Elastin-derived peptides favor type 2 innate lymphoid cells in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airway inflammation and obstruction, primarily caused by tobacco smoking. Although the involvement of immune cells in COPD pathogenesis is well established, the contribution of innate lymphoid cells (ILCs) remains poorly understood. ILCs are a type of innate immune cells that participate in tissue remodeling processes, but their specific role in COPD has not been fully elucidated. During COPD, the breakdown of pulmonary elastin generates elastin peptides that elicit biological activities on immune cells. This study aimed to investigate the presence of ILC in patients with COPD and examine the impact of elastin peptides on their functionality. Our findings revealed an elevated proportion of ILC2 in the peripheral blood of patients with COPD, and a general activation of ILC as indicated by an increase in their cytokine secretion capacity. Notably, our study demonstrated that serum from patients with COPD promotes ILC2 phenotype, likely due to the elevated concentration of IL-5, a cytokine known to favor ILC2 activation. Furthermore, we uncovered that this increase in IL-5 secretion is partially attributed to its secretion by macrophages upon stimulation by elastin peptides, suggesting an indirect role of elastin peptides on ILC in COPD. These findings shed light on the involvement of ILC in COPD and provide insights into the potential interplay between elastin breakdown, immune cells, and disease progression. Further understanding of the mechanisms underlying ILC activation and their interaction with elastin peptides could contribute to the development of novel therapeutic strategies for COPD management.NEW & NOTEWORTHY Elastin-derived peptides, generated following alveolar degradation during emphysema in patients with COPD, are able to influence the response of type 2 innate lymphoid cells. We show that the orientation of innate lymphoid cells in patients with COPD is shifted toward a type 2 profile and that elastin peptides are indirectly participating in that shift through their influence of macrophages, which in turn impact innate lymphoid cells.

Identification of CD160-TM as a tumor target on triple negative breast cancers: possible therapeutic applications.

BACKGROUND: Despite major therapeutic advances, triple-negative breast cancer (TNBC) still presents a worth prognosis than hormone receptors-positive breast cancers. One major issue relies in the molecular and mutational heterogeneity of TNBC subtypes that is reinforced by the absence of reliable tumor-antigen that could serve as a specific target to further promote efficient tumor cell recognition and depletion. CD160 is a receptor mainly expressed by NK lymphocytes and presenting two isoforms, namely the GPI-anchored form (CD160-GPI) and the transmembrane isoform (CD160-TM). While CD160-GPI is constitutively expressed on resting cells and involved in the generation of NK cells' cytotoxic activity, CD160-TM is neo-synthesized upon activation and promotes the amplification of NK cells' killing ability. METHODS: CD160 expression was assessed by immunohistochemistry (IHC) and flow cytometry on TNBC patient biopsies or cell lines, respectively. Antibody (Ab)-mediated tumor depletion was tested in vitro by performing antibody-dependent cell cytotoxicity (ADCC) and phagocytosis (ADCP) assays, and in vivo on a TNBC mouse model. RESULTS: Preliminary data obtained by IHC on TNBC patients' tumor biopsies revealed an unconventional expression of CD160 by TNBC tumor cells. By using a specific but conformation-dependent anti-CD160-TM Ab, we established that CD160-TM, but not CD160-GPI, was expressed by TNBC tumor cells. A conformation-independent anti-CD160-TM mAb (22B12; muIgG2a isotype) was generated and selected according to pre-defined specificity and functional criterions. In vitro functional assays demonstrated that ADCC and ADCP could be induced in the presence of 22B12, resulting in TNBC cell line apoptosis. The ability of 22B12 to exert an in vivo anti-tumor activity was also demonstrated on a TNBC murine model. CONCLUSIONS: Our data identify CD160-TM as a tumor marker for TNBC and provide a rational for the use of anti-CD160-TM antibodies as therapeutic tools in this tumor context.

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant.

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE-/- ) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1ß at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes-and not epithelial cells-and was dependent on NLRP3 inflammasome activation and IL1ß-signalling. In females, CFA-induced TSLP was independent of IL1ß and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR-/- mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE-/- and ApoE-/- /TSLPR-/- mice with either CFA/IFA or PBS. ApoE-/- mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE-/- /TSLPR-/- mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.

