A major player in the development of inflammatory diseases, such as atherosclerosis, is vascular macrophages, which are primarily derived from infiltrating monocytes in an integrin-dependent process. Mac-1 (CD11b/CD18, ?M?2 or CR3), a major integrin expressed on macrophages, is generally considered as a proinflammatory receptor. Surprisingly, we find that Mac-1 deficiency on an LDLR-/- background exacerbates atherosclerosis, which contradicts published observations obtained using bone marrow transplantation approaches. To resolve this discrepancy, we performed reciprocal bone marrow transplantation experiments, and our new data agree with the published results. Unexpectedly, they implicate the existence of a subset of Mac-1+ resident cells with atheroprotective functions. Also contradicting the paradigm of Mac-1 being a proinflammatory receptor, we discovered that Mac-1 possesses a novel anti-inflammatory activity (J Exp Med 2007). In support of our original finding, genome-wide association studies have linked loss-of-function Mac-1 variants in human population to a greater risk of lupus, an autoimmune disease that correlates with accelerated atherosclerosis. Thus, Mac-1 appears to both promote and inhibit the development of inflammatory diseases; however, the molecular mechanism that confers Mac-1 with these diametrically opposing activities is unknown. In our preliminary studies, we screened several existing homolog-scanning Mac-1 mutants and identified a novel Mac-1 variant (D289A290?KH) that abolishes Mac-1?s proinflammatory activity without compromising its anti-inflammatory functions. Subsequently, we generated a knock-in mouse, Mac-1NA, using the CRISPR/Cas9 technology, in which the homologous residues (N289A290) of murine Mac-1 were mutated. Our preliminary study revealed that Mac-1-/- and Mac-1NA mice exhibit opposite patterns of inflammatory responses in vitro and in vivo. Based on these exciting preliminary data, we hypothesize that Mac-1 expressed on a subset of aortic resident cells plays an atheroprotective role by suppressing vascular inflammation. To test our hypothesis, we generated a GFP-coupled Mac-1flox mouse, which was subsequently crossed with the tamoxifen-inducible CX3CR1-CreERT2 mouse. Using this novel CX3CR1-CreERT2;Mac-1flox strain, we successfully tagged resident macrophages but not monocytes with GFP and simultaneously deleted their Mac-1 expression. We propose to use this novel mouse strain to identify the atheroprotective subset of Mac-1-expressing resident cells. We will also establish molecular mechanisms underlying the differential ability of Mac-1-/- and Mac-1NA to regulate macrophage proliferation, intracellular signaling, and foam cell differentiation during the development of atherosclerosis. Finally, we will investigate novel strategies to selectively target Mac-1?s proinflammatory functions and test their effectiveness to reduce atherosclerosis. Completion of this project will provide new insights into the mechanism by which Mac-1 confers macrophages with pro- or anti-inflammatory properties. It can also assist us in the design of Mac-1-targeting agents to prevent and treat inflammatory diseases.

Public Health Relevance

Atherosclerosis (the hardening and narrowing of artery) is the underlying cause of cardiovascular diseases, which account for nearly 801,000 deaths annually or about 1 of every 3 deaths in the US, with an estimated global cost of $863 billion in 2010. Despite the advance of modern medicine, no effective intervention is currently available. Completion of this project will develop novel therapeutic strategies to specifically target the proinflammatory activities of a major subset of leukocytes (macrophages), without compromising their anti- inflammatory functions. This information can assist us in the design of more effective therapeutic agents to prevent and treat atherosclerosis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Kirby, Ruth
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University of Maryland Baltimore
Internal Medicine/Medicine
Schools of Medicine
United States
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