A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense.

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

Inflammation, but not recruitment, of adipose tissue macrophages requires signalling through Mac-1 (CD11b/CD18) in diet-induced obesity (DIO).

Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, αMß2) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that a genetic deficiency and a therapeutic modulation of Mac-1 regulate adipose tissue inflammation in a mouse model of diet-induced obesity (DIO). C57Bl6/J mice genetically deficient (Mac-1-/-) or competent for Mac-1 (WT) consumed a high fat diet for 20 weeks. Surprisingly, Mac-1-/- mice presented with increased diet-induced weight gain, decreased insulin sensitivity in skeletal muscle and in the liver in insulin-clamps, insulin secretion deficiency and elevated glucose levels in fasting animals, and dyslipidaemia. Unexpectedly, accumulation of adipose tissue macrophages (ATMs) was unaffected, while gene expression indicated less inflamed adipose tissue and macrophages in Mac-1-/- mice. In contrast, inflammatory gene expression at distant locations, such as in skeletal muscle, was not changed. Treatment of ATMs with an agonistic anti-Mac-1 antibody, M1/70, induced pro-inflammatory genes in cell culture. In vivo, treatment with M1/70 induced a hyper-inflammatory phenotype with increased expression of IL-6 and MCP-1, whereas accumulation of ATMs did not change. Finally, inhibition of Mac-1's adhesive interaction to CD40L by the peptide inhibitor cM7 did not affect myeloid cell accumulation in adipose tissue. We present the surprising finding that adhesive properties of the leukocyte integrin Mac-1 are not required for macrophage accumulation in adipose tissue. Instead, Mac-1 modulates inflammatory gene expression in macrophages. These findings question the net effect of integrin blockade in cardio-metabolic disease.

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 ß-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.