Ly6Chigh Monocytes Research Articles

Diet Rich in Animal Protein Promotes Pro-inflammatory Macrophage Response and Exacerbates Colitis in Mice.

Diet is a major factor determining gut microbiota composition and perturbances in this complex ecosystem are associated with the inflammatory bowel disease (IBD). Here, we used gnotobiotic approach to analyze, how interaction between diet rich in proteins and gut microbiota influences the sensitivity to intestinal inflammation in murine model of ulcerative colitis. We found that diet rich in animal protein (aHPD) exacerbates acute dextran sulfate sodium (DSS)-induced colitis while diet rich in plant protein (pHPD) does not. The deleterious effect of aHPD was also apparent in chronic DSS colitis and was associated with distinct changes in gut bacteria and fungi. Therefore, we induced acute DSS-colitis in germ-free mice and transferred gut microbiota from aCD or aHPD fed mice to find that this effect requires presence of microbes and aHPD at the same time. The aHPD did not change the number of regulatory T cells or Th17 cells and still worsened the colitis in immuno-deficient RAG2 knock-out mice suggesting that this effect was not dependent on adaptive immunity. The pro-inflammatory effect of aHPD was, however, abrogated when splenic macrophages were depleted with clodronate liposomes. This treatment prevented aHPD induced increase in colonic Ly-6Chigh pro-inflammatory monocytes, but the ratio of resident Ly-6C−/low macrophages was not changed. These data show that the interactions between dietary protein of animal origin and gut microbiota increase sensitivity to intestinal inflammation by promoting pro-inflammatory response of monocytes.

Cyclooxygenase-2 Activity Regulates Recruitment of VEGF-Secreting Ly6Chigh Monocytes in Ventilator-Induced Lung Injury.

Mechanical ventilation is usually required for saving lives in critically ill patients; however, it can cause ventilator-induced lung injury (VILI). As VEGF-secreting Ly6Chigh monocytes are involved in VILI pathogenesis, we investigated whether cyclooxygenase-2 (COX-2) activity regulates the recruitment of VEGF-secreting Ly6Chigh monocytes during VILI. The clinically relevant two-hit mouse model of VILI, which involves the intravenous injection of lipopolysaccharide prior to high tidal volume (HTV)-mechanical ventilation, was used in this study. To investigate the role of COX-2 in the recruitment of VEGF-secreting Ly6Chigh monocytes during VILI, celecoxib, which is a clinical COX-2 inhibitor, was administered 1 h prior to HTV-mechanical ventilation. Pulmonary vascular permeability and leakage, inflammatory leukocyte infiltration, and lung oxygenation levels were measured to assess the severity of VILI. HTV-mechanical ventilation significantly increased the recruitment of COX-2-expressing Ly6Chigh, but not Ly6Clow, monocytes. Celecoxib significantly diminished the recruitment of Ly6Chigh monocytes, attenuated the levels of VEGF and total protein in bronchoalveolar lavage fluid, and restored pulmonary oxygenation during VILI. Our findings demonstrate that COX-2 activity is important in the recruitment of VEGF-secreting Ly6Chigh monocytes, which are involved in VILI pathogenesis, and indicate that the suppression of COX-2 activity might be a useful strategy in mitigating VILI.

NUP98-HBO1–fusion generates phenotypically and genetically relevant chronic myelomonocytic leukemia pathogenesis

AbstractChronic myelomonocytic leukemia (CMML) constitutes a hematopoietic stem cell (HSC) disorder characterized by prominent monocytosis and myelodysplasia. Although genome sequencing has revealed the CMML mutation profile, the mechanism of disease development remains unclear. Here we show that aberrant histone acetylation by nucleoporin-98 (NUP98)-HBO1, a newly identified fusion in a patient with CMML, is sufficient to generate clinically relevant CMML pathogenesis. Overexpression of NUP98-HBO1 in murine HSC/progenitors (HSC/Ps) induced diverse CMML phenotypes, such as severe leukocytosis, increased CD115+ Ly6Chigh monocytes (an equivalent subpopulation to human classical CD14+ CD16− monocytes), macrocytic anemia, thrombocytopenia, megakaryocyte-lineage dysplasia, splenomegaly, and cachexia. A NUP98-HBO1–mediated transcriptional signature in human CD34+ cells was specifically activated in HSC/Ps from a CMML patient cohort. Besides critical determinants of monocytic cell fate choice in HSC/Ps, an oncogenic HOXA9 signature was significantly activated by NUP98-HBO1 fusion through aberrant histone acetylation. Increased HOXA9 gene expression level with disease progression was confirmed in our CMML cohort. Genetic disruption of NUP98-HBO1 histone acetyltransferase activity abrogated its leukemogenic potential and disease development in human cells and a mouse model. Furthermore, treatment of azacytidine was effective in our CMML mice. The recapitulation of CMML clinical phenotypes and gene expression profile by the HBO1 fusion suggests our new model as a useful platform for elucidating the central downstream mediators underlying diverse CMML-related mutations and testing multiple compounds, providing novel therapeutic potential.

