LSECtin Positively Regulates LPS-induced TLR4 Signaling Pathway And The Immune Response

As an important class of pattern recognition receptors (PRRs), C-type lectin receptors (CLRs) have played crucial roles in pathogens recognition and infection. Some CLRs can directly induce intracellular signal transduction and activation of NF-КB pathway, leading to the production of inflammatory cytokines. It is noteworthy that there are many CLRs synergistic or antagonistic in various ways in Toll-like receptors (TLRs) signaling, inducting immune responses against microbial infections or interfering with TLR signal transduction, leading to microbial immune escape.Liver sinusoidal endothelial cell lectin (LSECtin) has been originally cloned by Dr. Liu. LSECtin belongs to the CLR family including known molecules DC-SIGN, DC-SIGNR and CD23. The family member DC-SIGN plays an important role in microbial infection and immunity through direct induction of intracellular signaling or regulation of intracellular TLR signaling. We investigate whether LSECtin contributes to regulation of the innate immune response in the body. In this paper, we have made the following discoveries: (1) LSECtin is a novel molecule for bacteria recognition. We have confirmed that LSECtin binds with E. coli and Salmonella typhimurium and this binding can be inhibited by Ca2+ chelator EGTA in the bacterial adhesion assay. Because E. coli and Salmonella typhimurium are Gram-negative bacteria and lipopolysaccharide (LPS) is the main component, we speculate that LSECtin may interacts with the LPS and plays a role in LPS-mediated signaling pathways and the immune response. (2) LSECtin expresses in macrophages. Using PCR, we confirmed that LSECtin expresses in mouse macrophage cell line RAW264.7 cells and mouse primary bone marrow-derived macrophages cell (BMMφ). This expression can be up-regulated by LPS stimulation. (3) LSECtin positively regulates LPS-induced TLR4 signaling pathway and the immune response. In LPS-induced shock model, compared with WT mice, LSECtin-/- mice produced significantly lower levels of inflammatory cytokines, had less lung injury, leading to decreased mortality significantly. After knocking down the expression of LSECtin in RAW264.7 cells, LPS-induced RAW264.7 cells secreted less inflammatory cytokines. After knocking out the expression of LSECtin in BMMφcells, LPS-induced secretion of inflammatory cytokines in BMMφcells also decreased significantly. LSECtin knock-down or knock-out slowed down the degradation of IκB and decreases p38 and JNK1/2 phosphorylation in LPS-stimulated RAW264.7 cells and BMMφcells, respectively. In contrast, the overexpression of LSECtin in HEK293 cells accelerated the degradation of the IκB and increased p38 and JNK1/2 phosphorylation levels. (4) LSECtin promotes the surface localization of TLR4 in the macrophage cells, increases the recruitment of TLR4 adapter molecule MyD88, which regulates NF-κB signaling pathway and the production of immune effector molecules. We demonstrated that LSECtin interacted with TLR4, CD14 and MD-2 through its CRD domain by co-immunoprecipitation experiments. Further, we confirmed that TLR4 expression and mean fluorescence intensity of LSECtin-/- BMMφcells were significantly decreased after LPS stimulation compared with WT BMMφ, suggesting that LSECtin promotes TLR4 localization in the BMMφcell surface. LSECtin knock-down or knock-out resulted in decreased expression of MyD88 in LPS-stimulated RAW264.7 cells and BMMφcells, respectively. In contrast, the overexpression of LSECtin in HEK293 cells increased the expression of MyD88. Therefore, LSECtin promotes the recruitment of MyD88 adapter molecule. (5) LSECtin may play a role in the membrane form rather than in the soluble form. Intriguingly, we found that soluble protein could not rescue the decreased production of cytokines by LSECtin-/- BMMφstimuted with LPS. LSECtin soluble protein can not reversed the decreased mortality in LSECtin-/- mice with LPS-induced shock as well. Therefore, we hypothesis that LSECtin itself must be anchored in the membrane and then promote TLR4 positioning.In summary, this study demonstrated that LSECtin positively regulates LPS-induced TLR4 signaling pathway and the immune response.