Monocyte Priming Research Articles

Hematopoietic NADPH Oxidase 4 Prevents Atherogenesis by Inhibiting High Calorie Diet-Induced Monocyte Priming and the Overrecruitment of Dysfunctional Monocyte-Derived Macrophages

Hematopoietic NADPH Oxidase 4 Prevents Atherogenesis by Inhibiting High Calorie Diet-Induced Monocyte Priming and the Overrecruitment of Dysfunctional Monocyte-Derived Macrophages

Helicobacter pylori induces a novel form of innate immune memory via accumulation of NF-кB proteins.

Helicobacter pylori is a widespread Gram-negative pathogen involved in a variety of gastrointestinal diseases, including gastritis, ulceration, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. Immune responses aimed at eradication of H. pylori often prove futile, and paradoxically play a crucial role in the degeneration of epithelial integrity and disease progression. We have previously shown that H. pylori infection of primary human monocytes increases their potential to respond to subsequent bacterial stimuli - a process that may be involved in the generation of exaggerated, yet ineffective immune responses directed against the pathogen. In this study, we show that H. pylori-induced monocyte priming is not a common feature of Gram-negative bacteria, as Acinetobacter lwoffii induces tolerance to subsequent Escherichia coli lipopolysaccharide (LPS) challenge. Although the increased reactivity of H. pylori-infected monocytes seems to be specific to H. pylori, it appears to be independent of its virulence factors Cag pathogenicity island (CagPAI), cytotoxin associated gene A (CagA), vacuolating toxin A (VacA) and γ-glutamyl transferase (γ-GT). Utilizing whole-cell proteomics complemented with biochemical signaling studies, we show that H. pylori infection of monocytes induces a unique proteomic signature compared to other pro-inflammatory priming stimuli, namely LPS and the pathobiont A. lwoffii. Contrary to these tolerance-inducing stimuli, H. pylori priming leads to accumulation of NF-кB proteins, including p65/RelA, and thus to the acquisition of a monocyte phenotype more responsive to subsequent LPS challenge. The plasticity of pro-inflammatory responses based on abundance and availability of intracellular signaling molecules may be a heretofore underappreciated form of regulating innate immune memory as well as a novel facet of the pathobiology induced by H. pylori.

Monocyte priming during acute myocardial infarction

Abstract Introduction Purpose Myocardial infarction (MI) inflicts a sterile wound on the heart which recruits hundreds of thousands of monocytes from the blood. The bone marrow (BM) responds to cardiac ischemia with an increase in monocyte production, hence meeting the high demand of these cells. Apart from this quantitative response, little is known whether the BM supplies qualitatively different monocytes during the course of MI. Methods & Results We isolated BM Ly6Chigh monocytes from non-infarcted mice and from mice which were infarcted 72h prior (surgically induced by permanent coronary ligation) and contrasted monocytic transcriptomes using RNA sequencing. We found 672 differentially expressed genes (DEG) of which 289 were up- and 383 down-regulated (log2FoldChange³±1.0, adjusted p<0.05). Since most DEG were enriched in "cell adhesion and recruitment" pathways, we tested the functional relevance of our findings in the context of monocyte recruitment to the heart using competitive adoptive transfer experiments. Our data revealed that the BM provides monocytes that are primed for better recruitment (relative recruitment: 56.1% 12h after MI vs. 43.9% steady state, p<0.01) at the early inflammatory phase following coronary ligation (12h after MI). In contrast, monocytes displayed worse recruitment capacities (relative recruitment: 30.4% 72h after MI vs. 65.8% steady state, p<0.01) at the end of the inflammatory phase (72h after MI). We next sought to identify key players in this BM-controlled cardiac recruitment process. Our RNA sequencing data revealed that – in monocytes with poor recruitment capacities – the cell adhesion molecule CD209 was among the most significantly downregulated genes at 72h after MI over steady state. To investigate the molecular role of CD209 in the process of leukocyte recruitment to the myocardium during MI, we cloned human CD209 from a cDNA library into an overexpression plasmid system. After ectopic overexpression of CD209, THP-1 cells (a cell line derived from immortalized human blood monocytes) showed enhanced adhesion to endothelial cells (HUVECs) compared to THP-1 cells under control conditions. Finally, we infarcted wild type (CD209+/+) and CD209 knockout (CD209-/-) mice and found decreased numbers of Ly6Chigh monocytes in infarcted hearts of CD209-/- mice in comparison to CD209+/+ mice (491+52 vs. 915+180 Ly6Chigh monocytes/mg infarct tissue, mean+SEM, p<0.05) while numbers of blood Ly6Chigh monocytes were unchanged. Conclusions The BM responds to MI and provides an altered monocyte repertoire in terms of numbers and function and hence remotely controls monocyte/macrophage accumulation in the ischemic heart. Consequently, the heart–bone marrow axis may represent a novel therapeutic target to beneficially modify post-MI cardiac remodeling, a major contributor of MI-related morbidity and mortality.