Role of Monocyte-Derived MicroRNA106b∼25 in Resilience to Social Stress

BackgroundClinical studies suggest that heightened peripheral inflammation contributes to the pathogenesis of stress-related disorders, including major depressive disorder. However, the molecular mechanisms within peripheral immune cells that mediate enhanced stress vulnerability are not well known. Because microRNAs (miRs) are important regulators of immune response, we sought to examine their role in mediating inflammatory and behavioral responses to repeated social defeat stress (RSDS), a mouse model of stress vulnerability that produces susceptible and resilient phenotypes. MethodsWe isolated Ly6chigh monocytes via fluorescence-activated cell sorting in the blood of susceptible and resilient mice following RSDS and profiled miR expression via quantitative real-time polymerase chain reaction. Bone marrow chimeric mice were generated to confirm a causal role of the miR-106b∼25 cluster in bone marrow–derived leukocytes in mediating stress resilience versus susceptibility. ResultsWe found that RSDS produces an increase in circulating Ly6chigh inflammatory monocytes in both susceptible and resilient mice. We next investigated whether intrinsic leukocyte posttranscriptional mechanisms contribute to individual differences in stress response and the resilient phenotype. Of the miRs profiled in our panel, eight were significantly regulated by RSDS within Ly6chigh monocytes, including miR-25-3p, a member of the miR-106b∼25 cluster. Selective knockout of the miR-106b∼25 cluster in peripheral leukocytes promoted behavioral resilience to RSDS. ConclusionsOur results identify the miR-106b∼25 cluster as a key regulator of stress-induced inflammation and depression that may represent a novel therapeutic target for drug development.

Endothelial Sox17 promotes allergic airway inflammation

IL-33, levels of which are known to be increased in patients with eosinophilic asthma and which is suggested as a therapeutic target for it, activates endothelial cells in which Sry-related high-mobility-group box (Sox) 17, an endothelium-specific transcription factor, was upregulated. We investigated the relationship between Sox17 and IL-33 and the possible role of Sox17 in the pathogenesis of asthma using a mouse model of airway inflammation. We used ovalbumin (OVA) to induce airway inflammation in endothelium-specific Sox17 null mutant mice and used IL-33 neutralizing antibody to evaluate the interplay between IL-33 and Sox17. We evaluated airway inflammation and measured levels of various cytokines, chemokines, and adhesion molecules. We also carried out loss- or gain-of-function experiments for Sox17 in human endothelial cells. Levels of IL-33 and Sox17 were significantly increased in the lungs of OVA-challenged mice. Anti-IL-33 neutralizing antibody treatment attenuated not only OVA-induced airway inflammation but also Sox17 expression in pulmonary endothelial cells. Importantly, endothelium-specific deletion of Sox17 resulted in significant alleviation of various clinical features of asthma, including airway inflammation, immune cell infiltration, cytokine/chemokine production, and airway hyperresponsiveness. Sox17 deletion also resulted in decreased densities of Ly6chigh monocytes and inflammatory dendritic cells in the lungs. In IL-33-stimulated human endothelial cells, Sox17 showed positive correlation with CCL2 and intercellular adhesion molecule 1 levels. Lastly, Sox17 promoted monocyte adhesion to endothelial cells and upregulated the extracellular signal-regulated kinase-signal transducer and activator of transcription 3 pathway. Sox17 was regulated by IL-33, and its genetic ablation in endothelial cells resulted in alleviation of asthma-related pathophysiologic features. Sox17 might be a potential target for asthma management.

Nanoparticle incorporating Toll-like receptor 4 inhibitor attenuates myocardial ischaemia-reperfusion injury by inhibiting monocyte-mediated inflammation in mice.