MicroRNA-21 Deficiency Alters the Survival of Ly-6Clo Monocytes in ApoE-/- Mice and Reduces Early-Stage Atherosclerosis-Brief Report.

Objective- To determine the role of microRNA-21 (miR-21) on the homeostasis of monocyte subsets and on atherosclerosis development in ApoE-/- (apolipoprotein E) mice. Approach and Results- In ApoE-/- mice, miR-21 expression was increased in circulating Ly-6Clo nonclassical monocytes in comparison to Ly-6Chi monocytes. The absence of miR-21 significantly altered the survival and number of circulating Ly-6Clo nonclassical monocytes in ApoE-/- mice. In the early stages of atherosclerosis, the absence of miR-21 limited lesion development both in the aortic sinus (by almost 30%) and in the aorta (by almost 50%). This was associated with less monocyte availability in circulation and increased apoptosis of local macrophages in plaques. At later stages of atherosclerosis, lesion size in the aortic root was similar in ApoE-/- and ApoE-/- miR-21-/- mice, but plaques showed a less stable phenotype (larger necrotic cores) in the latter. The loss of protection in advanced stages was most likely because of excessive inflammatory apoptosis related to an impairment of local efficient efferocytosis. Conclusions- Gene deletion of miR-21 in ApoE-/- mice alters Ly-6Clo nonclassical monocytes homeostasis and contribute to limit early-stage atherosclerosis.

The Dendritic Cell Receptor DNGR-1 Promotes the Development of Atherosclerosis in Mice.

RATIONALE: Necrotic core formation during the development of atherosclerosis is associated with a chronic inflammatory response and promotes accelerated plaque development and instability. However, the molecular links between necrosis and the development of atherosclerosis are not completely understood. Clec9a (C-type lectin receptor) or DNGR-1 (dendritic cell NK lectin group receptor-1) is preferentially expressed by the CD8α+ subset of dendritic cells (CD8α+ DCs) and is involved in sensing necrotic cells. We hypothesized that sensing of necrotic cells by DNGR-1 plays a determinant role in the inflammatory response of atherosclerosis. OBJECTIVE: We sought to address the impact of total, bone marrow-restricted, or CD8α+ DC-restricted deletion of DNGR-1 on atherosclerosis development. METHODS AND RESULTS: We show that total absence of DNGR-1 in Apoe (apolipoprotein e)-deficient mice (Apoe-/-) and bone marrow-restricted deletion of DNGR-1 in Ldlr (low-density lipoprotein receptor)-deficient mice (Ldlr-/-) significantly reduce inflammatory cell content within arterial plaques and limit atherosclerosis development in a context of moderate hypercholesterolemia. This is associated with a significant increase of the expression of interleukin-10 (IL-10). The atheroprotective effect of DNGR-1 deletion is completely abrogated in the absence of bone marrow-derived IL-10. Furthermore, a specific deletion of DNGR-1 in CD8α+ DCs significantly increases IL-10 expression, reduces macrophage and T-cell contents within the lesions, and limits the development of atherosclerosis. CONCLUSIONS: Our results unravel a new role of DNGR-1 in regulating vascular inflammation and atherosclerosis and potentially identify a new target for disease modulation.

Limited Macrophage Positional Dynamics in Progressing or Regressing Murine Atherosclerotic Plaques-Brief Report.

Objective- Macrophages play important roles in the pathogenesis of atherosclerosis, but their dynamics within plaques remain obscure. We aimed to quantify macrophage positional dynamics within progressing and regressing atherosclerotic plaques. Approach and Results- In a stable intravital preparation, large asymmetrical foamy macrophages in the intima of carotid artery plaques were sessile, but smaller rounded cells nearer plaque margins, possibly newly recruited monocytes, mobilized laterally along plaque borders. Thus, to test macrophage dynamics in plaques over a longer period of time in progressing and regressing disease, we quantified displacement of nondegradable phagocytic particles within macrophages for up to 6 weeks. In progressing plaques, macrophage-associated particles appeared to mobilize to deeper layers in plaque, whereas in regressing plaques, the label was persistently located near the lumen. By measuring the distance of the particles from the floor of the plaque, we discovered that particles remained at the same distance from the floor regardless of plaque progression or regression. The apparent deeper penetration of labeled cells in progressing conditions could be attributed to monocyte recruitment that generated new superficial layers of macrophages over the labeled phagocytes. Conclusions- Although there may be individual exceptions, as a population, newly differentiated macrophages fail to penetrate significantly deeper than the limited depth they reside on initial entry, regardless of plaque progression, or regression. These limited dynamics may prevent macrophages from escaping areas with unfavorable conditions (such as hypoxia) and pose a challenge for newly recruited macrophages to clear debris through efferocytosis deep within plaque.

