Christophe Combadière

Combined Inhibition of CCL2, CX3CR1, and CCR5 Abrogates Ly6Chi and Ly6Clo Monocytosis and Almost Abolishes Atherosclerosis in Hypercholesterolemic Mice

Background— Monocytes are critical mediators of atherogenesis. Deletion of individual chemokines or chemokine receptors leads to significant but only partial inhibition of lesion development, whereas deficiency in other signals such as CXCL16 or CCR1 accelerates atherosclerosis. Evidence that particular chemokine pathways may cooperate to promote monocyte accumulation into inflamed tissues, particularly atherosclerotic arteries, is still lacking. Methods and Results— Here, we show that chemokine-mediated signals critically determine the frequency of monocytes in the blood and bone marrow under both noninflammatory and atherosclerotic conditions. Particularly, CCL2-, CX3CR1-, and CCR5-dependent signals differentially alter CD11b+ Ly6G− 7/4hi (also known as Ly6Chi) and CD11b+ Ly6G− 7/4lo (Ly6Clo) monocytosis. Combined inhibition of CCL2, CX3CR1, and CCR5 in hypercholesterolemic, atherosclerosis-susceptible apolipoprotein E–deficient mice leads to abrogation of bone marrow monocytosis and to additive reduction in circulating monocytes despite persistent hypercholesterolemia. These effects are associated with a marked and additive 90% reduction in atherosclerosis. Interestingly, lesion size highly correlates with the number of circulating monocytes, particularly the CD11b+ Ly6G− 7/4lo subset. Conclusions— CCL2, CX3CR1, and CCR5 play independent and additive roles in atherogenesis. Signals mediated through these pathways critically determine the frequency of circulating monocyte subsets and thereby account for almost all macrophage accumulation into atherosclerotic arteries.

Decreased Atherosclerotic Lesion Formation in CX3CR1/Apolipoprotein E Double Knockout Mice

Background—Fractalkine (CX3CL1), a CX3C chemokine, is expressed in the vessel wall and mediates the firm adhesion and chemotaxis of leukocytes expressing its receptor, CX3CR1. A polymorphism in the CX3CR1 gene is associated with low CX3CR1 expression and reduced risk of acute coronary disease in humans. Methods and Results—We generated CX3CR1-deficient mice (CX3CR1−/−) by targeted gene disruption and crossed them with the proatherogenic apolipoprotein E-deficient mice (apoE−/−). Here we show that the extent of lipid-stained lesions in the thoracic aorta was reduced by 59% in CX3CR1/apoE double knockout mice compared with their CX3CR1+/+/apoE−/− littermates. The development of atherosclerosis in the aortic sinus was also markedly altered in the double knockout mice, with 50% reduction in macrophage accumulation. Although lesions of CX3CR1−/− mice were smaller in size, they retained a substantial accumulation of smooth muscle cells and collagen, features consistent with a stable plaque phenotype. Finally, CX3CR1+/−/apoE−/− mice showed the same reduction in atherosclerosis as the CX3CR1−/−/apoE−/− mice. Conclusions—The CX3CR1-CX3CL1 pathway seems to play a direct and critical role in monocyte recruitment and atherosclerotic lesion development in a mouse model of human atherosclerosis.

CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration

The role of retinal microglial cells (MCs) in age-related macular degeneration (AMD) is unclear. Here we demonstrated that all retinal MCs express CX3C chemokine receptor 1 (CX3CR1) and that homozygosity for the CX3CR1 M280 allele, which is associated with impaired cell migration, increases the risk of AMD. In humans with AMD, MCs accumulated in the subretinal space at sites of retinal degeneration and choroidal neovascularization (CNV). In CX3CR1-deficient mice, MCs accumulated subretinally with age and albino background and after laser impact preceding retinal degeneration. Raising the albino mice in the dark prevented both events. The appearance of lipid-bloated subretinal MCs was drusen-like on funduscopy of senescent mice, and CX3CR1-dependent MC accumulation was associated with an exacerbation of experimental CNV. These results show that CX3CR1-dependent accumulation of subretinal MCs evokes cardinal features of AMD. These findings reveal what we believe to be a novel pathogenic process with important implications for the development of new therapies for AMD.