Myocardial ischaemia-reperfusion (IR) injury hampers the therapeutic effect of revascularization in patients with acute myocardial infarction (AMI). Innate immunity for damage-associated protein patterns promotes the process of IR injury; however, the blockade of Toll-like receptor 4 (TLR4) in myocardial IR injury has not been translated into clinical practice. Therefore, we aimed to examine whether the nanoparticle-mediated administration of TAK-242, a chemical inhibitor of TLR4, attenuates myocardial IR injury in a clinically feasible protocol in a mouse model. We have prepared poly-(lactic-co-glycolic acid) nanoparticles containing TAK-242 (TAK-242-NP). TAK-242-NP significantly enhanced the drug delivery to monocytes/macrophages in the spleen, blood, and the heart in mice. Intravenous administration of TAK-242-NP (containing 1.0 or 3.0 mg/kg TAK-242) at the time of reperfusion decreased the infarct size, but the TAK-242 solution did not even when administered at a dosage of 10.0 mg/kg. TAK-242-NP inhibited the recruitment of Ly-6Chigh monocytes to the heart, which was accompanied by decreased circulating HMGB1, and NF-κB activation and cytokine expressions in the heart. TAK-242-NP did not decrease the infarct size further in TLR4-deficient mice, confirming the TLR4-specific mechanism in the effects of TAK-242-NP. Furthermore, TAK-242-NP did not decrease the infarct size further in CCR2-deficient mice, suggesting that monocyte/macrophage-mediated inflammation is the primary therapeutic target of TAK-242-NP. The nanoparticle-mediated delivery of TAK-242-NP represent a novel and clinical feasible strategy in patients undergone coronary revascularization for AMI by regulating TLR4-dependent monocytes/macrophages-mediated inflammation.

3280 Mycobacterium bovis Bacille-Calmette-Guérin infection aggravates atherosclerosis

OBJECTIVES/SPECIFIC AIMS: The study aimed at assessing whether M. bovis BCG infection and inflammation exacerbates the development of atherosclerosis in Ldlr-/- mice. METHODS/STUDY POPULATION: Twelve-week old male Ldlr-/- mice (n=10) were infected with M. bovis BCG (0.3–3.0x10^6 colony-forming units (CFUs)) via the intranasal route, to simulate a natural respiratory route of infection. Mice were subsequently fed a western-type diet (WD) containing 21% fat and 0.2% cholesterol for 16 weeks. Age-matched uninfected Ldlr-/- mice (n=10) fed with an identical WD served as controls. Mice were euthanized after 16 weeks of WD to examine atherosclerotic lesions in aortic root sections and en face aorta using Oil Red O staining. Plasma cholesterol and triglyceride levels were measured by enzymatic assays and lipoprotein distribution was assessed using fast protein liquid chromatography. Because of the important role of T cells and monocytes in atherosclerosis development, we assessed these cell subsets in blood using flow cytometry at 8 and 16 weeks. Experiments were conducted in duplicate. We used unpaired Student’s t-test for group comparisons of numeric variables and flow cytometry data. RESULTS/ANTICIPATED RESULTS: M. bovis BCG infection significantly increased atherosclerotic lesions in en face aorta (plaque size per aorta area ratio; 0.15±0.13 vs. 0.06±0.02; P<0.01), but not in the aortic root. There were no significant differences in plasma cholesterol (1,160 mg/dL vs. 1,278 mg/dL; P = 0.36), triglycerides (340 mg/dL vs. 413 mg/dL; P = 0.28), or lipoprotein profiles between infected vs. uninfected mice at 16 weeks. M. bovis BCG increased circulating T lymphocytes (1,490 cells/uL vs. 1,227 cells/uL; P = 0.03) and monocytes (901 cells/uL vs. 414 cells/uL; P<0.01) within 8 weeks post-infection. When we assessed T lymphocyte subsets, M. bovis BCG infection increased total CD4+ T cell counts (556 cells/uL vs. 416 cells/uL; P<0.01) but not CD8+ T cells. No differences in the proportion of CD44+CD25+ activated T lymphocytes were noted between groups. When we assessed monocyte subsets, M. bovis BCG infection increased the numbers of Ly6Chigh (709 cells/uL vs. 362 cells/uL; P<0.01) and Ly6Clow (145 cells/uL vs. 35 cells/uL; P<0.01) monocytes. Infection was associated with an increased proportion of Ly6Clow monocytes at week 8 (17% vs. 8%; P<0.01) and week 16 (19% vs. 5%; P<0.01), compared to uninfected mice. DISCUSSION/SIGNIFICANCE OF IMPACT: M. bovis BCG infection increased the extent of atherosclerosis formation in the aortas of WD-fed hyperlipidemic Ldlr-/- mice after 16 weeks. Lipid profiles were similar between infected and uninfected mice, and therefore do not explain the observed differences in atherosclerosis. Compared to uninfected controls, M. bovis BCG-infected mice exhibited increased CD4+ T cell and monocyte driven inflammation. Interestingly, M. bovis BCG-infected mice had a higher proportion of non-classical Ly6Clow monocytes, suggesting a pro-atherogenic contribution of these cells in our model. Overall, our results support a pathogenic role of mycobacterial infection in atherosclerosis development and ASCVD.

