Integrin β3 interacts with Toll-like receptor 4 to up-regulate CD14 expression in macrophages and contributes to sepsis induced acute lung injury

Abstract

Sepsis is the major challenge in clinical practice with high mortality.1 Toll-like receptors (TLRs) play a fundamental role in sepsis. CD14 is a co-receptor of many Toll-like receptors.2 In our previous study, we found integrin β3 deficiency decreased sepsis induced CD14 expression and attenuated acute lung injury. In this study we used a cecal ligation and puncture (CLP) induced sepsis model and LPS-treated macrophages to uncover the mechanism of integrin β3 modulated CD14 expression under inflammatory conditions. In vivo studies. To define the role of integrin β3 in macrophages in a septic mouse, macrophages were derived from bone marrow and retransfusion, with or without prior depletion of macrophages by clodronate-containing liposomes. To detect morphology changes, lungs were obtained at 24 h after CLP and fixed with 4% paraformaldehyde (PFA) for haematoxylin–eosin (HE) stain. To detect the inflammatory response, bronchoalveolar lavage fluid (BALF) was obtained and tested for the levels of tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-β via enzyme-linked immunosorbent assay (ELISA). To detect CD14 expression tissue, the lung was homogenised and lysed for western blot. In vitro studies. To further investigate if integrin β3 is directly or indirectly involved in CD14 expression, we used (1) wild type macrophages pretreated with integrin β3 inhibitor P11 or anti-antibody or (2) integrin β3–/– macrophages stimulated with LPS for different times and measured the levels of TNF-α and IL-6 via ELISA and CD14 via western blot and flow cytometry. Secondly, integrin β3–/– macrophages treated with recombinant CD14 protein and LPS for different time points were used and TNF-α and IL-6 secretion was measured by ELISA. Thirdly, macrophages were isolated from wild type mice and pretreated with polymyxin B (PMB) or TAK-242 or isolated from C3He/J mice and stimulated with LPS for 24 h to detect CD14 expression via western blot or flow cytometry. Lastly, co-transfected 293T cells with integrin β3-HA and TLR4-Flag plasmids were then stimulated with LPS for 30 or 60 min, and the interaction by co-immunoprecipitation was assessed. In wild type mice with prior depletion of macrophages followed by retransfusion of wild type macrophages, lung injury, inflammatory cytokines, and CD14 expression was significantly increased compared with retransfusion of integrin β3 deficient macrophages. In integrin β3 deficient mice with prior depletion of macrophages and retransfusion of wild type macrophages, the lung injury, inflammatory cytokines, and CD14 expression was increased compared with retransfusion of integrin β3 deficient macrophages. Blocking integrin β3 pathway by P11 or anti-antibody inhibited LPS-induced TNF-α and IL-6 release at early but not late times, but had no effect on CD14 expression in wild type macrophages. In integrin β3 deficient macrophages, LPS-induced TNF-α and IL-6 release was significantly decreased from 4 to 24 h as well as CD14 expression. Recombination mCD14 reversed integrin β3 deficiency decreased TNF-α and IL-6 release. Blocking LPS by PMB or inhibition of LPS–Toll-like receptor 4 (TLR4) pathway by TAK-242 or TLR4 mutant macrophages, LPS-induced CD14 expression was abolished. Co-IP results showed that TLR4 and integrin β3 (slightly) interacted during the resting state, and the interaction was increased after LPS stimulation (see Fig. 1). Integrin β3 is involved in TLR activation-induced CD14 up-regulation and contributed to sepsis-induced acute lung injury. Deletion of integrin β3 in macrophages not only inhibited integrin β3 pathway activation-induced inflammatory cytokine release but also decreased TLR4 activation-induced CD14 up-regulation and reduced CD14 effect on TLR recognition and enhanced inflammation. Specifically targeting integrin β3/TLR4-CD14 signalling may be a potential treatment strategy in polymicrobial sepsis.