Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression.

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DCs) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage-tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aortic intima resident macrophages (MacAIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although MacAIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaques is overtaken by recruited monocytes, which induce MacAIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Early intermittent hyperlipidaemia alters tissue macrophages to fuel atherosclerosis.

Hyperlipidaemia is a major risk factor of atherosclerotic cardiovascular disease (ASCVD). Risk of cardiovascular events depends on cumulative lifetime exposure to low-density lipoprotein cholesterol (LDL-C) and, independently, on the time course of exposure to LDL-C, with early exposure being associated with a higher risk1. Furthermore, LDL-C fluctuations are associated with ASCVD outcomes2-4. However, the precise mechanisms behind this increased ASCVD risk are not understood. Here we find that early intermittent feeding of mice on a high-cholesterol Western-type diet (WD) accelerates atherosclerosis compared with late continuous exposure to the WD, despite similar cumulative circulating LDL-C levels. We find that early intermittent hyperlipidaemia alters the number and homeostatic phenotype of resident-like arterial macrophages. Macrophage genes with altered expression are enriched for genes linked to human ASCVD in genome-wide association studies. We show that LYVE1+ resident macrophages are atheroprotective, and identify biological pathways related to actin filament organization, of which alteration accelerates atherosclerosis. Using the Young Finns Study, we show that exposure to cholesterol early in life is significantly associated with the incidence and size of carotid atherosclerotic plaques in mid-adulthood. In summary, our results identify early intermittent exposure to cholesterol as a strong determinant of accelerated atherosclerosis, highlighting the importance of optimal control of hyperlipidaemia early in life, and providing insights into the underlying biological mechanisms. This knowledge will be essential to designing effective therapeutic strategies to combat ASCVD.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

Cytokine Circuits in Cardiovascular Disease.

Arterial inflammation is a hallmark of atherosclerosis, and appropriate management of this inflammation represents a major unmet therapeutic need for cardiovascular disease patients. Here, we review the diverse contributions of immune cells to atherosclerosis, the mechanisms of immune cell activation in this context, and the cytokine circuits that underlie disease progression. We discuss the recent application of these insights in the form of immunotherapy to treat cardiovascular disease and highlight how studies on the cardiovascular co-morbidity that arises in autoimmunity might reveal additional roles for cytokines in atherosclerosis. Currently, data point to interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and IL-17 as cytokines that, at least in some settings, are effective targets to reduce cardiovascular disease progression.

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages.

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6+ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1+ stromal cells reduced the frequency of GATA6+ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1+ mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

Ferroptosis Mediated Inflammation Promotes Pulmonary Hypertension.

BACKGROUND: Mitochondrial dysfunction, characterized by impaired lipid metabolism and heightened reactive oxygen species generation, results in lipid peroxidation and ferroptosis. Ferroptosis is an inflammatory mode of cell death that promotes complement activation and macrophage recruitment. In pulmonary arterial hypertension (PAH), pulmonary arterial endothelial cells exhibit cellular phenotypes that promote ferroptosis. Moreover, there is ectopic complement deposition and inflammatory macrophage accumulation in the pulmonary vasculature. However, the effects of ferroptosis inhibition on these pathogenic mechanisms and the cellular landscape of the pulmonary vasculature are incompletely defined. METHODS: Multiomics and physiological analyses evaluated how ferroptosis inhibition-modulated preclinical PAH. The impact of adeno-associated virus 1-mediated expression of the proferroptotic protein ACSL (acyl-CoA synthetase long-chain family member) 4 on PAH was determined, and a genetic association study in humans further probed the relationship between ferroptosis and pulmonary hypertension. RESULTS: Ferrostatin-1, a small-molecule ferroptosis inhibitor, mitigated PAH severity in monocrotaline rats. RNA-sequencing and proteomics analyses demonstrated that ferroptosis was associated with PAH severity. RNA-sequencing, proteomics, and confocal microscopy revealed that complement activation and proinflammatory cytokines/chemokines were suppressed by ferrostatin-1. In addition, ferrostatin-1 combatted changes in endothelial, smooth muscle, and interstitial macrophage abundance and gene activation patterns as revealed by deconvolution RNA-sequencing. Ferroptotic pulmonary arterial endothelial cell damage-associated molecular patterns restructured the transcriptomic signature and mitochondrial morphology, promoted the proliferation of pulmonary artery smooth muscle cells, and created a proinflammatory phenotype in monocytes in vitro. Adeno-associated virus 1-Acsl4 induced an inflammatory PAH phenotype in rats. Finally, single-nucleotide polymorphisms in 6 ferroptosis genes identified a potential link between ferroptosis and pulmonary hypertension severity in the Vanderbilt BioVU repository. CONCLUSIONS: Ferroptosis promotes PAH through metabolic and inflammatory mechanisms in the pulmonary vasculature.

