Almudena Ortega-Gomez

Resolution of inflammation: an integrated view

Resolution of inflammation is a coordinated and active process aimed at restoration of tissue integrity and function. This review integrates the key molecular and cellular mechanisms of resolution. We describe how abrogation of chemokine signalling blocks continued neutrophil tissue infiltration and how apoptotic neutrophils attract monocytes and macrophages to induce their clearance. Uptake of apoptotic neutrophils by macrophages reprograms macrophages towards a resolving phenotype, a key event to restore tissue homeostasis. Finally, we highlight the therapeutic potential that derives from understanding the mechanisms of resolution.

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.

Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis

Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsis.

Nanomedicine-based strategies for treatment of atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall that arises from an imbalanced lipid metabolism and a maladaptive inflammatory response. Despite intensive research on mechanisms underlying atherosclerotic lesion formation and progression during the past decade, translation of this knowledge into the clinic is scarce. Although developments have primarily been made in the area of antitumor therapy, recent advances have shown the potential of nanomedicine-based treatment strategies for atherosclerosis. Here we describe the features of currently available nanomedical formulations that have been optimized for atherosclerosis treatment, and we further describe how they can be instructed to target inflammatory processes in the arterial wall. Despite their limitations, nanomedical applications might hold promise for personalized medicine, and further efforts are needed to improve atherosclerosis-specific targeting.

Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5

Disrupting the HNP1 and CCL5 heteromer between neutrophils and platelets blocks monocyte recruitment to inflammatory sites. Anti-inflammatory reaches for the SKY Inflammation aids the body’s response to infection or injury, but can cause damage if excessive or unresolved. Alard et al. now examine how two early inflammatory mediators—neutrophils and platelets—cooperate to enhance inflammation. They found that human neutrophil peptide 1 (HNP1), which is secreted from neutrophils, forms a heteromer with CCL5 on platelets, resulting in stimulated monocyte adhesion and an increase in inflammation. Disrupting this interaction with a peptide (SKY) decreased inflammation and blocked monocyte recruitment in a mouse model of myocardial infarction. If these results hold true in humans, they could form the basis for a new specific therapeutic in inflammation-associated diseases. In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation

Functional synergism and inhibitory effects of chemokine heterodimers can be selectively targeted by specific peptides in models of inflammation. Hampering heterodimers interrupts inflammation Inflammation is dependent on the recruitment of cells responding to chemokines. Von Hundelshausen et al. cataloged how human chemokines interact with each other and found that certain kinds of chemokine pairs can activate or inhibit receptor signaling. These chemokine heterodimers were shown to be active in mouse models of acute and chronic inflammation, which were ameliorated by treatment with a peptide designed to disrupt the chemokine pairing. Patients suffering from inflammatory conditions such as atherosclerosis could benefit from these kinds of therapeutics. Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting.

Neutrophil-macrophage interplay in atherosclerosis: protease-mediated cytokine processing versus NET release

Neutrophil-macrophage interplay in atherosclerosis: protease-mediated cytokine processing versus NET release -

Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment

Background: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment. Methods: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe–/–and CatG-deficient mice (Apoe–/–Ctsg–/–) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy. Results: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice. Conclusions: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.

Chrono-pharmacological Targeting of the CCL2-CCR2 Axis Ameliorates Atherosclerosis

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.

Functional ex-vivo Imaging of Arterial Cellular Recruitment and Lipid Extravasation

The main purpose of this sophisticated and highly versatile method is to visualize and quantify structural vessel wall properties, cellular recruitment, and lipid/dextran extravasation under physiological conditions in living arteries. This will be of interest for a broad range of researchers within the field of inflammation, hypertension, atherosclerosis, and even the pharmaceutical industry. Currently, many researchers are using in vitro techniques to evaluate cellular recruitment, like transwell or flow chamber systems with cultured cells, with unclear physiological comparability. The here introduced method describes in detail the use of a sophisticated and flexible method to study arterial wall properties and leukocyte recruitment in fresh and viable murine carotid arteries ex vivo under arterial flow conditions. This model mimics the in vivo situation and allows the use of cells and arteries isolated from two different donors (for example, wildtype vs. specific knockouts) to be combined into one experiments, thereby providing information on both leukocyte and/or endothelial cell properties of both donors. As such, this model can be considered an alternative for the complicated and invasive in vivo studies, such as parabiotic experiments.