Myeloid cannabinoid CB1 receptor deletion confers atheroprotection in male mice by reducing macrophage proliferation in a sex-dependent manner.

AIMS: Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signaling in atherosclerosis. METHODS AND RESULTS: Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1 deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on estrogen receptor (ER)α-estradiol signaling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology. CONCLUSION: Impaired macrophage CB1 signaling is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming in male mice. The importance of macrophage CB1 signaling appears to be sex-dependent.

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays, antigen-specific antibody assays and cellular assays.

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax™), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M-052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.

Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) drives atherosclerosis in mice and is associated with an unstable plaque phenotype and cerebrovascular events in humans.

AIMS: GITR-a co-stimulatory immune checkpoint protein-is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). METHODS AND RESULTS: GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr-/-Apoe-/- mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr-/-Apoe-/- and Apoe-/- monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr-/-Apoe-/- monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity. CONCLUSION: Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.

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.

Identification of an Arg-Leu-Arg tripeptide that contributes to the binding interface between the cytokine MIF and the chemokine receptor CXCR4.

MIF is a chemokine-like cytokine that plays a role in the pathogenesis of inflammatory and cardiovascular disorders. It binds to the chemokine-receptors CXCR2/CXCR4 to trigger atherogenic leukocyte migration albeit lacking canonical chemokine structures. We recently characterized an N-like-loop and the Pro-2-residue of MIF as critical molecular determinants of the CXCR4/MIF binding-site and identified allosteric agonism as a mechanism that distinguishes CXCR4-binding to MIF from that to the cognate ligand CXCL12. By using peptide spot-array technology, site-directed mutagenesis, structure-activity-relationships, and molecular docking, we identified the Arg-Leu-Arg (RLR) sequence-region 87-89 that - in three-dimensional space - 'extends' the N-like-loop to control site-1-binding to CXCR4. Contrary to wildtype MIF, mutant R87A-L88A-R89A-MIF fails to bind to the N-terminal of CXCR4 and the contribution of RLR to the MIF/CXCR4-interaction is underpinned by an ablation of MIF/CXCR4-specific signaling and reduction in CXCR4-dependent chemotactic leukocyte migration of the RLR-mutant of MIF. Alanine-scanning, functional competition by RLR-containing peptides, and molecular docking indicate that the RLR residues directly participate in contacts between MIF and CXCR4 and highlight the importance of charge-interactions at this interface. Identification of the RLR region adds important structural information to the MIF/CXCR4 binding-site that distinguishes this interface from CXCR4/CXCL12 and will help to design MIF-specific drug-targeting approaches.

Structure-activity relationship of synthetic toll-like receptor 4 agonists.

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators.

Characterization of monoclonal antibody HTA125 with specificity for human TLR4.

Binding of monoclonal antibody HTA125 to human toll-like receptor 4 (TLR4) was characterized by flow cytometry using MonoMac6 human monocytic cells. Data were obtained using direct binding to cell surface TLR4 by labeled HTA125, as well as inhibition of direct binding using purified reagents, and by two-step binding. HTA125 bound weakly to human TLR4, and could be inhibited by mouse Ig, mouse IgG Fc, and mouse IgG2a. In addition, purified human IgG Fc and purified human immunoglobulin of isotypes IgG1 and IgG4 could block binding of HTA125 to MonoMac6 cells. Furthermore, a mouse IgG1 monoclonal antibody possessing specificity for human CD64, which is a high affinity IgG Fc receptor, partially inhibited binding of HTA125 to MonoMac6 cells. Finally, co-stimulation via TLR4 and Fc receptor, resulted in cytokine production by MonoMac6 cells different than that induced via TLR4 alone. Therefore, the utility of HTA125 remains as a weak detector of human TLR4, and as an agent to block TLR4 ligands with an understanding that Fc receptor may be engaged also.

Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529.

MPL (Corixa) adjuvant is a chemically modified derivative of lipopolysaccharide that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of lipopolysaccharide. MPL adjuvant has been used extensively in clinical trials as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. With over 33,000 doses administered to date, MPL adjuvant has emerged as a safe and effective vaccine adjuvant. Recently, scientists at Corixa Corporation have developed a library of synthetic lipid A mimetics (aminoalkyl glucosaminide 4-phosphates) with demonstrated immunostimulatory properties. Similar to MPL adjuvant, these synthetic compounds signal through Toll-like receptor 4 to stimulate the innate immune system. One of these compounds, Ribi.529 (RC-529), has emerged as a leading adjuvant with a similar efficacy and safety profile to MPL adjuvant in both preclinical and clinical studies.

Adaptation of commercial immunoassays to analysis of complex biological substances.

An Intracellular Adhesion Molecule I (ICAM-1) immunoassay from R and D Systems, and a Melanoma Inhibitory Activity (MIA) immunoassay from Roche Diagnostics were tested for accurate quantitation within complex biological substances such as cell lysates. Prior to assay, lysates of melanoma cells were treated with detergents to obtain soluble antigens. Maximum ICAM-1 and maximum MIA were detected after treatment using 0.8% Triton X-100. Two key aspects of assay accuracy were: 1) determining the dilutions of test sample that provided accurate quantitation (sample range), and 2) performing spiking experiments at these dilutions to determine absence or presence of a "matrix" effect due to biological complexity of the sample. A high degree of accuracy was found by diluting this particular cellular extract 50-fold prior to ICAM-1 assay, or only 5-fold prior to MIA assay. In addition, the bicinchoninic acid protein assay was analyzed to test the accuracy of protein quantitation of cellular lysates. Precision, limits of detection, and quantitation, robustness, linearity, and specificity also were tested for the immunoassays.

Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529.

Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood. The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells. Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case. Similarly, the intravenous administration of these AGPs induced resistance to L. monocytogenes infection as measured by survival or reduction of bacteria in the spleen. Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes. Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.