Background: Lipopolysaccharide (LPS) activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. Materials and methods: Human neutrophils and monocytes, isolated from buffy coat, were stimulated with LPS and then disrupted in a nitrogen bomb to prepare protein extracts with preserved integrity and association. Lysates were then subjected to WB, immunoprecipitation or co-immunoprecipitation using specific antibodies raised against TBK1, TRIF, TRAF3,NAP1, HSP-90 and SHP-2. Results: In a previous study (Tamassia N. et al., J Immunol.2007; 178(11):7344-56), we have demonstrated that human neutrophils are unable to mobilize the MyD88-independent path-way, as revealed by the lack of inducibility neither of IFNb(the principal MyD88-independent gene) nor of IFNb-dependent genes in LPS-treated cells. Consistent with these latter results, we have also found that IRF3, a critical transcription factor for IFNb gene induction, and TBK1, an IRF3-phosphory-lating kinase, are both not activated by LPS in human neutrophils. We are currently investigating the molecular mechanisms that, ultimately, may explain why in neutrophils the TLR4-mediated, MyD88-independent pathway is impaired. Specifically, we are testing whether the interactions of TBK1with its various regulatory proteins, such as TRIF, TRAF3,NAP1, HSP-90 and SHP-2, appropriately occur or not inhuman neutrophils. Conclusions: Our results will elucidate the molecular bases of the disconnected activation of the signaling pathways down-stream of TLR4 in key cellular components of the inflammatory and immune responses. They will also help to better understand the primordial role of neutrophils in host defence against non-viral infections.
Understanding why LPS is unable to mobilize the MyD88-independent pathway in human neutrophils
TAMASSIA, Nicola;CASSATELLA, Marco Antonio
2010-01-01
Abstract
Background: Lipopolysaccharide (LPS) activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. Materials and methods: Human neutrophils and monocytes, isolated from buffy coat, were stimulated with LPS and then disrupted in a nitrogen bomb to prepare protein extracts with preserved integrity and association. Lysates were then subjected to WB, immunoprecipitation or co-immunoprecipitation using specific antibodies raised against TBK1, TRIF, TRAF3,NAP1, HSP-90 and SHP-2. Results: In a previous study (Tamassia N. et al., J Immunol.2007; 178(11):7344-56), we have demonstrated that human neutrophils are unable to mobilize the MyD88-independent path-way, as revealed by the lack of inducibility neither of IFNb(the principal MyD88-independent gene) nor of IFNb-dependent genes in LPS-treated cells. Consistent with these latter results, we have also found that IRF3, a critical transcription factor for IFNb gene induction, and TBK1, an IRF3-phosphory-lating kinase, are both not activated by LPS in human neutrophils. We are currently investigating the molecular mechanisms that, ultimately, may explain why in neutrophils the TLR4-mediated, MyD88-independent pathway is impaired. Specifically, we are testing whether the interactions of TBK1with its various regulatory proteins, such as TRIF, TRAF3,NAP1, HSP-90 and SHP-2, appropriately occur or not inhuman neutrophils. Conclusions: Our results will elucidate the molecular bases of the disconnected activation of the signaling pathways down-stream of TLR4 in key cellular components of the inflammatory and immune responses. They will also help to better understand the primordial role of neutrophils in host defence against non-viral infections.
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