Inhibition of microRNA-92a prevents endothelial dysfunction and atherosclerosis in mice.

RATIONALE FOR STUDY: MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression at post-transcriptional level. We hypothesized that a specific pool of endothelial miRNAs could be selectively regulated by flow conditions and inflammatory signals, and as such be involved in the development of atherosclerosis. OBJECTIVE: To identify miRNAs, called atheromiRs, which are selectively regulated by shear stress and oxidized low-density lipoproteins (oxLDL), and to determine their role in atherogenesis. METHODS AND RESULTS: Large-scale miRNA profiling in HUVECs identified miR-92a as an atheromiR candidate, whose expression is preferentially upregulated by the combination of low shear stress (SS) and atherogenic oxLDL. Ex vivo analysis of atheroprone and atheroprotected areas of mouse arteries and human atherosclerotic plaques demonstrated the preferential expression of miR-92a in atheroprone low SS regions. In Ldlr(-/-) mice, miR-92a expression was markedly enhanced by hypercholesterolemia, in particular in atheroprone areas of the aorta. Assessment of endothelial inflammation in gain- and loss-of-function experiments targeting miR-92a expression revealed that miR-92a regulated endothelial cell activation by oxLDL, more specifically under low SS conditions, which was associated with modulation of Kruppel-like factor 2 (KLF2), Kruppel-like factor 4 (KLF4), and suppressor of cytokine signaling 5. miR-92a expression was regulated by signal transducer and activator of transcription 3 in SS- and oxLDL-dependent manner. Furthermore, specific in vivo blockade of miR-92a expression in Ldlr(-/-) mice reduced endothelial inflammation and altered the development of atherosclerosis, decreasing plaque size and promoting a more stable lesion phenotype. CONCLUSIONS: Upregulation of miR-92a by oxLDL in atheroprone areas promotes endothelial activation and the development of atherosclerotic lesions. Therefore, miR-92a antagomir seems as a new atheroprotective therapeutic strategy.

Angiotensin II mobilizes spleen monocytes to promote the development of abdominal aortic aneurysm in Apoe-/- mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is widespread among elderly people and results in progressive expansion and rupture of the aorta with high mortality. Macrophages, which are the main population observed within the site of aneurysm, are thought to derive from circulating monocytes although no direct evidence has been provided to date. In this study, we were particularly interested in understanding the trafficking behavior of monocyte subsets in AAA and their role in disease pathogenesis. APPROACH AND RESULTS: Using bone marrow transplantation in Apoe(-/-) mice, we showed that circulating monocytes give rise to abdominal aortic macrophages in hypercholesterolemic mice submitted to angiotensin II (AngII). Detailed monitoring of monocyte compartmentalization revealed that lymphocyte antigen 6C(high) and lymphocyte antigen 6C(low) monocytes transiently increase in blood early after AngII infusion and differentially infiltrate the abdominal aorta. The splenic reservoir accounted for the mobilization of the 2 monocyte subsets after 3 days of AngII infusion. Spleen removal or lymphocyte deficiency in Apoe(-/-) Rag2(-/-) mice similarly impaired early monocyte increase in blood in response to AngII and protected against AAA development, independently of blood pressure. Reconstitution of Apoe(-/-) Rag2(-/-) mice with total splenocytes but not with B-cell-depleted splenocytes restored monocyte mobilization in response to AngII and enhanced susceptibility to AAA. CONCLUSIONS: Taken together, the data show that lymphocyte antigen 6C(high) and lymphocyte antigen 6C(low) monocytes are mobilized from the spleen in response to AngII. Intriguingly, the process is dependent on the presence of B cells and significantly contributes to the development of AAA and the occurrence of aortic rupture.