Immediate-release niacin and a monounsaturated fatty acid-rich meal on postprandial inflammation and monocyte characteristics in men with metabolic syndrome

When considered separately, long-term immediate-release niacin and fatty meals enriched in monounsaturated fatty acids (MUFA) decrease postprandial triglycerides, but their effects on postprandial inflammation, which is common in individuals with metabolic syndrome, are less known. Moreover, successful combination is lacking and its impact on acute disorders of the innate immune cells in the metabolic syndrome remains unclear. Here, we aimed to establish the effects from combination with niacin of different fats [butter, enriched in saturated fatty acids (SFA), olive oil, enriched in MUFA, and olive oil supplemented with eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids] on plasma inflammatory markers and circulating monocyte subsets, activation and priming at the postprandial period in individuals with metabolic syndrome. A random-order within-subject crossover experiment was performed, in which 16 individuals with metabolic syndrome and 16 age-matched healthy volunteers took 2g immediate-release niacin together with the corresponding fatty meal or a meal with no fat as control. In total, 128 postprandial curves were analysed. We sampled hourly over 6h for plasma concentrations of soluble inflammatory markers and triglycerides. Circulating monocyte subsets (CD14/CD16 balance), activation (CCL2/CCR2 axis) and priming (M1/M2-like phenotype) at the time of postprandial hypertriglyceridemic peak were also addressed. Dietary SFA (combined with niacin) promote postprandial excursions of circulating IL-6, IL-1β, TNF-α and CD14/CCR2-rich monocytes with a pro-inflammatory M1-like phenotype, particularly in individuals with metabolic syndrome. In contrast, dietary MUFA (combined with niacin) postprandially increased circulating CD16-rich monocytes with an anti-inflammatory M2-like phenotype. Omega-3 PUFA did not add to the effects of MUFA. The co-administration of a single-dose of immediate-release niacin with a fatty meal rich in MUFA, in contrast to SFA, suppresses postprandial inflammation at the levels of both secretory profile and monocyte response in individuals with metabolic syndrome. These findings highlight a potential role of combining niacin and dietary MUFA for the homeostatic control of inflammation and the innate immune system, identifying a new search direction for the management of disorders associated with the metabolic syndrome.

Monocyte priming during acute myocardial infarction alterations of the bone marrow monocyte reservoir secondary to myocardial infarction

Monocyte priming during acute myocardial infarction alterations of the bone marrow monocyte reservoir secondary to myocardial infarction

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

Adjuvants and Vaccines Used in Allergen-Specific Immunotherapy Induce Neutrophil Extracellular Traps.

Aluminum hydroxide (alum) and monophosphoryl-lipid A (MPLA) are conventional adjuvants in vaccines for allergen-specific immunotherapy (AIT). Alum triggers the release of neutrophil extracellular traps (NETs) by neutrophils. NETs contain expelled decondensed chromatin associated with granular material and may act as danger-associated molecular patterns and activate antigen-presenting cells. We investigated whether adjuvant-induced NETs contribute to innate responses to AIT-vaccines. Human neutrophils were incubated with alum, MPLA and adjuvant-containing AIT-vaccine preparations. NETs were verified by time-lapse and confocal fluorescence microscopy and quantitatively assessed by DNA and elastase release and ROS production. In contrast to MPLA, alum represented a potent trigger for NET release. Vaccine formulations containing alum resulted in less NET release than alum alone, whereas the vaccine containing MPLA induced stronger NET responses than MPLA alone. NETs and alum alone and synergistically increased the expression of molecules involved in antigen presentation, i.e., CD80, CD86 and CD83, by peripheral blood monocytes. Monocyte priming with NETs resulted in individually differing IL-1β- and IL-6-responses. Thus, NETs induced by adjuvants in AIT-vaccines can provide autonomous and cooperative effects on early innate responses. The high diversity of individual innate responses to adjuvants and AIT-vaccines may affect their therapeutic efficacy.

