Z. Chen1, Z. Shao1, Q. Li1 1East Hospital,Tongji University School Of Medicine,Shanghai, SHANGHAI, China
Introduction:
Sepsis is one of the life-threatening diseases worldwide. It is characterized by inappropriate amplified systemic inflammatory response that may promote multiple organ dysfunction and mortality. TLR activation plays a key role in sepsis.CD14 is necessary for TLRs dependent induction of pro-inflammatory cytokines. Recent studies indicated that integrinβ3 involved in TLR-triggered innate immunity. Our previously study found that integrin inhibitor RGDs alleviated sepsis induced lung injury and improved survival rate. However, the interaction between integrinβ3 and TLR activation in sepsis remains largely unknown. This study aims to uncover the mechanism of integrinβ3 and TLR activation in in vivo and in vitro study.
Methods:
In vivo: WT, TLR4 and integirnβ3-/-mice undergoing cecal ligation and puncture (CLP) induced sepsis were sacrificed at indicated time. Serum was collected to detected inflammatory cytokines via ELISA. Tissue was fixed with 4% paraformaldehyde (PFA) for HE stain or immunohistochemical. Tissue lysate for CD14 expression was detected by Western blot.
In vitro: Peritoneal macrophages isolated from WT , TLR4 and integirnβ3-/-mice were stimulated with LPS at different concentration or indicated time, then assayed CD14 expression by Wb and flow cytometry. Medium was collected for TNF-α and IL-6 release via ELISA. Pretreated WT macrophages with TAK or anti-β3 Ab then stimulated with LPS and detected CD14 expression by Wb and flow cytometry. 293T cells were cotransfected with Flag-TLR4 and HA-integrinβ3 for 24h and stimulated with LPS for 30 and 60min then assayed by co-immunoprecipitation.
Results:
Integrinβ3−/− mice are resistant to CLP induced sepsis. Sepsis induced CD14 up-regulation of the tissue was suppressed in TLR4-/- and integrinβ3−/− mice. LPS induced CD14 expression was blocked in PMB or TAK pretreated WT macrophages and TLR4 mutant macrophages. LPS increased CD14 expression was suppressed in integrinβ3−/− macrophages. Integrinβ3 inhbition via anti-β3 or P11did not affect CD14 expression in LPS stimulated macrophages. Activation integrinβ3 by vitronectin also had no effect in CD14 expression in macrophages. Both integrinβ3 inhibition and integrinβ3 depletion attenuated LPS induced pro-inflammatory cytokines production. TLR4 and integrinβ3 interaction at rest condition and increased under LPS stimulation.
Conclusion:
Integrinβ3 positively regulates TLR4-triggered inflammatory responses through two different ways. Integrinβ3 signaling activation involved in LPS induced inflammatory cytokines production. Integrinβ3 interacted with TLR4 contributed to LPS induced CD14 expression. Specifically targeting integrinβ3/TLR4-CD14 signaling may represent a novel strategy in polymicrobial sepsis.