M1 macrophages act as LTβR-independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis

Aims The goal of this study was to characterize the role of inflammatory macrophages in the induction of the vascular smooth muscle cell (VSMC)-mediated formation of aortic tertiary lymphoid organs (TLOs). Methods and results Mouse bone marrow-derived M1 macrophages acted as lymphoid tissue inducer cells. Indeed, they expressed high levels of tumour necrosis factor (TNF)-alpha and membrane-bound lymphotoxin (LT)-alpha, two inducing cytokines that triggered expression of the chemokines CCL19, CCL20, and CXCL16, as did M1 supernatant. The blockade of LT beta R signalling with LT beta R-Ig had no effect, whereas that of TNFR1/ 2 signalling reduced chemokine expression by VSMCs in both wild-type (WT) and LT beta R KO mice, demonstrating that LT beta R signalling is dispensable for the M1-inducing effect. This effect was corroborated by the development of TLOs observed in LT beta R KO->apolipoprotein E knockout (ApoE KO) aortic segments after orthotopic transplantation. Furthermore, treatment of ApoE KOmice with anti-TNF-alpha antibody decreased the number and incidence of aortic TLOs. Finally, lymphoid nodules composed of T and B cells formed in in vivo-implanted scaffolds seeded with VSMCs previously stimulated ex vivo by M1-conditioned medium. Conclusions These results are the first to identify M1 macrophages as inducer cells that trigger the expression of chemokines by VSMCsindependently of LT beta R signalling. We propose that the dialogue betweenmacrophages andVSMCs-established across the vascular wall-contributes to the formation of aortic TLOs.