Rory R. Koenen

MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment

The cytokine macrophage migration inhibitory factor (MIF) plays a critical role in inflammatory diseases and atherogenesis. We identify the chemokine receptors CXCR2 and CXCR4 as functional receptors for MIF. MIF triggered Gαi- and integrin-dependent arrest and chemotaxis of monocytes and T cells, rapid integrin activation and calcium influx through CXCR2 or CXCR4. MIF competed with cognate ligands for CXCR4 and CXCR2 binding, and directly bound to CXCR2. CXCR2 and CD74 formed a receptor complex, and monocyte arrest elicited by MIF in inflamed or atherosclerotic arteries involved both CXCR2 and CD74. In vivo, Mif deficiency impaired monocyte adhesion to the arterial wall in atherosclerosis-prone mice, and MIF-induced leukocyte recruitment required Il8rb (which encodes Cxcr2). Blockade of Mif but not of canonical ligands of Cxcr2 or Cxcr4 in mice with advanced atherosclerosis led to plaque regression and reduced monocyte and T-cell content in plaques. By activating both CXCR2 and CXCR4, MIF displays chemokine-like functions and acts as a major regulator of inflammatory cell recruitment and atherogenesis. Targeting MIF in individuals with manifest atherosclerosis can potentially be used to treat this condition.

Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice

Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.

MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages

Macrophages in atherosclerotic plaques drive inflammatory responses, degrade lipoproteins, and phagocytose dead cells. MicroRNAs (miRs) control the differentiation and activity of macrophages by regulating the signaling of key transcription factors. However, the functional role of macrophage-related miRs in the immune response during atherogenesis is unknown. Here, we report that miR-155 is specifically expressed in atherosclerotic plaques and proinflammatory macrophages, where it was induced by treatment with mildly oxidized LDL (moxLDL) and IFN-γ. Leukocyte-specific Mir155 deficiency reduced plaque size and number of lesional macrophages after partial carotid ligation in atherosclerotic (Apoe-/-) mice. In macrophages stimulated with moxLDL/IFN-γ in vitro, and in lesional macrophages, loss of Mir155 reduced the expression of the chemokine CCL2, which promotes the recruitment of monocytes to atherosclerotic plaques. Additionally, we found that miR-155 directly repressed expression of BCL6, a transcription factor that attenuates proinflammatory NF-κB signaling. Silencing of Bcl6 in mice harboring Mir155-/- macrophages enhanced plaque formation and CCL2 expression. Taken together, these data demonstrated that miR-155 plays a key role in atherogenic programming of macrophages to sustain and enhance vascular inflammation.

Platelet Microparticles: A Transcellular Delivery System for RANTES Promoting Monocyte Recruitment on Endothelium

Objective—Platelet activation mediates multiple cellular responses, including secretion of chemokines such as RANTES (CCL5), and formation of platelet microparticles (PMPs). We studied the role of PMPs in delivering RANTES and promoting monocyte recruitment. Methods and Results—Here we show that PMPs contain substantial amounts of RANTES and deposit RANTES on activated endothelium or murine atherosclerotic carotid arteries. RANTES deposition is facilitated by flow conditions and more efficient than that conferred by PMP supernatants. Interactions of PMPs with activated endothelium in flow were mostly characterized by rolling. RANTES deposition showed a diffuse distribution pattern and was rarely colocalized with firmly adherent PMPs, substantiating that RANTES deposition occurs during transient interactions. Importantly, preperfusion with PMPs enhanced monocyte arrest on activated endothelium or atherosclerotic carotid arteries, which could be inhibited by a blocking antibody or a RANTES receptor antagonist. Blockade or deficiency of PMP-expressed adhesion receptors demonstrated differential requirement of P-selectin, glycoprotein Ib (GPIb), GPIIb/IIIa, and junctional adhesion molecule-A for PMP interactions with endothelium, PMP-dependent RANTES deposition, and subsequent monocyte arrest. Conclusion—Circulating PMPs may serve as a finely tuned transcellular delivery system for RANTES, triggering monocyte arrest to inflamed and atherosclerotic endothelium, introducing a novel mechanism for platelet-dependent monocyte recruitment in inflammation and atherosclerosis.

Auto-Antigenic Protein-DNA Complexes Stimulate Plasmacytoid Dendritic Cells to Promote Atherosclerosis

Background— Inflammation has been closely linked to auto-immunogenic processes in atherosclerosis. Plasmacytoid dendritic cells (pDCs) are specialized to produce type-I interferons in response to pathogenic single-stranded nucleic acids, but can also sense self-DNA released from dying cells or in neutrophil extracellular traps complexed to the antimicrobial peptide Cramp/LL37 in autoimmune disease. However, the exact role of pDCs in atherosclerosis remains elusive. Methods and Results— Here we demonstrate that pDCs can be detected in murine and human atherosclerotic lesions. Exposure to oxidatively modified low-density lipoprotein enhanced the capacity of pDCs to phagocytose and prime antigen-specific T cell responses. Plasmacytoid DCs can be stimulated to produce interferon-&agr; by Cramp/DNA complexes, and we further identified increased expression of Cramp and formation of neutrophil extracellular traps in atherosclerotic arteries. Whereas Cramp/DNA complexes aggravated atherosclerotic lesion formation in apolipoprotein E–deficient mice, pDC depletion and Cramp-deficiency in bone marrow reduced atherosclerosis and anti–double-stranded DNA antibody titers. Moreover, the specific activation of pDCs and interferon-&agr; treatment promoted plaque growth, associated with enhanced anti–double-stranded–DNA antibody titers. Accordingly, anti–double-stranded DNA antibodies were elevated in patients with symptomatic versus asymptomatic carotid artery stenosis. Conclusions— Self-DNA (eg, released from dying cells or in neutrophil extracellular traps) and an increased expression of the antimicrobial peptide Cramp/LL37 in atherosclerotic lesions may thus stimulate a pDC-driven pathway of autoimmune activation and the generation of anti–double-stranded-DNA antibodies, critically aggravating atherosclerosis lesion formation. These key factors may thus represent novel therapeutic targets.