Suppressor of Cytokine Signaling 1 is Involved in Gene Regulation Which Controls the Survival of Ly6Clow Monocytes in Mice.

Inflammatory processes are controlled by the fine-tuned balance of monocyte subsets. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C that is highly expressed on inflammatory monocytes (Ly6Chigh) and to a lesser extent on patrolling monocytes (Ly6Clow). Our previous study revealed an accumulation of Ly6Chigh monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (SOCS)-1 leading to an increased atherosclerotic burden. To decipher the underlying mechanisms, we performed a genome-wide analysis of SOCS-1-dependent gene regulation in Ly6Chigh and Ly6Clow monocytes. In monocyte subsets from SOCS-1competent and -deficient mice differentially regulated genes were identified using an Illumina mRNA microarray (45,200 transcripts), which were randomly validated by qPCR. Principal component analysis was performed to further characterize mRNA profiles in monocyte subsets. To unravel potential regulatory mechanisms behind the differential mRNA expression, in silico analysis of a transcription factor (TF) network correlating with SOCS-1-dependent mRNA expression was carried out and combined with a weighted correlation network analysis (WGCNA). mRNA analysis in monocyte subsets revealed 46 differentially regulated genes by 2-fold or more. Principal component analysis illustrated a distinct separation of mRNA profiles in monocyte subsets from SOCS-1-deficient mice. Notably, two cell surface receptors crucially involved in the determination of monocyte differentiation and survival, C-X3-C chemokine receptor 1 (CX3CR1) and colony stimulating factor 1 receptor (CSF1R), were identified to be regulated by SOCS-1. Moreover, in silico analysis of a TF network in combination with the WGCNA revealed genes coding for PPAR-γ, NUR77 and several ETSdomain proteins that act as pivotal inflammatory regulators. Our study reveals that SOCS-1 is implicated in a TF network regulating the expression of central transcription factors like PPAR-γ and NUR77 thereby influencing the expression of CX3CR1 and CSF1R that are known to be pivotal for the survival of Ly6Clow monocytes.

Sleep modulates haematopoiesis and protects against atherosclerosis.

Sleep is integral to life1. Although insufficient or disrupted sleep increases the risk of multiple pathological conditions, including cardiovascular disease2, we know little about the cellular and molecular mechanisms by which sleep maintains cardiovascular health. Here we report that sleep regulates haematopoiesis and protects against atherosclerosis in mice. We show that mice subjected to sleep fragmentation produce more Ly-6Chigh monocytes, develop larger atherosclerotic lesions and produce less hypocretin-a stimulatory and wake-promoting neuropeptide-in the lateral hypothalamus. Hypocretin controls myelopoiesis by restricting the production of CSF1 by hypocretin-receptor-expressing pre-neutrophils in the bone marrow. Whereas hypocretin-null and haematopoietic hypocretin-receptor-null mice develop monocytosis and accelerated atherosclerosis, sleep-fragmented mice with either haematopoietic CSF1 deficiency or hypocretin supplementation have reduced numbers of circulating monocytes and smaller atherosclerotic lesions. Together, these results identify a neuro-immune axis that links sleep to haematopoiesis and atherosclerosis.

Circulating CD14+CD16− classical monocytes do not associate with a vulnerable plaque phenotype, and do not predict secondary events in severe atherosclerotic patients

AimsMouse studies have established distinct monocyte subtypes that participate in the process of atherosclerotic lesion formation. The pro-inflammatory Ly6Chigh monocyte subtype actively contributes to murine plaque progression and destabilization. Also in humans, different peripheral monocyte subtypes have been identified, of which the CD14+CD16− classical monocyte is suggested to display similar pro-atherosclerotic properties as the murine Ly6Chigh subtype. We aimed to investigate if circulating CD14+CD16− classical monocytes associate with characteristics of a vulnerable carotid atherosclerotic plaque and if they associate with the risk of secondary adverse manifestations of atherosclerotic disease. Methods and resultsWe enrolled 175 carotid endarterectomy patients of the Athero-Express biobank in our study. Just prior to surgical procedure, blood was collected and peripheral blood mononuclear cells were isolated. Characterization of monocyte subsets was performed by flow cytometry. Plaque characteristics were semi-quantitatively scored for the presence of fat, collagen, intraplaque hemorrhage and calcification. Vessel density, smooth muscle cells and macrophages were assessed quantitatively on a continuous scale. All features of a vulnerable plaque phenotype, including low amounts of collagen and smooth muscle cells, and increased fat content, vessel density, intraplaque hemorrhage and plaque macrophages were not significantly associated with differential levels of peripheral classical CD14+CD16− monocytes or other monocyte subsets. Using Cox regression models to evaluate the prognostic value of circulating monocyte subtypes, we found that total counts of peripheral monocytes, as well as CD14+CD16− classical and other monocyte subtypes were not associated with the risk of secondary cardiovascular events during 3 years follow-up. ConclusionCirculating classical CD14+CD16− monocytes do not associate with specific vulnerable plaque characteristics. In addition, they do not predict secondary adverse manifestations. This suggests that in patients with established carotid artery disease, the circulating monocytes do not reflect plaque characteristics and have no value in identifying patients at risk for future cardiovascular events.

Everolimus depletes plaque macrophages, abolishes intraplaque neovascularization and improves survival in mice with advanced atherosclerosis

Background and aimsInhibition of the mechanistic target of rapamycin (mTOR) is a promising approach to halt atherogenesis in different animal models. This study evaluated whether the mTOR inhibitor everolimus can stabilize pre-existing plaques, prevent cardiovascular complications and improve survival in a mouse model of advanced atherosclerosis. MethodsApoE−/-Fbn1C1039G+/− mice (n = 24) were fed a Western diet (WD) for 12 weeks. Subsequently, mice were treated with everolimus (1.5 mg/kg daily) or vehicle for another 12 weeks while the WD continued. ResultsDespite hypercholesterolemia, everolimus treatment was associated with a reduction in circulating Ly6Chigh monocytes (15 vs. 28% of total leukocytes, p = 0.046), a depletion of plaque macrophages (2.1 vs. 4.1%, p = 0.040) and an abolishment of intraplaque neovascularization, which are all indicative of a more stable plaque phenotype. Moreover, everolimus reduced hypoxic brain damage and improved cardiac function, which led to increased survival (100 vs. 67% of animals, p = 0.038). ConclusionsEverolimus enhances features of plaque stability and counters cardiovascular complications in ApoE−/-Fbn1C1039G+/− mice, even when administered at a later stage of the disease.

TNF, but not hyperinsulinemia or hyperglycemia, is a key driver of obesity-induced monocytosis revealing that inflammatory monocytes correlate with insulin in obese male mice.

Inflammation contributes to obesity‐related hyperinsulinemia and insulin resistance, which often precede type 2 diabetes. Inflammation is one way that obesity can promote insulin resistance. It is not clear if the extent of obesity, hyperinsulinemia, or hyperglycemia, underpins changes in cellular immunity during diet‐induced obesity. In particular, the requirement for obesity or directionality in the relationship between insulin resistance and monocyte characteristics is poorly defined. Inflammatory cytokines such as tumor necrosis factor (TNF) can contribute to insulin resistance. It is unclear if TNF alters monocytosis or specific markers of cellular immunity in the context of obesity. We measured bone marrow and blood monocyte characteristics in WT and TNF −/− mice that were fed obesogenic, high fat (HF) diets. We also used hyperglycemic Akita mice and mice implanted with insulin pellets in order to determine if glucose or insulin were sufficient to alter monocyte characteristics. We found that diet‐induced obesity in male mice increased the total number of monocytes in blood, but not in bone marrow. Immature, inflammatory (Ly6Chigh) monocytes decreased within the bone marrow and increased within peripheral blood of HF‐fed mice. We found that neither hyperinsulinemia nor hyperglycemia was sufficient to induce the observed changes in circulating monocytes in the absence of diet‐induced obesity. In obese HF‐fed mice, antibiotic treatment lowered insulin and insulin resistance, but did not alter circulating monocyte characteristics. Fewer Ly6Chigh monocytes were present within the blood of HF‐fed TNF −/− mice in comparison to HF‐fed wild‐type (WT) mice. The prevalence of immature Ly6Chigh monocytes in the blood correlated with serum insulin and insulin resistance irrespective of the magnitude of adipocyte or adipose tissue hypertrophy in obese mice. These data suggest that diet‐induced obesity instigates a TNF‐dependent increase in circulating inflammatory monocytes, which predicts increased blood insulin and insulin resistance independently from markers of adiposity or adipose tissue expansion.

Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155.