Impact of Cholesterol Metabolism in Immune Cell Function and Atherosclerosis.

Cholesterol, the most important sterol in mammals, helps maintain plasma membrane fluidity and is a precursor of bile acids, oxysterols, and steroid hormones. Cholesterol in the body is obtained from the diet or can be de novo synthetized. Cholesterol homeostasis is mainly regulated by the liver, where cholesterol is packed in lipoproteins for transport through a tightly regulated process. Changes in circulating lipoprotein cholesterol levels lead to atherosclerosis development, which is initiated by an accumulation of modified lipoproteins in the subendothelial space; this induces significant changes in immune cell differentiation and function. Beyond lesions, cholesterol levels also play important roles in immune cells such as monocyte priming, neutrophil activation, hematopoietic stem cell mobilization, and enhanced T cell production. In addition, changes in cholesterol intracellular metabolic enzymes or transporters in immune cells affect their signaling and phenotype differentiation, which can impact on atherosclerosis development. In this review, we describe the main regulatory pathways and mechanisms of cholesterol metabolism and how these affect immune cell generation, proliferation, activation, and signaling in the context of atherosclerosis.

Fingolimod (FTY720) Activates Protein Phosphatase‐2A in Human Monocytes and Inhibits Uric Acid Crystal‐Induced Interleukin‐1 Beta Production

IntroductionGout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in articular joints. Phagocytosis of MSU crystals by monocytes/macrophages results in downstream expression and production of interleukin‐1 beta (IL‐1β). The two‐step process of gout inflammation involves 1) priming of monocytes with danger signals e.g. lipopolysaccharide (LPS) or soluble uric acid (UA) and 2) MSU phagocytosis and activation of the NLRP3 inflammasome in monocytes. Protein‐phosphatase‐2A (PP2A) is a serine/threonine phosphatase, and a reduction in intracellular macrophage PP2A activity was shown to aggravate LPS‐induced proinflammatory cytokine production. The prodrug Fingolimod (FTY720) and its phosphorylated active metabolite (p‐FTY720) are efficacious in multiple sclerosis and their mechanism of action involves activation of intracellular PP2A. The objective of this study is to investigate the role of PP2A in regulating monocyte priming and activation by MSU crystals and whether intracellular PP2A activation exerts an anti‐inflammatory effect in MSU‐stimulated monocytes.MethodsHuman THP‐1 monocytes (ATCC; 500,000 cells per well) were treated with UA (50 mg/dL) and LPS (10ng/mL) for 24 hours. Subsequently, cells were treated with MSU crystals (100ug/mL) and MSU phagocytosis was determined at 4 hours using flow cytometry and the percentage of cells displaying side‐scattering above a threshold value, indicative of crystal phagocytosis, were determined. Intracellular PP2A activity was determined at 6 hours following MSU stimulation using the PP2A Immunoprecipitation Phosphatase Assay Kit (Fisher Scientific). IL‐1β expression and protein levels at 6 hours were determined using quantitative polymerase chain reaction and ELISA, respectively. THP‐1 monocytes were treated with FTY720 or p‐FTY720 (2.5mM for both treatments; Cayman Chemicals) and PP2A activity was determined at 1, 3, 6 and 24 hours. THP‐1 monocytes were treated with UA+LPS followed by MSU ± p‐FTY720 (2.5mM for 6 hours) and IL‐1β expression and protein levels were determined. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey’s post‐hoc test.ResultsUA+LPS pre‐treatment increased MSU phagocytosis by THP‐1 monocytes (fig. 1A and 1B; p<0.001) and IL‐1β expression (fig. 1C; p<0.001) and production (fig. 1D; p<0.001). This effect was associated with a reduction in intracellular PP2A activity (fig. 1E; p<0.001). FTY720 and p‐FTY720 increased intracellular PP2A activity in monocytes (fig. 2A; p<0.001) with a higher peak effect achieved at 1 hour for p‐FTY720 (p<0.01). p‐FTY720 treatment reduced IL‐1β expression (fig. 2B; p<0.001) and mature IL‐1β production (fig. 2C; p<0.001) in UA+LPS pre‐treated human monocytes following MSU stimulation.ConclusionUA and LPS enhanced MSU phagocytosis, expression and production of IL‐1β, potentially via a reduction in intracellular PP2A activity. FTY720 and p‐FTY720 activated PP2A in monocytes with an onset of effect of 1 hour and correspondingly reduced IL‐1β expression and production. PP2A is a novel therapeutic target for gout treatment.Impact of soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatments on THP‐1 monocyte phagocytic activity towards monosodium urate (MSU) crystals, interleukin‐1 beta (IL‐1β) expression and production and association with intracellular protein phosphatase‐2A (PP2A) activity. A) Representative flow cytometry scatter plots showing enhanced MSU crystal phagocytosis (as shown by more cells with increased side‐scatter in P2 region of interest) by THP‐1 monocytes following UA+LPS pre‐treatment. B) UA and UA+LPS pre‐treatment increased the percentage of THP‐1 monocytes positive for MSU phagocytosis. C) UA+LPS pre‐treatment increased IL‐1β expression by THP‐1 monocytes following MSU challenge. D) UA+LPS pre‐treatment increased IL‐1β production by THP‐1 monocytes following MSU challenge. E) Intracellular PP2A activity was reduced in UA or UA+LPS pre‐treated THP‐1 monocytes. *p<0.001; **p<0.01; n.s.: non significant.Figure 1Impact of FTY720 (Fingolimod; prodrug) and phosphorylated FTY720 (p‐FTY‐720; active metabolite) treatments on protein phosphatase‐2A (PP2A) activity in human THP‐1 monocytes and impact of p‐FTY720 treatment on interleukin‐1 beta (IL‐1β) expression and production by monosodium urate (MSU) crystal stimulated THP‐1 monocytes ± soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatment. Treatments were conducted using 2.5μM concentrations. A) FTY720 and p‐FTY720 increased intracellular PP2A activity in THP‐1 monocytes. B) p‐ FTY720 reduced IL‐1β expression in THP‐1 monocytes. C) p‐FTY720 reduced IL‐1β production in THP‐1 monocytes. *p<0.001; **p<0.01; n.s.: non significant.Figure 2

