Hui Yin1, Huaxin Zhou2, Yi Kang1, Xiaoju Zhang1, Xiaoxian Duan2, Ridab Alnabhan2, Shuang Liang2, David A Scott2, Richard J Lamont2, Jia Shang3, Huizhi Wang4. 1. Department of Infectious Diseases, Henan Provincial People's Hospital (Zhengzhou University People's Hospital), Zhengzhou, Henan 450001, China. 2. Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY 40202, USA. 3. Department of Infectious Diseases, Henan Provincial People's Hospital (Zhengzhou University People's Hospital), Zhengzhou, Henan 450001, China. Electronic address: shangjia666@126.com. 4. Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY 40202, USA. Electronic address: hhwang01@louisville.edu.
Abstract
BACKGROUND: While Syk has been shown to associate with TLR4, the immune consequences of Syk-TLR interactions and related molecular mechanisms are unclear. METHODS: Gain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting. RESULTS: Syk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr-Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K-Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses. CONCLUSIONS: Syk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K-Akt and NF-κB signaling pathways. GENERAL SIGNIFICANCE: Syk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.
BACKGROUND: While Syk has been shown to associate with TLR4, the immune consequences of Syk-TLR interactions and related molecular mechanisms are unclear. METHODS: Gain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting. RESULTS:Syk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr-Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K-Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses. CONCLUSIONS:Syk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K-Akt and NF-κB signaling pathways. GENERAL SIGNIFICANCE: Syk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.
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