Abstract
The outcomes of the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial have unequivocally proven that inflammation is a key driver of atherosclerosis and that targeting ...inflammation, in this case by using an anti-interleukin-1β antibody, improves cardiovascular disease (CVD) outcomes. This is especially true for CVD patients with a pro-inflammatory constitution. Although CANTOS has epitomized the importance of targeting inflammation in atherosclerosis, treatment with canakinumab did not improve CVD mortality, and caused an increase in infections. Therefore, the identification of novel drug targets and development of novel therapeutics that block atherosclerosis-specific inflammatory pathways and exhibit limited immune-suppressive side effects, as pursued in our collaborative research centre, are required to optimize immunotherapy for CVD. In this review, we will highlight the potential of novel immunotherapeutic targets that are currently considered to become a future treatment for CVD.
Acute myocardial infarction (MI) is the leading cause of mortality worldwide. Anti-inflammatory strategies to reduce neutrophil-driven acute post-MI injury have been shown to limit acute cardiac ...tissue damage. On the other hand, whether neutrophils are required for resolving post-MI inflammation and repair is unknown.
We show that neutrophil-depleted mice subjected to MI had worsened cardiac function, increased fibrosis, and progressively developed heart failure. Flow cytometry of blood, lymphoid organs and digested hearts revealed reduced numbers of Ly6Chigh monocytes in infarcts of neutrophil-depleted mice, whereas the number of macrophages increased, which was paralleled by reduced splenic Ly6Chigh monocyte mobilization but enhanced proliferation of cardiac macrophages. Macrophage subtype analysis revealed reduced cardiac expression of M1 markers, whereas M2 markers were increased in neutrophil-depleted mice. Surprisingly, we found reduced expression of phagocytosis receptor myeloid-epithelial-reproductive tyrosine kinase, a marker of reparative M2c macrophages which mediate clearance of apoptotic cells. In agreement with this finding, neutrophil-depleted mice had increased numbers of TUNEL-positive cells within infarcts. We identified neutrophil gelatinase-associated lipocalin (NGAL) in the neutrophil secretome as a key inducer of macrophages with high capacity to engulf apoptotic cells. The cardiac macrophage phenotype in neutrophil-depleted mice was restored by administration of neutrophil secretome or NGAL.
Neutrophils are crucially involved in cardiac repair after MI by polarizing macrophages towards a reparative phenotype. Therapeutic strategies to reduce acute neutrophil-driven inflammation after MI should be carefully balanced as they might interfere with the healing response and cardiac remodelling.
The pericardial adipose tissue (AT) contains a high density of lymphoid clusters. It is unknown whether these clusters play a role in post-myocardial infarction (MI) inflammatory responses and ...cardiac outcome.
Lymphoid clusters were examined in epicardial AT of humans with or without coronary artery disease. Murine pericardial lymphoid clusters were visualized in mice subjected to coronary artery ligation. To study the relevance of pericardial clusters during inflammatory responses after MI, we surgically removed the pericardial AT and performed B-cell depletion and granulocyte-macrophage colony-stimulating factor blockade. Leukocytes in murine hearts, pericardial AT, spleen, mediastinal lymph nodes, and bone marrow were quantified by flow cytometry. Cannabinoid receptor CB2 (CB2
) mice were used as a model for enhanced B-cell responses. The effect of impaired dendritic cell (DC) trafficking on pericardial AT inflammatory responses was tested in CCR7
mice subjected to MI. Cardiac fibrosis and ventricular function were assessed by histology and echocardiography.
We identified larger B-cell clusters in epicardial AT of human patients with coronary artery disease in comparison with controls without coronary artery disease. Infarcted mice also had larger pericardial clusters and 3-fold upregulated numbers of granulocyte-macrophage colony-stimulating factor-producing B cells within pericardial AT, but not spleen or lymph nodes. This was associated with higher DC and T-cell counts in pericardial AT, which outnumbered DCs and T cells in lymph nodes. Analysis of DC maturation markers, tracking experiments with fluorescently labeled cells, and use of CCR7-deficient mice suggested that activated DCs migrate from infarcts into pericardial AT via CCR7. B-cell depletion or granulocyte-macrophage colony-stimulating factor neutralization inhibited DC and T-cell expansion within pericardial AT, and translated into reduced bone marrow granulopoiesis and cardiac neutrophil infiltration 3 days after MI. The relevance of the pericardial AT in mediating all these effects was confirmed by removal of pericardial AT and ex vivo coculture with pericardial AT and granulocyte progenitors. Finally, enhanced fibrosis and worsened ejection fraction in CB2
mice were limited by pericardial AT removal.