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

MTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes.

Introduction: Cells of the innate immune system particularly monocytes and macrophages have been recognized as pivotal players both during the initial insult as well as the chronic phase of atherosclerosis. It has recently been shown that oxidized low-density lipoprotein (oxLDL) induces a long-term pro-inflammatory response in monocytes due to epigenetic and metabolic reprogramming, an emerging new concept called trained innate immunity. Changes in the cellular redox state are crucial events in the regulation of many physiologic functions in macrophages including transcription, differentiation and inflammatory response. Here we have analyzed the role of reactive oxygen species (ROS) in regulating this proinflammatory monocyte priming in response to oxLDL-treatment.Methods and Results: Human monocytes were isolated and incubated with oxLDL for 24 h. After 5 days of resting, oxLDL treated cells produced significantly more inflammatory cytokines upon restimulation with the TLR2-agonist Pam3cys. Furthermore, oxLDL incubation induced persistent mTOR activation, ROS formation, HIF1α accumulation and HIF1α target gene expression, while pharmacologic mTOR inhibition or siRNA mediated inhibition of the mTORC1 subunit Raptor prevented ROS formation and proinflammatory priming. mTOR dependent ROS formation was associated with increased expression of NAPDH oxidases and necessary for the emergence of the primed phenotype as antioxidant treatment blocked oxLDL priming. Inhibition of cytosolic ROS formation could also block mTOR activation and HIF1α accumulation suggesting a positive feedback loop between mTOR and cytosolic ROS. Although mitochondrial ROS scavenging did not block HIF1α-accumulation at an early time point (24 h), it was persistently reduced on day 6. Therefore, mitochondrial ROS formation appears to occur initially downstream of the mTOR-cytoROS-HIF1α feedback loop but seems to be a crucial factor that controls the long-term activation of the mTOR-HIF1α-axis.Conclusion: In summary, our data demonstrate that mTOR dependent ROS production controls the oxLDL-induced trained innate immunity phenotype in human monocyte derived macrophages. Pharmacologic modulation of these pathways might provide a potential approach to modulate inflammation, associated with aberrant monocyte activation, during atherosclerosis development.