MicroRNA (miR) miR-155 modulates microglial activation and polarization, but its role in activation of microglia during bacterial brain infection is unclear. We studied miR-155 expression in brains of C57BL/6 (B6.WT) mice infected i.p. with the neuro-invasive bacterial pathogen Listeria monocytogenes (L. monocytogenes). Infected mice were treated with ampicillin starting 2 days (d) post-infection (p.i.) and analyzed 3d, 7d, and 14d p.i. Virulent L. monocytogenes strains EGD and 10403s upregulated miR-155 in whole brain 7 d p.i. whereas infection with avirulent, non-neurotropic Δhly or ΔactA L. monocytogenes mutants did not. Similarly, infection with virulent but not mutated bacteria upregulated IFN-γ mRNA in the brain at 7 d p.i. Upregulation of miR-155 in microglia was confirmed by qPCR of flow cytometry-sorted CD45intCD11bpos brain cells. Subsequently, brain leukocyte influxes and gene expression in sorted microglia were compared in L. monocytogenes-infected B6.WT and B6.Cg-Mir155tm1.1Rsky/J (B6.miR-155−/−) mice. Brain influxes of Ly-6Chigh monocytes and upregulation of IFN-related genes in microglia were similar to B6.WT mice at 3 d p.i. In contrast, by d 7 p.i. expressions of microglial IFN-related genes, including markers of M1 polarization, were significantly lower in B6.miR-155−/− mice and by 14 d p.i., influxes of activated T-lymphocytes were markedly reduced. Notably, CD45highCD11bpos brain cells from B6.miR-155−/− mice isolated at 7 d p.i. expressed 2-fold fewer IFN-γ transcripts than did cells from B6.WT mice suggesting reduced IFN-γ stimulation contributed to dampened gene expression in B6.miR-155−/− microglia. Lastly, in vitro stimulation of 7 d p.i. brain cells with heat-killed L. monocytogenes induced greater production of TNF in B6.miR-155−/− microglia than in B6.WT microglia. Thus, miR-155 affects brain inflammation by multiple mechanisms during neuroinvasive L. monocytogenes infection. Peripheral miR-155 promotes brain inflammation through its required role in optimal development of IFN-γ-secreting lymphocytes that enter the brain and activate microglia. Microglial miR-155 promotes M1 polarization, and also inhibits inflammatory responses to stimulation by heat-killed L. monocytogenes, perhaps by targeting Tab2.

Endothelial repair is dependent on CD11c+ leukocytes to establish regrowing endothelial sheets with high cellular density.

Endothelial injury makes the vessel wall vulnerable to cardiovascular diseases. Injured endothelium regenerates by collective sheet migration, that is, the endothelial cells coordinate their motion and regrow as a sheet of cells with retained cell-cell contacts into the wounded area. Leukocytes appear to be involved in endothelial repair in vivo; however, little is known about their identity and role in the reparative sheet migration process. To address these questions, we developed a high-quality en face technique that enables visualizing of leukocytes and endothelial cells simultaneously following an endoluminal scratch wound injury of the mouse carotid artery. We discovered that regrowing endothelium forms a broad proliferative front accompanied by CD11c+ leukocytes. Functionally, the leukocytes were dispensable for the initial migratory response of the regrowing endothelial sheet, but critical for the subsequent formation and maintenance of a front zone with high cellular density. Marker expression analyses, genetic fate mapping, phagocyte targeting experiments, and mouse knock-out experiments indicate that the CD11c+ leukocytes were mononuclear phagocytes with an origin from both Ly6Chigh and Ly6Clow monocytes. In conclusion, CD11c+ mononuclear phagocytes are essential for a proper endothelial regrowth following arterial endoluminal scratch injury. Promoting the endothelial-preserving function of CD11c+ leukocytes may be a strategy to enhance endothelial repair following surgical and endovascular procedures.

CD18 Regulates Monocyte Hematopoiesis and Promotes Resistance to Experimental Schistosomiasis.

Infection with Schistosoma mansoni causes a chronic parasitic disease that progress to severe liver and gastrointestinal damage, and eventually death. During its development into mammalian hosts, immature schistosomula transit through the lung vasculature before they reach the liver to mature into adult worms. A low grade inflammatory reaction is induced during this process. However, molecules that are required for efficient leukocyte accumulation in the lungs of S. mansoni-infected subjects are unknown. In addition, specific leukocyte subsets that mediate pulmonary response during S. mansoni migration through the lung remain to be elucidated. β2 integrins are fundamental regulators of leukocyte trans-endothelial migration and function. Therefore, we investigated their role during experimental schistosomiasis. Mice that express low levels of CD18 (the common β2 integrin subunit) and wild type C57BL/6 mice were subcutaneously infected with S. mansoni cercariae. Cellular profiles of lungs and livers were evaluated in different time points after infection by flow cytometry. Low levels of CD18 affected the accumulation of patrolling Ly6Clow, intermediate Ly6Cinter monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells in the lungs 7 days after infection. This correlated with increased TNF-α levels. Strikingly, low CD18 expression resulted in monocytopenia both in the peripheral blood and bone marrow during acute infection. After 48 days, S. mansoni worm burdens were higher in the hepatic portal system of CD18low mice, which also displayed reduced hepatic accumulation of patrolling Ly6Clow and intermediate Ly6Cinter, but not inflammatory Ly6Chigh monocytes. Higher parasite burden resulted in increased granulomatous lesions in the liver, increased egg deposition and enhanced mortality. Overall, our data point for a fundamental role of CD18 for monocyte hematopoiesis during infection, which promotes an efficient host response against experimental schistosomiasis.