Role of splenic monocytes in atherosclerosis.

PURPOSE OF REVIEW: This review relates recent findings that highlight the role of the spleen as an active donor of monocytes during inflammation, with a special focus on atherosclerosis. RECENT FINDINGS: The contribution of hypercholesterolemia and monocytes/macrophages to atherosclerotic lesion formation is undisputable. The origin of plaque macrophages is, however, still a subject of debate as to whether they derive from local amplification of (resident) macrophages or from continuous recruitment and differentiation of monocytes. Recently, the spleen has emerged as an important reservoir of monocytes that contributes to lesion growth. The regulation of monocyte mobilization from the splenic compartment has, therefore, raised a keen interest in understanding the cellular and molecular mechanisms involved in this process. SUMMARY: Impaired regulation of cholesterol metabolism increases the proliferation of hematopoietic stem and progenitor cells in both the bone marrow and the spleen. Recent findings identified the implication of angiotensin II, red pulp macrophages and B-lymphocytes as partners of monocyte expansion in, and mobilization from the spleen. Future studies will help in understanding the mechanisms of monocyte mobilization and its precise roles in atherosclerosis, and whether modulation of the splenic components may become a promising future direction in the prevention and treatment of cardiovascular diseases.

Monocyte trafficking in acute and chronic inflammation.

Environmental signals at the site of inflammation mediate rapid monocyte mobilization and dictate differentiation programs whereby these cells give rise to macrophages or dendritic cells. Monocytes participate in tissue healing, clearance of pathogens and dead cells, and initiation of adaptive immunity. However, recruited monocytes can also contribute to the pathogenesis of infection and chronic inflammatory disease, such as atherosclerosis. Here, we explore monocyte trafficking in the context of acute inflammation, relying predominantly on data from microbial infection models. These mechanisms will be compared to monocyte trafficking during chronic inflammation in experimental models of atherosclerosis. Recent developments suggest that monocyte trafficking shares common themes in diverse inflammatory diseases; however, important differences exist between monocyte migratory pathways in acute and chronic inflammation.

Natural regulatory T cells limit angiotensin II-induced aneurysm formation and rupture in mice.

OBJECTIVE: Abdominal aortic aneurysm is an inflammatory disease leading to destructive vascular remodeling and ultimately to lethal aortic rupture. Despite its frequent association with atherosclerosis, compelling studies have shown striking differences and potentially opposite roles of T-cell helper responses in aneurysm as compared with atherosclerosis, casting doubt on the relevance and suitability of T-cell-targeted therapies in this context. APPROACH AND RESULTS: Here, we show that selective depletion of T regulatory (Treg) cells using a CD25-specific monoclonal antibody significantly enhances the susceptibility of C57Bl/6 mice to angiotensin II-induced abdominal aortic aneurysm and promotes aortic rupture (n=25-44 mice/group). Similar results are observed in angiotensin II-treated Cd80(-/-)/Cd86(-/-) or Cd28(-/-) mice with impaired Treg cell homeostasis (n=18-23 mice/group). Treg cell depletion is associated with increased immune cell activation and a blunted interleukin (IL)-10 anti-inflammatory response, suggesting an immunoinflammatory imbalance. Interestingly, Il-10(-/-) mice (n=20 mice/group) show increased susceptibility to angiotensin II-induced abdominal aortic aneurysm and aortic rupture and are insensitive to Treg cell depletion. Finally, reconstitution of Cd28(-/-) Treg-deficient mice with Treg cells (n=22 mice/group) restores a balance in the immunoinflammatory response, rescues the animals from increased susceptibility to aneurysm, and prevents aortic dissection. CONCLUSIONS: These results identify a critical role for Treg cells and IL-10 in the control of aneurysm formation and its progression to rupture and suggest that therapies targeting Treg responses may be most suited to treat aneurysmal disease.

Natural regulatory T cells control the development of atherosclerosis in mice.

Atherosclerosis is an immunoinflammatory disease elicited by accumulation of lipids in the artery wall and leads to myocardial infarction and stroke. Here, we show that naturally arising CD4(+)CD25(+) regulatory T cells, which actively maintain immunological tolerance to self and nonself antigens, are powerful inhibitors of atherosclerosis in several mouse models. These results provide new insights into the immunopathogenesis of atherosclerosis and could lead to new therapeutic approaches that involve immune modulation using regulatory T cells.