20 - PKM2 S-glutathionylation promotes the reprogramming of macrophage metabolism and activation states

Introduction Pyruvate Kinase Isoform M2 (PKM2) regulates glycolysis in monocytes and macrophages and is induced and S-glutathionylated in response to diet-induced metabolic stress. We hypothesized that PKM2 S-glutathionylation promotes PKM2 tetramer dissociation into glycolytically-inactive dimers and that nuclear translocation of these dimers activates HIF1a-dependent gene expression, inducing both glycolytic enzymes as well and pro-inflammatory proteins, reprogramming macrophages to a hyper-inflammatory phenotype. Methods Bone marrow derived macrophages (BMDM) overexpressing glutaredoxin-1-EGFP (Grx1) or EGFP only were exposed for 36 h to metabolic stress (high glucose plus native human LDL). PKM2 expression and phosphorylation state (Tyr105) were assessed in cell lysates by Western blot analysis. PKM2 S-glutathionylation was quantified using the Biotin-switch assay. PKM2 oligomerization state was measured by non-reducing SDS-PAGE and Western blot analysis. To confirm the role of PKM2 S-glutathionylation on metabolic reprogramming of monocytes in vivo, we conducted lentiviral gene transfer experiments in mice using CD68 promoter-driven Grx1 and EGFP expression. Mice were fed a HFD for 10 week to induce monocyte priming. We purified blood monocytes from EGFPMactg and Grx1Mactg mice and determined by single-cell Western blot PKM2 induction and phosphorylation, GLUT1, 14-3-3z and IL-1b expression. Results PKM2 induction, -glutathionylation, and phosphorylation were all enhanced in metabolically stressed BMDMs but prevented in Grx1-overexpressing BMDMs. The dimer/tetramer ratio was increased in response to metabolic stress as was HIF1a-dependent expression of GLUT1 and IL-1b. Compared to monocytes isolated from HFD-fed mice, monocyte from Grx1Mactg mice showed reduced priming, reduced expression of PKM2, GLUT1 and IL-1b and diminished PKM2 phosphorylation. Conclusion Metabolic stress-induced PKM2 S-glutathionylation promotes PKM2 tetramer dissociation, phosphorylation and subsequent nuclear translocation, which, via HIF1a-dependent expression of glycolytic enzymes and inflammatory cytokines, reprograms monocyte-derived macrophages into a hyper-inflammatory phenotype. This mechanism may contribute to chronic inflammatory diseases associated with metabolic disorders.

Dietary 23–hydroxy ursolic acid protects against atherosclerosis and obesity by preventing dyslipidemia-induced monocyte priming and dysfunction

Background and aimsWe demonstrated that dietary ursolic acid (UA) reduces atherosclerotic lesion size and improves kidney function in diabetic mice. Based on structure-function analyses of naturally occurring UA analogs, we synthesized 23-hydroxy ursolic acid (23-OHUA), a compound with structural features predicted to enhance its bioavailability and anti-atherogenic properties compared to UA. The goal of this study was to determine the anti-obesogenic and atheroprotective properties of 23-OHUA and its mechanism of action. MethodsWe performed chemotaxis assays to determine IC50 of phytochemicals on primed THP-1 monocytes. We fed 12-week old female LDLR−/− mice a high-fat diet (HFD) or a HFD supplemented with either 0.05% UA or 0.05% 23-OHUA, and measured monocyte priming, weight gain and atherosclerotic lesion size after 6 and 20 weeks. ResultsBoth dietary UA and 23-OHUA prevented dyslipidemia-induced loss of MKP-1 activity, and hyper-chemotactic activity, hallmarks of blood monocytes priming and dysfunction, but they did not affect plasma lipids or blood glucose levels nor WBC and monocyte counts. After 20 weeks, mice fed 23-OHUA showed 11% less weight gain compared to HFD-fed control mice and a 40% reduction in atherosclerotic plaque size, whereas UA reduced lesion size by only 19% and did not reduce weight gain. ConclusionsDietary 23-OHUA reduces weight gain and attenuates atherogenesis in mice by protecting monocytes against metabolic stress-induced priming and dysfunction. Based on its mechanism of action, 23-OHUA may represent a novel therapeutic approach for the prevention and treatment of obesity and atherosclerosis.

