PD-L1 expression on nonclassical monocytes reveals their origin and immunoregulatory function.

The role of nonclassical monocytes (NCMs) in health and disease is emerging, but their location and function within tissues remain poorly explored. Imaging of NCMs has been limited by the lack of an established single NCM marker. Here, we characterize the immune checkpoint molecule PD-L1 (CD274) as an unequivocal marker for tracking NCMs in circulation and pinpoint their compartmentalized distribution in tissues by two-photon microscopy. Visualization of PD-L1+ NCMs in relation to bone marrow vasculature reveals that conversion of classical monocytes into NCMs requires contact with endosteal vessels. Furthermore, PD-L1+ NCMs are present in tertiary lymphoid organs (TLOs) under inflammatory conditions in both mice and humans, and NCMs exhibit a PD-L1-dependent immunomodulatory function that promotes T cell apoptosis within TLOs. Our findings establish an unambiguous tool for the investigation of NCMs and shed light on their origin and function.

Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation.

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.

Human Neutrophil Peptide 1 Limits Hypercholesterolemia-induced Atherosclerosis by Increasing Hepatic LDL Clearance.

Increases in plasma LDL-cholesterol have unequivocally been established as a causal risk factor for atherosclerosis. Hence, strategies for lowering of LDL-cholesterol may have immediate therapeutic relevance. Here we study the role of human neutrophil peptide 1 (HNP1) in a mouse model of atherosclerosis and identify its potent atheroprotective effect both upon transgenic overexpression and therapeutic delivery. The effect was found to be due to a reduction of plasma LDL-cholesterol. Mechanistically, HNP1 binds to apolipoproteins enriched in LDL. This interaction facilitates clearance of LDL particles in the liver via LDL receptor. Thus, we here identify a non-redundant mechanism by which HNP1 allows for reduction of LDL-cholesterol, a process that may be therapeutically instructed to lower cardiovascular risk.

Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice.

RATIONALE: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin αIIbß3-mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. OBJECTIVE: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. METHODS AND RESULTS: JAM-A-deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On αIIbß3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e (apoe(-/-)) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A(-/-)apoe-/- mice showed increased aortic plaque formation when compared with trJAM-A(+/+) apoe(-/-) controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A(-/-) apoe(-/-) animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A(-/-) apoe(-/-)mice, and JAM-A-deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A(-/-) apoe(-/-)mice. Notably, these proinflammatory effects of JAM-A-deficient platelets could be abolished by the inhibition of αIIbß3 signaling in vitro. CONCLUSIONS: Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease.

Identification and characterization of circulating variants of CXCL12 from human plasma: effects on chemotaxis and mobilization of hematopoietic stem and progenitor cells.

Mobilization of hematopoietic stem and progenitor cells (HPCs) is induced by treatment with granulocyte-colony stimulating factor, chemotherapy, or irradiation. We observed that these treatments are accompanied by a release of chemotactic activity into the blood. This plasma activity is derived from the bone marrow, liver, and spleen and acts on HPCs via the chemokine receptor CXCR4. A human blood peptide library was used to characterize CXCR4-activating compounds. We identified CXCL12[22-88] and N-terminally truncated variants CXCL12[24-88], CXCL12[25-88], CXCL12[27-88], and CXCL12[29-88]. Only CXCL12[22-88] could effectively bind to CXCR4 and induce intracellular calcium flux and chemotactic migration of HPCs. CXCL12[25-88] and CXCL12[27-88] revealed neither agonistic nor antagonistic activities in vitro, whereas CXCL12[29-88] inhibited CXCL12[22-88]-induced chemotactic migration. Since binding to glycosaminoglycans (GAG) modulates the function of CXCL12, binding to heparin was analyzed. Surface plasmon resonance kinetic analysis showed that N-terminal truncation of Arg22-Pro23 increased the dissociation constant KD by one log10 stage ([22-88]: KD: 5.4 ± 2.6 µM; [24-88]: KD: 54 ± 22.4 µM). Further truncation of the N-terminus decreased the KD ([25-88] KD: 30 ± 4.8 µM; [27-88] KD: 23 ± 1.6 µM; [29-88] KD: 19 ± 5.4 µM), indicating increasing competition for heparin binding. Systemic in vivo application of CXCL12[22-88] as well as CXCL12[27-88] or CXCL12[29-88] induced a significant mobilization of HPCs in mice. Our findings indicate that plasma-derived CXCL12 variants may contribute to the regulation of HPC mobilization by modulating the binding of CXCL12[22-88] to GAGs rather than blocking the CXCR4 receptor and, therefore, may have a contributing role in HPC mobilization.

Touch of chemokines.

Chemoattractant cytokines or chemokines constitute a family of structurally related proteins found in vertebrates, bacteria, or viruses. So far, 48 chemokine genes have been identified in humans, which bind to around 20 chemokine receptors. These receptors belong to the seven transmembrane G-protein-coupled receptor family. Chemokines and their receptors were originally studied for their role in cellular trafficking of leukocytes during inflammation and immune surveillance. It is now known that they exert different functions under physiological conditions such as homeostasis, development, tissue repair, and angiogenesis but also under pathological disorders including tumorigenesis, cancer metastasis, inflammatory, and autoimmune diseases. Physicochemical properties of chemokines and chemokine receptors confer the ability to homo- and hetero-oligomerize. Many efforts are currently performed in establishing new therapeutically compounds able to target the chemokine/chemokine receptor system. In this review, we are interested in the role of chemokines in inflammatory disease and leukocyte trafficking with a focus on vascular inflammatory diseases, the operating synergism, and the emerging therapeutic approaches of chemokines.

Inhibition of human initiator caspase 8 and effector caspase 3 by cross-class inhibitory bovSERPINA3-1 and A3-3.

Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb:Q9TTE1) binds tighly to human CASP3 (uniprotkb:P42574) and CASP8 (uniprotkb:Q14790) with k(ass) of 4.2x10(5) and 1.4x10(6) M(-1)s(-1), respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb:Q3ZEJ6) was also observed with k(ass) of 1.5x10(5) and 2.7x10(6) M(-1)s(-1), respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp(371) as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed.