Dongqiang Yang1, ShuJian Li2, Xiaoxian Duan3, Junling Ren4, Shuang Liang3, Lan Yakoumatos3, Yi Kang1, Silvia M Uriarte3, Jia Shang1, Wei Li2, Huizhi Wang5. 1. Department of Infectious Diseases, Henan Provincial People's Hospital (People's Hospital of Zhengzhou University), Zhengzhou 450003, China. 2. Department of Neurology, Henan Provincial People's Hospital (People's Hospital of Zhengzhou University), Zhengzhou 450003, China. 3. Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY 40202, USA. 4. VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, VA, USA. 5. VCU Philips Institute for Oral Health Research, Virginia Commonwealth University School of Dentistry, Department of Oral and Craniofacial Molecular Biology, Richmond, VA, USA. Electronic address: wangh3@vcu.edu.
Abstract
BACKGROUND: Accumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain. METHODS: Gain- and Loss- of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemia mouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration. RESULTS: LPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces β-catenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding activity. These results were confirmed by multiple gain- and loss- of function approaches including specific siRNA and ectopic expression of Dvl3, GSK3β, and β-catenin in monocytes. Moreover, in vivo relevance was established in a murine endotoxin model, in which Wnt3a inhibition enhances the inflammatory responses by augmenting the systemic pro-inflammatory cytokine levels and neutrophil infiltration. CONCLUSIONS: TLR4 activation promotes Wnt3a-Dvl3 signaling, which acts as rheostats to restrain the intensity of inflammation through regulating GSK3β-β-catenin signaling and NF-κB activity. GENERAL SIGNIFICANCE: Wnt3a-Dvl3-β-catenin signaling axis could be a potential interventional target for manipulating the direction and intensity of inflammatory responses. Published by Elsevier Ltd.
BACKGROUND: Accumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain. METHODS: Gain- and Loss- of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemiamouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration. RESULTS:LPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces β-catenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding activity. These results were confirmed by multiple gain- and loss- of function approaches including specific siRNA and ectopic expression of Dvl3, GSK3β, and β-catenin in monocytes. Moreover, in vivo relevance was established in a murine endotoxin model, in which Wnt3a inhibition enhances the inflammatory responses by augmenting the systemic pro-inflammatory cytokine levels and neutrophil infiltration. CONCLUSIONS:TLR4 activation promotes Wnt3a-Dvl3 signaling, which acts as rheostats to restrain the intensity of inflammation through regulating GSK3β-β-catenin signaling and NF-κB activity. GENERAL SIGNIFICANCE: Wnt3a-Dvl3-β-catenin signaling axis could be a potential interventional target for manipulating the direction and intensity of inflammatory responses. Published by Elsevier Ltd.