MicroRNA 33 Regulates the Population of Peripheral Inflammatory Ly6Chigh Monocytes through Dual Pathways.

ABSTRACTMicroRNA 33 (miR-33) targets ATP-binding cassette transporter A1 (ABCA1), and its deficiency increases serum high-density lipoprotein (HDL)-cholesterol (HDL-C) and ameliorates atherosclerosis. Although we previously reported that miR-33 deficiency increased peripheral Ly6Chigh monocytes on an ApoE-deficient background, the effect of miR-33 on the monocyte population has not been fully elucidated, especially in a wild-type (WT) background. We found that Ly6Chigh monocytes in miR-33−/− mice were decreased in peripheral blood and increased in bone marrow (BM). Expansion of myeloid progenitors and decreased apoptosis in Lin− Sca1+ c-Kit+ (LSK) cells were observed in miR-33−/− mice. A BM transplantation study and competitive repopulation assay revealed that hematopoietic miR-33 deficiency caused myeloid expansion and increased peripheral Ly6Chigh monocytes and that nonhematopoietic miR-33 deficiency caused reduced peripheral Ly6Chigh monocytes. Expression of high-mobility group AT-hook 2 (HMGA2) targeted by miR-33 increased in miR-33-deficient LSK cells, and its knockdown abolished the reduction of apoptosis. Transduction of human apolipoprotein A1 and ABCA1 in WT mouse liver increased HDL-C and reduced peripheral Ly6Chigh monocytes. These data indicate that miR-33 deficiency affects distribution of inflammatory monocytes through dual pathways. One pathway involves the enhancement of Hmga2 expression in hematopoietic stem cells to increase Ly6Chigh monocytes, and the other involves the elevation of HDL-C to decrease peripheral Ly6Chigh monocytes.

Gr-1+ Cells Other Than Ly6G+ Neutrophils Limit Virus Replication and Promote Myocardial Inflammation and Fibrosis Following Coxsackievirus B3 Infection of Mice

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. An early and abundant neutrophil accumulation in the myocardium is a hallmark of early CVB3 infection. Yet the relative contribution of neutrophils to host susceptibility to CVB3 myocarditis remains largely unknown. Herein, peripheral neutrophil depletion was implemented in a BALB/c mouse model of acute CVB3 myocarditis using the specific 1A-8 (anti-Ly6G) or a RB6-8C5 (anti-Gr-1) mAb covering a wide range. Anti-Ly6G treatment led to systemic neutropenia throughout the disease, but did not alter virus replication, disease susceptibility and histopathological changes in the heart and pancreas of mice. In contrast, depletion of both neutrophils and monocytes/macrophages by anti-Gr-1 mAb prior to and after infection significantly promoted susceptibility of mice to CVB3 infection which was associated with exacerbated cardiac and pancreatic viral load. However, depletion of Gr1+ cells significantly suppressed acute myocarditis and pancreatic acini destruction at day 7 post infection via reducing Ly6Chigh monocyte population in the circulation. Additionally, cardiac interstitial fibrosis was not affected by neutrophil depletion, whereas Gr-1+ cells other than neutrophils increased cardiac fibrosis at day 21 p.i. by increasing cardiac expression of profibrotic cytokine TNF-α and TGF-β. Thus, Neutrophil function is most likely not essential for CVB3 control and peripheral neutrophils play dispensable role in the pathogenesis of acute myocarditis and pancreatitis during CVB3 infection. Whereas Gr-1+ cells other than neutrophils play a major role in limiting viral replication while promoting myocardial and pancreatic inflammatory injury and fibrosis.