TREM2 macrophages mediate the beneficial effects of bariatric surgery against MASH.

BACKGROUND AND AIMS: For patients with obesity and metabolic syndrome, bariatric procedures such as vertical sleeve gastrectomy (VSG) have a clear benefit in ameliorating metabolic dysfunction-associated steatohepatitis (MASH). While the effects of bariatric surgeries have been mainly attributed to nutrient restriction and malabsorption, whether immuno-modulatory mechanisms are involved remains unclear. APPROACH AND RESULT: Using murine models, we report that VSG ameliorates MASH progression in a weight loss-independent manner. Single-cell RNA sequencing revealed that hepatic lipid-associated macrophages (LAMs) expressing the triggering receptor expressed on myeloid cells 2 (TREM2) repress inflammation and increase their lysosomal activity in response to VSG. Remarkably, TREM2 deficiency in mice ablates the reparative effects of VSG, suggesting that TREM2 is required for MASH resolution. Mechanistically, TREM2 prevents the inflammatory activation of macrophages and is required for their efferocytic function. CONCLUSIONS: Overall, our findings indicate that bariatric surgery improves MASH through a reparative process driven by TREM2+ macrophages, providing insights into the mechanisms of disease reversal that may result in new therapies and improved surgical interventions.

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability-Brief Report.

BACKGROUND: Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. METHODS: To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. RESULTS: AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. CONCLUSIONS: Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.

Loss of TREM2 diminishes CAA despite an overall increase of amyloid load in Tg-SwDI mice.

INTRODUCTION: The microglial receptor triggering receptor expressed on myeloid cells 2 (TREM2) is a major risk factor for Alzheimer's disease (AD). Experimentally, Trem2 deficiency affects parenchymal amyloid beta (Aß) deposition. However, the role of TREM2 in cerebrovascular amyloidosis, especially cerebral amyloid angiopathy (CAA), remains unexplored. METHODS: Tg-SwDI (SwDI) mice, a CAA-prone model of AD, and Trem2 knockout mice were crossed to generate SwDI/TWT, SwDI/THet, and SwDI/TKO mice, followed by pathological and biochemical analyses at 16 months of age. RESULTS: Loss of Trem2 led to a dramatic decrease in CAA and microglial association, despite a marked increase in overall brain Aß load. Single nucleus RNA sequencing analysis revealed that in the absence of Trem2, microglia were activated but trapped in transition to the fully reactive state, with distinct responses of vascular cells. DISCUSSION: Our study provides the first evidence that TREM2 differentially modulates parenchymal and vascular Aß pathologies, offering significant implications for both TREM2- and Aß-targeting therapies for AD. HIGHLIGHTS: Triggering receptor expressed on myeloid cells 2 (TREM2) differentially modulates brain parenchymal and vascular amyloidosis. Loss of Trem2 markedly reduces cerebral amyloid angiopathy despite an overall increase of amyloid beta load in Tg-SwDI mice. Microglia are trapped in transition to the fully reactive state without Trem2. Perivascular macrophages and other vascular cells have distinct responses to Trem2 deficiency. Balanced TREM2-targeting therapies may be required for optimal outcomes.

FFAR4 regulates cardiac oxylipin balance to promote inflammation resolution in HFpEF secondary to metabolic syndrome.

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome, but a predominant subset of HFpEF patients has metabolic syndrome (MetS). Mechanistically, systemic, nonresolving inflammation associated with MetS might drive HFpEF remodeling. Free fatty acid receptor 4 (Ffar4) is a GPCR for long-chain fatty acids that attenuates metabolic dysfunction and resolves inflammation. Therefore, we hypothesized that Ffar4 would attenuate remodeling in HFpEF secondary to MetS (HFpEF-MetS). To test this hypothesis, mice with systemic deletion of Ffar4 (Ffar4KO) were fed a high-fat/high-sucrose diet with L-NAME in their water to induce HFpEF-MetS. In male Ffar4KO mice, this HFpEF-MetS diet induced similar metabolic deficits but worsened diastolic function and microvascular rarefaction relative to WT mice. Conversely, in female Ffar4KO mice, the diet produced greater obesity but no worsened ventricular remodeling relative to WT mice. In Ffar4KO males, MetS altered the balance of inflammatory oxylipins systemically in HDL and in the heart, decreasing the eicosapentaenoic acid-derived, proresolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE), while increasing the arachidonic acid-derived, proinflammatory oxylipin 12-hydroxyeicosatetraenoic acid (12-HETE). This increased 12-HETE/18-HEPE ratio reflected a more proinflammatory state both systemically and in the heart in male Ffar4KO mice and was associated with increased macrophage numbers in the heart, which in turn correlated with worsened ventricular remodeling. In summary, our data suggest that Ffar4 controls the proinflammatory/proresolving oxylipin balance systemically and in the heart to resolve inflammation and attenuate HFpEF remodeling.