Our findings unveil a new mechanism by which the pericardial AT coordinates immune cell activation, granulopoiesis, and outcome after MI.
MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to ...control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.
Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils ...adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.
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•Aged mice show high levels of neutrophil reverse transendothelial migration (rTEM)•Mast cells (MC) and MC-derived CXCL1 drive neutrophil rTEM in inflamed aged tissues•Intensified endothelial ACKR1-CXCL1 axis promotes neutrophil CXCR2 internalization•Aged lungs program rTEM neutrophils toward an activated and noxious phenotype
Aging is a critical risk factor for inflammatory disorders. Barkaway, Rolas et al. show that inflamed aged tissues present a high frequency of neutrophil reverse transendothelial migration (rTEM) back into the circulation in a mast cell-dependent manner. rTEM neutrophils are retained in aged lungs and programmed toward an activated phenotype, capable of inducing tissue damage.
Myocardial infarction (MI) is the leading cause of death in Western countries. Epidemiological studies show acute MI to be more prevalent in the morning and to be associated with a poorer outcome in ...terms of mortality and recovery. The mechanisms behind this association are not fully understood. Here, we report that circadian oscillations of neutrophil recruitment to the heart determine infarct size, healing, and cardiac function after MI. Preferential cardiac neutrophil recruitment during the active phase (Zeitgeber time, ZT13) was paralleled by enhanced myeloid progenitor production, increased circulating numbers of CXCR2hi neutrophils as well as upregulated cardiac adhesion molecule and chemokine expression. MI at ZT13 resulted in significantly higher cardiac neutrophil infiltration compared to ZT5, which was inhibited by CXCR2 antagonism or neutrophil‐specific CXCR2 knockout. Limiting exaggerated neutrophilic inflammation at this time point significantly reduced the infarct size and improved cardiac function.
Synopsis
Expression levels of chemokine receptor CXCR2 on circulating neutrophils exhibit diurnal oscillations. This causes time‐of‐day‐dependent variations in the number of neutrophils infiltrating the heart after myocardial infarction, with major consequences for infarction healing.
During the sleep‐to‐wake transition period, myeloid progenitor production in the bone marrow is enhanced and the heart expresses higher levels of neutrophil chemoattractants and adhesion molecules.
A myocardial infarction at this time point leads to excessive cardiac neutrophil recruitment, larger infarct size, and worsened heart function.
The enhanced cardiac neutrophil infiltration after myocardial infarction onset during the sleep‐to‐wake transition period is CXCR2‐dependent.
Expression levels of chemokine receptor CXCR2 on circulating neutrophils exhibit diurnal oscillations. This causes time‐of‐day‐dependent variations in the number of neutrophils infiltrating the heart after myocardial infarction, with major consequences for infarction healing.
Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. ...Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.
Synopsis
Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.
Chemokines and galectins can engage in cross talk by pairing, as exemplified by galectin‐3 and CXCL12.
The association of CXCL12 with galectin‐3 appears to have potential for modulating chemokine activity.
Galectin‐3 inhibits CXCL12‐induced chemotaxis of leukocytes and their recruitment to inflammation sites.
Galectin‐3 attenuates CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex.
Chemokines and galectins are simultaneously upregulated during inflammation and mediate leukocyte recruitment. A systematic screen now demonstrates their physical interaction as heterodimers, identifying several novel interacting pairs.
Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively ...abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.
Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor ...Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor β1 integrin and occurs in the absence of blood flow. Downregulation of β1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a β1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.
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•Loss of cerebral cavernous malformation proteins causes an upregulation of Klf2•Upregulation of Klf2 requires β1 integrin and occurs in the absence of blood flow•Klf2 positively regulates egfl7 expression and promotes angiogenesis•Aberrant angiogenesis signaling underlies ccm mutant cardiovascular defects
Renz et al. identify the CCM complex as a key regulator of a β1 integrin-Klf2-Egfl7 mechanotransduction pathway in zebrafish. This regulation prevents angiogenic overgrowth and ensures the quiescence and differentiation of endothelial and endocardial cells.
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