Leptospiral dissemination is restrained by liver macrophages through Clec4d-driven capture via C/EBPβ activation

Leptospirosis, caused by pathogenic Leptospira species, is a globally significant zoonotic disease with high morbidity and mortality. However, the organs or cells mainly involved in capturing circulating leptospires and the related mechanisms remain poorly understood. In this study, we firstly proved that the liver was the primary organ that captured leptospires during the very early intravascular phase of infection in mice. Then, we used unbiased flow sorting of leptospires-positive cells and intravital microscopy of mice infected with leptospires, and found that liver macrophages were the main leptospires-capturing immune cells. The depletion of liver macrophages abolished the ability of liver to capture leptospires and prompted leptospiral spread in other organs. The C-type lectin receptor signaling pathway and Clec4d were identified as the differential pathways and gene through RNA-seq analysis, respectively. The ectopic expression of Clec4d in HEK-293T cells or treatment with a Clec4d inhibitor, mannan proved that Clec4d functioned as a capture receptor of leptospires. Mechanistically, the transcription factor CCAAT/enhancer-binding protein beta (C/EBPβ) was activated and directly bound to the promoter of Clec4d to promote the expression of Clec4d in liver macrophages, thereby enhancing leptospiral capture. Mice treated with C/EBPβ inhibitor showed a significant inhibition of liver macrophages in capturing leptospires and increased leptospiral load in other organs. Our findings identify a novel mechanism by which the liver macrophages restrict leptospiral dissemination through C/EBPβ-Clec4d axis, and suggest a therapeutic strategy to prevent leptospiral dissemination through enhancing liver macrophages functions.