Abstract 20934: Chronic Polarization of Inflammatory Monocytes by Super-Low Grade Endotoxin Aggravates the Pathogenesis of Atherosclerosis

The subclinical levels of Gram negative bacterial endotoxin, LPS, circulating in the blood are prevalent in the individuals with adverse health conditions or lifestyles. LPS is a potent agent in the dynamic programming of monocytes, and altered innate immune homeostasis mediated by non-resolving inflammatory monocytes has long been suspected in the pathogenesis of atherosclerosis. However, the mechanisms responsible for the sustainment of inflammatory monocytes during atherosclerosis are poorly understood. We hypothesized that the selective modulation of molecular switches in monocytes by super-low dose LPS may facilitate the establishment of non-resolving inflammation during atherosclerosis. We challenged high-fat-diet (HFD) fed ApoE deficient mice with chronic LPS administration, and observed that LPS effectively induced expansion of Ly6C high pro-inflammatory monocytes with elevated levels of CCR5, MCP-1, and reduced SR-B1. Compare to PBS administration, chronic injection of super-low dose LPS significantly elevated the lipid deposition within the atherosclerotic plaques in HFD fed ApoE deficient mice, while induced remarkable reduction in collagen content of the plaques. To test whether the LPS reprogrammed monocytes, instead of systemic effects of LPS, may contribute the exacerbation of atherosclerosis, we performed adoptive transfer experiment. Bone-marrow monocytes harvested from ApoE deficient mice were primed in vitro with super-low dose LPS for 5 days, and then intravenously injected into HFD fed ApoE once weekly for 1 month. We observed significant increases of the plaque sizes as well as lipid deposition in atherosclerotic lesions, indicating that programmed monocytes directly aggravated atherosclerosis progression. At the molecular level, we observed that monocytes programmed by super-low dose LPS maintained higher levels of transcription factor IRF5 and reduced levels of a critical inflammatory repressor, Blimp-1. Intriguingly, the inflammatory monocyte priming by super-low dose LPS was dependent upon TRAM/TRIF but not MyD88. In conclusion, our data suggest super-low grade LPS may program sustained low-grade inflammatory monocytes that are conducive for the chronic pathogenesis of atherosclerosis.

Glutaredoxin 1 Deficiency Causes Late-Age Onset Metabolic Disease in Female C57BL/6 Mice, Associated with Monocyte and Macrophage Dysfunction

Glutaredoxin 1 (Grx1) reduces mixed disulfides between glutathione and protein thiols thereby controlling thiol-redox regulated cellular functions, including intracellular signaling, migration, angiogenesis, autophagy and apoptosis. Grx1 protects monocytes and macrophages from metabolic-stress-induced “priming’ and dysfunction. Here we report that female but not male Grx1-/- mice on a C57BL/6 background fed a normal chow diet become obese and hyperglycemic by 6 and 8 months of age respectively. At 18 months female Grx1-/- mice developed spontaneous atherosclerosis and late-age onset associated metabolic disease, characterized by mild hyperlipidemia, hyperglycemia and obesity. Atherosclerotic lesions in 18 month female Grx1-/- mice, measured by en-face analysis, were localized to both the ascending arch and abdominal aortic regions. In aged female Grx1-/- mice, monocyte priming was also measured in vivo using the Matrigel plug assay and ex vivo in multi-well Boyden chambers using peritoneal macrophages isolated from female Grx1-/- mice and age-matched WT mice. Furthermore, aged Grx1-/- female mice possessed increased numbers of circulating Ly6Chi inflammatory blood monocytes compared to male Grx1-/- and WT mice. Macrophages isolated from female Grx1-/- mice also displayed increased protein-S-glutathionylation, increased expression of pro-inflammatory “M1”-associated genes, increased susceptibility to ketocholesterol-induced apoptosis, and impaired autophagy. Together, these data underscore the critical role of protein-S-glutathionylation in monocyte macrophage dysfunction and the development of atherosclerosis and obesity and identify a protective role for Grx1 in these processes. Monocytic Grx1 and metabolic stress-induced protein-S glutathionylation may therefore represent a new mechanistic link between metabolic disorders and chronic inflammatory diseases.

Dyslipidemic Diet-Induced Monocyte "Priming" and Dysfunction in Non-Human Primates Is Triggered by Elevated Plasma Cholesterol and Accompanied by Altered Histone Acetylation.