Mild dyslipidemia accelerates tumorigenesis through expansion of Ly6Chi monocytes and differentiation to pro-angiogenic myeloid cells.

Cancer and cardiovascular disease (CVD) share common risk factors such as dyslipidemia, obesity and inflammation. However, the role of pro-atherogenic environment and its associated low-grade inflammation in tumor progression remains underexplored. Here we show that feeding C57BL/6J mice with a non-obesogenic high fat high cholesterol diet (HFHCD) for two weeks to induce mild dyslipidemia, increases the pool of circulating Ly6Chi monocytes available for initial melanoma development, in an IL-1ß-dependent manner. Descendants of circulating myeloid cells, which accumulate in the tumor microenvironment of mice under HFHCD, heighten pro-angiogenic and immunosuppressive activities locally. Limiting myeloid cell accumulation or targeting VEGF-A production by myeloid cells decrease HFHCD-induced tumor growth acceleration. Reverting the HFHCD to a chow diet at the time of tumor implantation protects against tumor growth. Together, these data shed light on cross-disease communication between cardiovascular pathologies and cancer.

Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) 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(-) 7/4(hi) (also known as Ly6C(hi)) and CD11b(+) Ly6G(-) 7/4(lo) (Ly6C(lo)) 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(-) 7/4(lo) 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.

Lactadherin deficiency leads to apoptotic cell accumulation and accelerated atherosclerosis in mice.

BACKGROUND: Atherosclerosis is an immunoinflammatory disease; however, the key factors responsible for the maintenance of immune regulation in a proinflammatory milieu are poorly understood. METHODS AND RESULTS: Here, we show that milk fat globule-EGF factor 8 (Mfge8, also known as lactadherin) is expressed in normal and atherosclerotic human arteries and is involved in phagocytic clearance of apoptotic cells by peritoneal macrophages. Disruption of bone marrow-derived Mfge8 in a murine model of atherosclerosis leads to substantial accumulation of apoptotic debris both systemically and within the developing lipid lesions. The accumulation of apoptotic material is associated with a reduction in interleukin-10 in the spleen but an increase in interferon-gamma production in both the spleen and the atherosclerotic arteries. In addition, we report a dendritic cell-dependent alteration of natural regulatory T-cell function in the absence of Mfge8. These events are associated with a marked acceleration of atherosclerosis. CONCLUSIONS: Lack of Mfge8 in bone marrow-derived cells enhances the accumulation of apoptotic cell corpses in atherosclerosis and alters the protective immune response, which leads to an acceleration of plaque development.

Indoleamine 2 3-dioxygenase knockout limits angiotensin II-induced aneurysm in low density lipoprotein receptor-deficient mice fed with high fat diet.

AIMS: Abdominal aortic aneurysm (AAA) is an age-associated disease characterized by chronic inflammation, vascular cell apoptosis and metalloproteinase-mediated extracellular matrix degradation. Despite considerable progress in identifying targets involved in these processes, therapeutic approaches aiming to reduce aneurysm growth and rupture are still scarce. Indoleamine 2-3 dioxygenase 1 (IDO) is the first and rate-limiting enzyme involved in the conversion of tryptophan (Trp) into kynurenine (Kyn) pathway. In this study, we investigated the role of IDO in two different models of AAA in mice. METHODS AND RESULTS: Mice with deficiencies in both low density receptor-deficient (Ldlr-/-) and IDO (Ldlr-/-Ido1-/-) were generated by cross-breeding Ido1-/- mice with Ldlr-/-mice. To induce aneurysm, these mice were infused with angiotensin II (Ang II) (1000 ng/min/kg) and fed with high fat diet (HFD) during 28 days. AAAs were present in almost all Ldlr-/- infused with AngII, but only in 50% of Ldlr-/-Ido1-/- mice. Immunohistochemistry at an early time point (day 7) revealed no changes in macrophage and T lymphocyte infiltration within the vessel wall, but showed reduced apoptosis, as assessed by TUNEL assay, and increased α-actin staining within the media of Ldlr-/-Ido1-/- mice, suggesting enhanced survival of vascular smooth muscle cells (VSMCs) in the absence of IDO. In another model of elastase-induced AAA in C57Bl/6 mice, IDO deficiency had no effect on aneurysm formation. CONCLUSION: Our study showed that the knockout of IDO prevented VSMC apoptosis in AngII -treated Ldlr-/- mice fed with HFD, suggesting a detrimental role of IDO in AAA formation and thus would be an important target for the treatment of aneurysm.

