGF-β1 signaling and regulation of glucocorticoid activity. We have shown that inhibition of CK1δ/ε with PF670462 reduces TGF-β1-induced fibrogenic signals and glucocorticoid insensitivity induced by TGF-β1 or viral infection, and also ameliorates experimental fibrosis. We now demonstrate for the first time that CK1δ/ε is a novel target with therapeutic potential to prevent viral infection-induced inflammatory responses.
Methods: CK1δ/ε expression was assessed by IHC in human asthmatic cohort. In vitro poly (I:C)-induced inflammatory cytokine production in BEAS-2B cells was assessed by RT-qPCR and ELISA. The potential involvement of CK1δ/ε in above experimental settings was ascertained using pharmacological (PF670462) and genetic (siRNA) approaches. In vivo inflammatory responses were established in BALB/c mice infected with respiratory syncytial virus (RSV) through intranasal insufflation. Mice were treated 2 days later with either dexamethasone (1mg/kg/day, ip), PF670462 (30mg/kg/day, ip), or the combination for 3 days. Protein content and cell number in bronchoalveolar lavage fluid (BALF) were determined. Inflammatory cytokine gene expression was determined in the whole lung extracts by RT-qPCR.
Results: Expression of CK1δ was elevated in the airway wall of severe asthmatics compared to well-controlled asthmatics and non-asthmatic controls. In BEAS-2B airway epithelial cells, poly (I:C)-induced pro-inflammatory cytokines (IL-6, IL-8 and GM-CSF) were significantly attenuated by inhibition of CK1δ/ε using 3μM PF670462 or CK1δ/ε-targeting siRNAs. RSV-induced increases in BALF protein content and IL-6 mRNA expression in the lung were significantly reduced by the combination of dexamethasone and PF670462. Increased inflammatory cell number in BALF was also reduced by the dexamethasone/PF670462 combination (from 1.58±0.19 x 106 to 0.92±0.11 x 106). Aerosolised PF670462 also protects from RSV induced lung damage in BALB/c mice.
Conclusions: Inhibition of CK1δ/ε both in vitro and in vivo suppresses viral infection-induced inflammatory responses. Targeting CK1δ/ε may thus be a novel therapeutic approach for sensitizing glucocorticoid activity for the treatment of viral exacerbation of asthma.
Author Block: M. Li, S. Y. Langenbach, C. Keenan, Y. C. Xia, T. Harris, A. Stewart; Pharmacology and Therapeutics, University of Melbourne, Melbourne, Australia.
Rationale: The key CLOCK component CK1delta/epsilon (CK1δ/ε) is implicated in TGF-β1 signaling and regulation of glucocorticoid activity. We have shown that inhibition of CK1δ/ε with PF670462 reduces TGF-β1-induced fibrogenic signals and glucocorticoid insensitivity induced by TGF-β1 or viral infection, and also ameliorates experimental fibrosis. We now demonstrate for the first time that CK1δ/ε is a novel target with therapeutic potential to prevent viral infection-induced inflammatory responses.
Methods: CK1δ/ε expression was assessed by IHC in human asthmatic cohort. In vitro poly (I:C)-induced inflammatory cytokine production in BEAS-2B cells was assessed by RT-qPCR and ELISA. The potential involvement of CK1δ/ε in above experimental settings was ascertained using pharmacological (PF670462) and genetic (siRNA) approaches. In vivo inflammatory responses were established in BALB/c mice infected with respiratory syncytial virus (RSV) through intranasal insufflation. Mice were treated 2 days later with either dexamethasone (1mg/kg/day, ip), PF670462 (30mg/kg/day, ip), or the combination for 3 days. Protein content and cell number in bronchoalveolar lavage fluid (BALF) were determined. Inflammatory cytokine gene expression was determined in the whole lung extracts by RT-qPCR.
Results: Expression of CK1δ was elevated in the airway wall of severe asthmatics compared to well-controlled asthmatics and non-asthmatic controls. In BEAS-2B airway epithelial cells, poly (I:C)-induced pro-inflammatory cytokines (IL-6, IL-8 and GM-CSF) were significantly attenuated by inhibition of CK1δ/ε using 3μM PF670462 or CK1δ/ε-targeting siRNAs. RSV-induced increases in BALF protein content and IL-6 mRNA expression in the lung were significantly reduced by the combination of dexamethasone and PF670462. Increased inflammatory cell number in BALF was also reduced by the dexamethasone/PF670462 combination (from 1.58±0.19 x 106 to 0.92±0.11 x 106). Aerosolised PF670462 also protects from RSV induced lung damage in BALB/c mice.
Conclusions: Inhibition of CK1δ/ε both in vitro and in vivo suppresses viral infection-induced inflammatory responses. Targeting CK1δ/ε may thus be a novel therapeutic approach for sensitizing glucocorticoid activity for the treatment of viral exacerbation of asthma.
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