Monocytes and the recruitment of monocyte-derived macrophages into sites of inflammation play a key role in atherogenesis and other chronic inflammatory diseases linked to cardiometabolic syndrome and obesity. Previous studies from our group have shown that metabolic stress promotes monocyte priming, i.e., enhanced adhesion and accelerated chemotaxis of monocytes in response to chemokines, both in vitro and in dyslipidemic LDLR−/− mice. We also showed that metabolic stress-induced monocyte dysfunction is, at least to a large extent caused by the S-glutathionylation, inactivation, and subsequent degradation of mitogen-activated protein kinase phosphatase 1. Here, we analyzed the effects of a Western-style, dyslipidemic diet (DD), which was composed of high levels of saturated fat, cholesterol, and simple sugars, on monocyte (dys)function in non-human primates (NHPs). We found that similar to mice, a DD enhances monocyte chemotaxis in NHP within 4 weeks, occurring concordantly with the onset of hypercholesterolemia but prior to changes in triglycerides, blood glucose, monocytosis, or changes in monocyte subset composition. In addition, we identified transitory decreases in the acetylation of histone H3 at the lysine residues 18 and 23 in metabolically primed monocytes, and we found that monocyte priming was correlated with the acetylation of histone H3 at lysine 27 after an 8-week DD regimen. Our data show that metabolic stress promotes monocyte priming and hyper-chemotactic responses in NHP. The histone modifications accompanying monocyte priming in primates suggest a reprogramming of the epigenetic landscape, which may lead to dysregulated responses and functionalities in macrophages derived from primed monocytes that are recruited to sites of inflammation.

Abstract 450: Monocytic Glutaredoxin 1 Protects Mice Against Obesity, Hyperglycemia and Atherosclerosis

Overexpression of glutaredoxin 1 (Grx1) protects monocytes from metabolic stress-induced priming, i.e. dysregulation and hypersensitization to chemokines (Ullevig et al. ATVB 2012). To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wild-type (WT) or Grx1 -/- donor mice into atherosclerosis-prone LDLR -/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1 Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1 Leuko -/- mice than in WT mice, but the differences did not reach statistical significance. However, after 20 weeks, Grx1 Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1 Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR-based gene array. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance of proinflammatory “crown-like” structures. In contrast, genes associated with inflammation resolving macrophages were significantly suppressed. Macrophages isolated from Grx1 -/- mice and stimulated with INFγ+TNFα also showed increased expression of pro-inflammatory M1-associated genes, whereas M2-associated genes were suppressed in Grx-1 -/- macrophages activated with IL-4. Furthermore, macrophages from Grx1 -/- mice exposed to metabolic stress also display increased protein S -glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to macrophages from wild-type mice. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming in response to HFD-induced metabolic stress and accelerates the infiltration of dysfunctional monocyte-derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects mice against obesity and atherogenesis.

96 - Monocytic Glutaredoxin 1 Protects Mice against Obesity, Hyperglycemia and Atherosclerosis

We previously reported that glutaredoxin 1 (Grx1) protects monocytes from metabolic-stress induced priming, dysregulation and hypersensitization to chemokines. To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wild-type (WT) or Grx1-/- (Grx1Leuko -/-) donor mice into atherosclerosis-prone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/- mice than in WT, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance proinflammatory “crown-like” structures. Gene expression analysis by qRT-PCR of peritoneal macrophages revealed that macrophages isolated from Grx1-/- mice also have increased expression of pro-inflammatory M1-associated genes and decreased M2-associated genes after activation with INFγ+TNFα and IL-4 respectively. Additionally, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein-S-glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to wild-type cells. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming and accelerates the infiltration of dysfunctional monocyte-derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects against obesity and atherosclerosis.

Hemolysis Controls Pro-Inflammatory Monocyte Priming Via Induction of and Interaction with Circadian Clock Protein Rev-Erbα in Sickle Cell Disease