Impairment in postischemic neovascularization in mice lacking the CXC chemokine receptor 3.

Inflammatory cell infiltration is a feature of postischemic neovascularization. However, mechanisms leading to leukocyte attraction to the site of neovascularization are still undefined. We hypothesized that the CXC chemokine receptor 3 (CXCR3) may contribute to leukocyte accumulation and subsequently to blood vessel growth in the ischemic area. Ischemia induced by femoral artery ligature improved the number of CXCR3-expressing cells and the level of its ligand, CXCL10. Angiographic score, blood flow recovery measurement, and capillary density analysis showed a significant decrease of ischemic/nonischemic leg ratio in CXCR3-deficient mice when compared with controls (P<0.05), at day 21 after ischemia. Interestingly, this impairment was as important as that observed in mice deficient for the well known CC-chemokine monocyte chemoattractant protein-1 (MCP-1). At day 7 of ischemic injury, the number of CD3-positive T cells and Mac-3-positive monocytes/macrophages was 38% and 45% lower, respectively, in the ischemic leg of CXCR3-deficient mice compared with the control group (P<0.05), suggesting an important role for CXCR3 in leukocyte recruitment into the ischemic area. VEGF protein content, a classical proangiogenic factor, was also markedly reduced (80% reduction) in ischemic leg of CXCR3-deficient mice (P<0.01). Injection of bone marrow-derived mononuclear cells (BM-MNCs) isolated from wild-type animals restored the neovascularization reaction in CXCR3-deficient mice whereas BM-MNCs from CXCR3-deficient mice was ineffective. In conclusion, CXCR3 plays a key role in neovascularization and provides novel information on the mechanisms leading to leukocyte infiltration in the vessel growth area.

Role of bone marrow-derived CC-chemokine receptor 5 in the development of atherosclerosis of low-density lipoprotein receptor knockout mice.

OBJECTIVE: CC chemokine receptor CCR5 is expressed by atheroma-associated cells and could mediate leukocyte attraction into developing lesions. We examined the role of bone marrow-derived CCR5 in the development of atherosclerotic lesions after 8, 12, or 35 weeks of high-fat diet. METHODS AND RESULTS: Low-density lipoprotein-receptor (LDLr)-deficient mice were lethally irradiated and transplanted with CCR5+/+ or CCR5-/- bone marrow. After 8 weeks of fat diet, CCR5 deficiency in leukocytes led to 30% decrease of macrophage accumulation within the fatty streak (P<0.05), with no change in lesion size. After 12 weeks of fat diet, CCR5 deficiency also resulted in 30% decrease of plaque-macrophage accumulation (P<0.005), associated with 16% reduction in lesion size in the aortic sinus (P=0.13), despite a significant increase in total cholesterol levels (P=0.03). Lesions with CCR5 deficiency showed 52% reduction in matrix metalloproteinase (MMP)-9 expression (P=0.02) and 2-fold increase in collagen accumulation (P<0.0001). These changes were associated with a significant increase of interleukin (IL)-10 mRNA expression in spleens of CCR5-/- mice compared with CCR5+/+ controls. In addition, we found enhanced IL-10 production by CCR5-deficient peritoneal macrophages and decreased tumor necrosis factor (TNF)-alpha production by CCR5-/- T cells in comparison with CCR5+/+ controls. CCR5-/- and CCR5+/+ reconstituted animals showed no differences in plaque size or composition after 35 weeks of high-fat diet despite the persistent absence of CCR5 in plaques of mice reconstituted with CCR5-/- bone marrow. CONCLUSIONS: Bone marrow-derived CCR5 favors the development of an inflammatory and collagen-poor plaque phenotype in association with decreased macrophage-derived IL-10 and enhanced T cell-derived TNF-alpha. These effects are not sustained in the very advanced stages of atherosclerosis.