Regulation of Dendritic Cell Migration by RANK Signalling in Lymphatic Endothelial Cells 2303575

Abstract

Abstract Introduction Lymph nodes (LNs) are key sites for initiating adaptive immune responses, enabling antigen-specific T cells to interact effectively with dendritic cells (DCs) and B cells. DCs migrate from peripheral tissues to draining LNs via lymphatic vessels lined by lymphatic endothelial cells (LECs), guided by chemokines and adhesion molecules. LEC-derived CCL21 binds CCR7 on activated DCs, while ACKR4 on subcapsular sinus floor LECs modulates the CCL21 gradient to steer DC migration. Recent findings implicate lymphotoxin β receptor (LTβR) signalling in regulating leukocyte trafficking through canonical and non-canonical NF-κB pathways, affecting expression of CCL21 and VCAM-1. However, the role of RANK (Receptor Activator of NF-κB) in LEC-mediated DC migration remains unclear. Methods We generated Prox1-CreERT2 Rank flox/flox mice and induced LEC-specific knockout via tamoxifen and confirmed gene deletion by PCR and flow cytometry. Bone marrow-derived DCs, matured with LPS and pulsed with OVA, were transferred into control or knockout mice. Migration to draining LNs and antigen presentation to CD4+ T cells were assessed. Results RANK-deficient mice showed significantly impaired DC migration, unlike LTβR-deficient mice. To dissect underlying mechanisms, we developed an in vitro LEC—DC interaction model using LECs isolated from wild-type, Rank-deficient, and Rank-overexpressing mice. Gene manipulation was achieved using Cre-expressing adeno-associated virus, and RANKL was administered to stimulate signalling. We observed a RANK-RANKL signalling-dependent MAdCAM-1 and VCAM-1 expression in LECs. We also observed ACKR4 alterations in the loss or overexpression of RANK. Conclusion We have shown that RANK activation affects DC transmigration across the lymphatic barrier and are currently investigating its molecular mechanism. Funding Source The project receives funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement number 101034170 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)