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5-/- mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

Disruption of Platelet-derived Chemokine Heteromers Prevents Neutrophil Extravasation in Acute Lung Injury

RATIONALE Acute lung injury (ALI) causes high mortality, but its molecular mechanisms and therapeutic options remain ill-defined. Gram-negative bacterial infections are the main cause of ALI, leading to lung neutrophil infiltration, permeability increases, deterioration of gas exchange, and lung damage. Platelets are activated during ALI, but insights into their mechanistic contribution to neutrophil accumulation in the lung are elusive. OBJECTIVES To determine mechanisms of platelet-mediated neutrophil recruitment in ALI. METHODS Interference with platelet-neutrophil interactions using antagonists to P-selectin and glycoprotein IIb/IIIa or a small peptide antagonist disrupting platelet chemokine heteromer formation in mouse models of ALI. MEASUREMENTS AND MAIN RESULTS In a murine model of LPS-induced ALI, we uncover important roles for neutrophils and platelets in permeability changes and subsequent lung damage. Furthermore, platelet depletion abrogated lung neutrophil infiltration, suggesting a sequential participation of platelets and neutrophils. Whereas antagonists to P-selectin and glycoprotein IIb/IIIa had no effects on LPS-mediated ALI, antibodies to the platelet-derived chemokines CCL5 and CXCL4 strongly diminished neutrophil eflux and permeability changes. The two chemokines were found to form heteromers in human and murine ALI samples, positively correlating with leukocyte influx into the lung. Disruption of CCL5-CXCL4 heteromers in LPS-, acid-, and sepsis-induced ALI abolished lung edema, neutrophil infiltration, and tissue damage, thereby revealing a causal contribution. CONCLUSIONS Taken together, our data identify a novel function of platelet-derived chemokine heteromers during ALI and demonstrate means for therapeutic interference.

Therapeutic targeting of chemokine interactions in atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall that is characterized by a disturbed equilibrium of immune responses and lipid accumulation, leading to the development of plaques. The atherogenic influx of mononuclear cells is orchestrated by chemokines and their receptors. Studies using gene-deficient mice and antagonists based on peptides and small molecules have generated insight into targeting chemokine–receptor axes for treating atherosclerosis, which might complement lipid-lowering strategies and risk factor modulation. Combined inhibition of multiple chemokine axes could interfere with the contributions of chemokines to disease progression at specific cells, stages or sites. In addition, the recently characterized heterophilic interactions of chemokines might present a novel target for the treatment and prevention of inflammatory diseases such as atherosclerosis.

Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1hi) or non‐classical (resident/Gr1lo) monocytes to dissect their differential role in atheroprogression under high‐fat diet (HFD). Apolipoprotein E‐deficient (Apoe−/−) mice lacking classical but not non‐classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1‐neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene‐deficient Apoe−/− mice, adoptive transfer of gene‐deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.

Structural determinants of MIF functions in CXCR2-mediated inflammatory and atherogenic leukocyte recruitment

We have recently identified the archaic cytokine macrophage migration inhibitory factor (MIF) as a non-canonical ligand of the CXC chemokine receptors CXCR2 and CXCR4 in inflammatory and atherogenic cell recruitment. Because its affinity for CXCR2 was particularly high, we hypothesized that MIF may feature structural motives shared by canonical CXCR2 ligands, namely the conserved N-terminal Glu-Leu-Arg (ELR) motif. Sequence alignment and structural modeling indeed revealed a pseudo-(E)LR motif (Asp-44-X-Arg-11) constituted by non-adjacent residues in neighboring loops but with identical parallel spacing as in the authentic ELR motif. Structure–function analysis demonstrated that mutation of residues R11, D44, or both preserve proper folding and the intrinsic catalytic property of MIF but severely compromises its binding to CXCR2 and abrogates MIF/CXCR2-mediated functions in chemotaxis and arrest of monocytes on endothelium under flow conditions. R11A-MIF and the R11A/D44A-MIF double-mutant exhibited a pronounced defect in triggering leukocyte recruitment to early atherosclerotic endothelium in carotid arteries perfused ex vivo and upon application in a peritonitis model. The function of D44A-MIF in peritoneal leukocyte recruitment was preserved as a result of compensatory use of CXCR4. In conjunction, our data identify a pseudo-(E)LR motif as the structural determinant for MIFs activity as a non-canonical CXCR2 ligand, epitomizing the structural resemblance of chemokine-like ligands with chemokines and enabling selective targeting of pro-inflammatory MIF/CXCR2 interactions.