Binding of CD40L to Mac-1's i-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis-but does not affect immunity and thrombosis in mice

Rationale: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr -/- mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.Fil: Wolf, Dennis. Albert-Ludwigs-Universität Freiburg; Alemania. Baker IDI Heart and Diabetes Institute; AustraliaFil: Hohmann, Jan David. Baker IDI Heart and Diabetes Institute; AustraliaFil: Wiedemann, Ansgar. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Bledzka, Kamila. Cleveland Clinic. Department of Molecular Cardiology; Estados UnidosFil: Blankenbach, Hermann. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Gutte, Katharina. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Zeschky, Katharina. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Bassler, Nicole. Baker IDI Heart and Diabetes Institute; AustraliaFil: Hoppe, Natalie. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Rodriguez, Alexandra Ortiz. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Herr, Nadine. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Hilgendorf, Ingo. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Stachon, Peter. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Willecke, Florian. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Duerschmied, Daniel. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: von zur Muhlen, Constantin. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Soloviev, Dmitry A.. Cleveland Clinic. Department of Molecular Cardiology; Estados UnidosFil: Zhang, Li. University of Maryland; Estados UnidosFil: Bode, Christoph. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Plow, Edward F.. Cleveland Clinic. Department of Molecular Cardiology; Estados UnidosFil: Libby, Peter. Harvard Medical School; Estados UnidosFil: Peter, Karlheinz. Baker IDI Heart and Diabetes Institute; AustraliaFil: Zirlik, Andreas. Albert-Ludwigs-Universität Freiburg; Alemani