Background: Patients with SCD tolerate a systemic pro-inflammatory vascular milieu created by chronic ischemia/reperfusion injury and profound erythrocyte hemolysis. In addition to this chronic low level inflammation, exposure to relatively innocuous, sub-clinical inflammatory stimuli appears to ignite an exaggerated, potentially fatal inflammatory response in patients. The etiology of this inflammatory hyper-reactivity is not well understood. There is ample evidence that, in steady state, a cadre of inflammatory cells, especially monocytes, exhibit a primed phenotype. Such priming, or propensity to activate, likely contributes to baseline inflammation, and is requisite for the inflated inflammatory response. Monocytes are quite unique amongst the leukocytes in that their inflammatory potential, including Il-6 release, is governed by the mammalian circadian clock. A role for the rhythmic oscillation of clock proteins as a controller of inflammation in SCD has never been demonstrated. However, a binding partner for heme, the nuclear receptor rev-erbα, is implicated as a regulator of clock controlled genes.Objective: To test the hypothesis that hemolysis, via heme-induced perturbation of the clock protein Rev-erbα, forms the basis for an enhanced inflammatory response in the monocyte.Methods: Intraperitoneal low dose lipopolysaccharide (LPS) was used to elicit an inflammatory response in the Townes mouse model of SCD. Plasma from the mice was acquired 6 hours after LPS injection. Analysis of 25 cytokines was accomplished using luminex methods. Monocytes were modeled in vitro using THP-1 cells. Simultaneous analysis of 84 induced inflammatory genes was conducted via qRT-PCR using the Qiagen RT Profiler PCR array. Inflammatory cytokine levels in cell supernatants were determined via ELISA.Results: We challenged the mice with low dose LPS (<10ng). Interrogation of the inflammatory cytokines in these mice revealed no change in any cytokine tested in the AA mice, but 20 out of 25 inflammatory cytokines were upregulated in mice with the SS genotype. The monocyte-based cytokines were clearly target of LPS activation in the SS mice. TNF-α and Il-1β were both upregulated 20 fold and 80 fold respectively in the SS mice. KC levels (the murine equivalent of Il-8) levels were increased 80 fold in the SS mice treated with LPS. Il-6 levels, however, were the most pronounced with a 40,000 fold increase over PBS injected SS mice. We then evaluated the role of hemolysis on monocyte inflammatory potential in vitro. Sustained monocyte exposure to physiological levels of heme in SCD alone could induce a low level of inflammatory gene expression and Il-6 release. However, sustained exposure to heme dramatically increased Il-6 release from the monocyte in response to LPS. Expression of the Il-6 gene was also increased, but the peak gene expression was time delayed compared to LPS treatment alone. In fact, we noted this phase shifting of inflammatory gene expression in the heme primed cells. LPS induced the release of significantly more TNF-α and Il-1β into the culture media in the presence of heme - consistent with the notion of heme setting a hyperactive threshold in response to LPS. We also noted that heme induced expression of the clock gene rev-erbα, and that antagonizing the activity of rev-erbα ablated the enhanced inflammatory response induced by LPS in the heme primed cells.Conclusion: These data provide evidence that hemolysis may play an important role in the hyper-inflammatory monocyte response via heme- induced dysregulation of the circadian clock. These novel observations provide entirely new avenues of anti-inflammatory therapy in SCD. DisclosuresNo relevant conflicts of interest to declare.

Structure‐Function Analysis of Ursolic Acid and its Analogs to Identify Their Anti‐inflammatory Mechanism of Action

We showed that ursolic acid (UA), a pentacyclic triterpenoid with anti‐inflammatory properties, reduces atherosclerotic lesion size and monocyte dysfunction in diabetic mice. We proposed that UA protects monocytes against metabolic stress‐induced monocyte dysfunction or “priming,” by preventing the induction of Nox4, the rate‐limiting step promoting hyper‐responsiveness to chemoattractants and accelerated chemotaxis induced by metabolic stress. We have also demonstrated that UA rescues MKP‐1 protein expression and activity in metabolically primed cells. The aim of this study was to investigate nine of UA's naturally occurring analogs and identify the structural features of UA required to inhibit monocyte priming and prevent Nox4 induction. We determined the doses (0.03 ‐ 3 μM) at which UA and its analogs blocked metabolic stress‐induced acceleration of chemotaxis in response to MCP‐1 in metabolically primed THP‐1 monocytes. Only the UA analogs hederagenin (IC50 = 0.64 μM), oleanolic acid (IC50 = 1.9 μM), and uvaol (IC50 = 2.46 μM), protected monocytes against metabolic priming. UA was the most potent analog (IC50 = 0.1 μM). All protective analogs also prevented Nox4 induction and rescued MKP‐1 from degradation. Our data suggests that hederagenin, oleanolic acid and uvaol prevent monocyte priming and dysfunction through the same mechanism as UA and thus may all be atheroprotective. Identifying the structural features responsible for UA's beneficial effects will enable us to develop more potent, dietary supplements for the prevention of atherosclerosis and other monocyte‐mediated diabetic complications.​&#x0D; &#x0D; Funding Source: Supported by a grant to RA from the NIH (R01‐AT006885)