Traumatic Brain Injury Leads to Accelerated Atherosclerosis in Apolipoprotein E Deficient Mice

Traumatic brain injury (TBI) has been associated with atherosclerosis and cardiovascular mortality in humans. However the causal relationship between TBI and vascular disease is unclear. This study investigated the direct role of TBI on vascular disease using a murine model of atherosclerosis. Apolipoprotein E deficient mice were placed on a western diet beginning at 10 weeks of age. Induction of TBI or a sham operation was performed at 14 weeks of age and mice were sacrificed 6 weeks later at 20 weeks of age. MRI revealed evidence of uniform brain injury in all mice subjected to TBI. There were no differences in total cholesterol levels or blood pressure between the groups. Complete blood counts and flow cytometry analysis performed on peripheral blood 6 weeks following TBI revealed a higher percentage of Ly6C-high monocytes in mice subjected to TBI compared to sham-treated mice. Mice with TBI also showed elevated levels of plasma soluble E-selectin and bone marrow tyrosine hydroxylase. Analysis of atherosclerosis at the time of sacrifice revealed increased atherosclerosis with increased Ly6C/G immunostaining in TBI mice compared to sham-treated mice. In conclusion, progression of atherosclerosis is accelerated following TBI. Targeting inflammatory pathways in patients with TBI may reduce subsequent vascular complications.

Cardiac (myo)fibroblasts modulate the migration of monocyte subsets

Cardiac fibroblasts play an important role in the regulation of the extracellular matrix and are newly recognized as inflammatory supporter cells. Interferon (IFN)-γ is known to counteract transforming growth factor (TGF)-ß1-induced myofibroblast differentiation. This study aims at investigating in vitro how IFN-γ affects TGF-ß1-induced monocyte attraction. Therefore, C4 fibroblasts and fibroblasts obtained by outgrowth culture from the left ventricle (LV) of male C57BL6/j mice were stimulated with TGF-β1, IFN-γ and TGF-β1 + IFN-γ. Confirming previous studies, IFN-γ decreased the TGF-ß1-induced myofibroblast differentiation, as obviated by lower collagen I, III, α-smooth muscle actin (α-SMA), lysyl oxidase (Lox)-1 and lysyl oxidase-like (LoxL)-2 levels in TGF-β1 + IFN-γ- versus TGF-ß1-stimulated cardiac fibroblasts. TGF-β1 + IFN-γ-stimulated C4 and cardiac fibroblasts displayed a higher CC-chemokine ligand (CCL) 2, CCL7 and chemokine C-X3-C motif ligand (Cx3CL1) release versus sole TGF-ß1-stimulated fibroblasts. Analysis of migrated monocyte subsets towards the different conditioned media further revealed that sole TGF-β1- and IFN-γ-conditioned media particularly attracted Ly6Clow and Ly6Chigh monocytes, respectively, as compared to control media. In line with theses findings, TGF-β1 + IFN-γ-conditioned media led to a lower Ly6Clow/Ly6Chigh monocyte migration ratio compared to sole TGF-ß1 treatment. These differences in monocyte migration reflect the complex interplay of pro-inflammatory cytokines and pro-fibrotic factors in cardiac remodelling and inflammation.

In vitro uptake of oligomannose-coated liposomes leads to differentiation of inflammatory monocytes into mature antigen-presenting cells that can activate T cells

Oligomannose-coated liposomes (OMLs), containing entrapped antigens, serve as effective antigen delivery vehicles and as a novel adjuvant to induce antigen-specific cellular immune responses. However, in vitro activation of antigen-presenting cells (APCs) by OMLs has not yet been demonstrated. In this paper, we found that OMLs can deliver the antigens and the stimulatory signals into inflammatory monocytes in vitro, leading to differentiation of the cells to mature APCs. When OMLs were co-cultured with peripheral blood mononuclear cells from C57BL/6 mice in the presence of mouse serum, OMLs were preferentially incorporated into both Ly6Chigh monocytes and Ly6Clow monocytes, which are referred to as murine inflammatory and resident monocytes, respectively. The expression of CD11c, CD80, CD86, CCR7, and MHC class II on the Ly6Chigh monocytes was significantly enhanced during the 24 h after OML uptake, whereas upregulation of these molecules on the Ly6Clow monocytes was limited. In addition, the antigenic peptide of OVA encased in OMLs was presented on MHC class I of only Ly6Chigh monocytes. Furthermore, OVA-encasing OML-ingesting monocytes can activate CD8+ T cells from OT-1 mice, suggesting that antigens encapsulated in OMLs were cross-presented in inflammatory monocytes. Adoptive transfer of the monocytes that engulf OVA-encasing OMLs led to induction of an antigen-specific Th1 immune response in mice. Taken together, mature APCs can be generated from inflammatory monocytes in peripheral blood by ex vivo treatment of the cells with OMLs without any additional stimuli.