International union of pharmacology. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145–176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

IDENTIFICATION OF CX3CR1 : A CHEMOTACTIC RECEPTOR FOR THE HUMAN CX3C CHEMOKINE FRACTALKINE AND A FUSION CORECEPTOR FOR HIV-1

Fractalkine is a multimodular human leukocyte chemoattractant protein and a member of the chemokine superfamily. Unlike other human chemokines, the chemokine domain of fractalkine has three amino acids between two conserved cysteines, referred to as the CX 3C motif. Both plasma membrane-associated and shed forms of fractalkine have been identified. Here, we show that the recombinant 76-amino acid chemokine domain of fractalkine is a potent and highly specific chemotactic agonist at a human orphan receptor previously named V28 or alternatively CMKBRL1 (chemokine β receptor-like 1), which was shown previously to be expressed in neutrophils, monocytes, T lymphocytes, and several solid organs, including brain. CMKBRL1/V28 also functioned with CD4 as a coreceptor for the envelope protein from a primary isolate of HIV-1 in a cell-cell fusion assay, and fusion was potently and specifically inhibited by fractalkine. Thus CMKBRL1/V28 is a specific receptor for fractalkine, and we propose to rename it CX 3CR1 (CX 3C chemokine receptor 1), according to an accepted nomenclature system.

TGF-β activity protects against inflammatory aortic aneurysm progression and complications in angiotensin II–infused mice

Complicated abdominal aortic aneurysm (AAA) is a major cause of mortality in elderly men. Ang II-dependent TGF-beta activity promotes aortic aneurysm progression in experimental Marfan syndrome. However, the role of TGF-beta in experimental models of AAA has not been comprehensively assessed. Here, we show that systemic neutralization of TGF-beta activity breaks the resistance of normocholesterolemic C57BL/6 mice to Ang II-induced AAA formation and markedly increases their susceptibility to the disease. These aneurysms displayed a large spectrum of complications on echography, including fissuration, double channel formation, and rupture, leading to death from aneurysm complications. The disease was refractory to inhibition of IFN-gamma, IL-4, IL-6, or TNF-alpha signaling. Genetic deletion of T and B cells or inhibition of the CX3CR1 pathway resulted in partial protection. Interestingly, neutralization of TGF-beta activity enhanced monocyte invasiveness, and monocyte depletion markedly inhibited aneurysm progression and complications. Finally, TGF-beta neutralization increased MMP-12 activity, and MMP-12 deficiency prevented aneurysm rupture. These results clearly identify a critical role for TGF-beta in the taming of the innate immune response and the preservation of vessel integrity in C57BL/6 mice, which contrasts with its reported pathogenic role in Marfan syndrome.

Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis.

Experimental cerebral malaria (ECM) resulting from Plasmodium berghei ANKA infection involves T lymphocytes. However, the mechanisms of T cell-mediated pathogenesis remain unknown. We found that, in contrast to ECM-susceptible C57BL6 mice, perforin-deficient (PFP-KO) mice were resistant to ECM in the absence of brain lesions, whereas cytoadherence of parasitized erythrocytes and massive accumulation of activated/effector CD8 lymphocytes were observed in both groups of mice. ECM is induced in PFP-KO mice after adoptive transfer of cytotoxic CD8+ cells from infected C57BL6 mice, which were directed to the brain of PFP-KO mice. This specific recruitment might involve chemokine/chemokine receptors, since their expression was up-regulated on activated CD8 cells, and susceptibility to ECM was delayed in CCR5-KO mice. Thus, lymphocyte cytotoxicity and cell trafficking are key players in ECM pathogenesis.

CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis

Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase- and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved.

Role of Chemokines and Chemokine Receptors in Shaping the Effector Phase of the Antitumor Immune Response

Immune system-mediated eradication of neoplastic cells requires induction of a strong long-lasting antitumor T-cell response. However, generation of tumor-specific effector T cells does not necessarily result in tumor clearance. CTL must first be able to migrate to the tumor site, infiltrate the tumor tissue, and interact with the target to finally trigger effector functions indispensable for tumor destruction. Chemokines are involved in circulation, homing, retention, and activation of immunocompetent cells. Although some of them are known to contribute to tumor growth and metastasis, others are responsible for changes in the tumor microenvironment that lead to extensive infiltration of lymphocytes, resulting in tumor eradication. Given their chemoattractive and activating properties, a role for chemokines in the development of the effector phase of the antitumor immune response has been suggested. Here, we emphasize the role of the chemokine-chemokine receptor network at multiple levels of the T-cell-mediated antitumor immune response. The identification of chemokine-dependent molecular mechanisms implicated in tumor-specific CTL trafficking, retention, and regulation of their in situ effector functions may offer new perspectives for development of innovative immunotherapeutic approaches to cancer treatment.

CCR2+ monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice

Atrophic age‐related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2+ inflammatory monocyte infiltration in patients. Using age‐ and light‐induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2+ monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.