Single Cell RNA Sequencing in Atherosclerosis Research.

Technological advances in characterizing molecular heterogeneity at the single cell level have ushered in a deeper understanding of the biological diversity of cells present in tissues including atherosclerotic plaques. New subsets of cells have been discovered among cell types previously considered homogenous. The commercial availability of systems to obtain transcriptomes and matching surface phenotypes from thousands of single cells is rapidly changing our understanding of cell types and lineage identity. Emerging methods to infer cellular functions are beginning to shed new light on the interplay of components involved in multifaceted disease responses, like atherosclerosis. Here, we provide a technical guide for design, implementation, assembly, and interpretations of current single cell transcriptomics approaches from the perspective of employing these tools for advancing cardiovascular disease research.

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2+ foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.

Neutrophils promote VLA-4-dependent B cell antigen presentation and accumulation within the meninges during neuroinflammation.

The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibody-independent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4-dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS.

Metabolic regulation of macrophage proliferation and function in atherosclerosis.

PURPOSE OF REVIEW: Macrophage accumulation within atherosclerotic plaque is a primary driver of disease progression. However, recent advances in both phenotypic and functional heterogeneity of these cells have allowed for improved insight into potential regulation of macrophage function within lesions. In this review, we will discuss recent insights on macrophage heterogeneity, lipid processing, metabolism, and proliferation in atherosclerosis. Furthermore, we will identify outstanding questions in the field that are pertinent to future studies. RECENT FINDINGS: With the recent development of single-cell RNA sequencing, several studies have highlighted the diverse macrophage populations within plaques, including pro-inflammatory, anti-inflammatory, lipid loaded and tissue resident macrophages. Furthermore, new data has suggested that differential activation of metabolic pathways, including glycolysis and fatty acid oxidation, may play a key role in determining function. Recent works have highlighted that different populations retain varying capacity to undergo proliferation; regulating the proliferation pathway may be highly effective in reducing plaque in advanced lesions. SUMMARY: Macrophage populations within atherosclerosis are highly heterogeneous; differences in cytokine production, lipid handling, metabolism, and proliferation are seen between subpopulations. Understanding the basic cellular mechanisms that drive this heterogeneity will allow for the development of highly specific disease modulating agents to combat atherosclerosis.

CC Chemokine Receptor 5 Targeted Nanoparticles Imaging the Progression and Regression of Atherosclerosis Using Positron Emission Tomography/Computed Tomography.

Chemokines and chemokine receptors play an important role in the initiation and progression of atherosclerosis by mediating the trafficking of inflammatory cells. Chemokine receptor 5 (CCR5) has major implications in promoting the development of plaques to advanced stage and related vulnerability. CCR5 antagonist has demonstrated the effective inhibition of atherosclerotic progression in mice, making it a potential biomarker for atherosclerosis management. To accurately determine CCR5 in vivo, we synthesized CCR5 targeted Comb nanoparticles through a modular design and construction strategy with control over the physiochemical properties and functionalization of CCR5 targeting peptide d-Ala-peptide T-amide (DAPTA-Comb). In vivo pharmacokinetic evaluation through 64Cu radiolabeling showed extended blood circulation of 64Cu-DAPTA-Combs conjugated with 10%, 25%, and 40% DAPTA. The different organ distribution profiles of the three nanoparticles demonstrated the effect of DAPTA on not only physicochemical properties but also targeting efficiency. In vivo positron emission tomography/computed tomography (PET/CT) imaging in an apolipoprotein E knockout mouse atherosclerosis model (ApoE-/-) showed that the three 64Cu-DAPTA-Combs could sensitively and specifically detect CCR5 along the progression of atherosclerotic lesions. In an ApoE-encoding adenoviral vector (AAV) induced plaque regression ApoE-/- mouse model, decreased monocyte recruitment, CD68+ macrophages, CCR5 expression, and plaque size were all associated with reduced PET signals, which not only further confirmed the targeting efficiency of 64Cu-DAPTA-Combs but also highlighted the potential of these targeted nanoparticles for atherosclerosis imaging. Moreover, the up-regulation of CCR5 and colocalization with CD68+ macrophages in the necrotic core of ex vivo human plaque specimens warrant further investigation for atherosclerosis prognosis.

Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine Models.

RATIONALE: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. OBJECTIVE: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. METHODS AND RESULTS: Single-cell RNA sequencing analysis of CD45+ leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1ß and many other inflammatory transcripts in atherosclerotic aorta. CONCLUSIONS: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.

Thermoneutrality but Not UCP1 Deficiency Suppresses Monocyte Mobilization Into Blood.

RATIONALE: Ambient temperature is a risk factor for cardiovascular disease. Cold weather increases cardiovascular events, but paradoxically, cold exposure is metabolically protective because of UCP1 (uncoupling protein 1)-dependent thermogenesis. OBJECTIVE: We sought to determine the differential effects of ambient environmental temperature challenge and UCP1 activation in relation to cardiovascular disease progression. METHODS AND RESULTS: Using mouse models of atherosclerosis housed at 3 different ambient temperatures, we observed that cold temperature enhanced, whereas thermoneutral housing temperature inhibited atherosclerotic plaque growth, as did deficiency in UCP1. However, whereas UCP1 deficiency promoted poor glucose tolerance, thermoneutral housing enhanced glucose tolerance, and this effect held even in the context of UCP1 deficiency. In conditions of thermoneutrality, but not UCP1 deficiency, circulating monocyte counts were reduced, likely accounting for fewer monocytes entering plaques. Reductions in circulating blood monocytes were also found in a large human cohort in correlation with environmental temperature. By contrast, reduced plaque growth in mice lacking UCP1 was linked to lower cholesterol. Through application of a positron emission tomographic tracer to track CCR2+ cell localization and intravital 2-photon imaging of bone marrow, we associated thermoneutrality with an increased monocyte retention in bone marrow. Pharmacological activation of ß3-adrenergic receptors applied to mice housed at thermoneutrality induced UCP1 in beige fat pads but failed to promote monocyte egress from the marrow. CONCLUSIONS: Warm ambient temperature is, like UCP1 deficiency, atheroprotective, but the mechanisms of action differ. Thermoneutrality associates with reduced monocyte egress from the bone marrow in a UCP1-dependent manner in mice and likewise may also suppress blood monocyte counts in man.

Limited Macrophage Positional Dynamics in Progressing or Regressing Murine Atherosclerotic Plaques-Brief Report.

Objective- Macrophages play important roles in the pathogenesis of atherosclerosis, but their dynamics within plaques remain obscure. We aimed to quantify macrophage positional dynamics within progressing and regressing atherosclerotic plaques. Approach and Results- In a stable intravital preparation, large asymmetrical foamy macrophages in the intima of carotid artery plaques were sessile, but smaller rounded cells nearer plaque margins, possibly newly recruited monocytes, mobilized laterally along plaque borders. Thus, to test macrophage dynamics in plaques over a longer period of time in progressing and regressing disease, we quantified displacement of nondegradable phagocytic particles within macrophages for up to 6 weeks. In progressing plaques, macrophage-associated particles appeared to mobilize to deeper layers in plaque, whereas in regressing plaques, the label was persistently located near the lumen. By measuring the distance of the particles from the floor of the plaque, we discovered that particles remained at the same distance from the floor regardless of plaque progression or regression. The apparent deeper penetration of labeled cells in progressing conditions could be attributed to monocyte recruitment that generated new superficial layers of macrophages over the labeled phagocytes. Conclusions- Although there may be individual exceptions, as a population, newly differentiated macrophages fail to penetrate significantly deeper than the limited depth they reside on initial entry, regardless of plaque progression, or regression. These limited dynamics may prevent macrophages from escaping areas with unfavorable conditions (such as hypoxia) and pose a challenge for newly recruited macrophages to clear debris through efferocytosis deep within plaque.

Signaling through FcRγ-associated receptors on dendritic cells drives IL-33-dependent TH2-type responses.

BACKGROUND: Although allergic sensitization can be generated against various allergens, it is unknown how such a diversity of antigens is able to promote TH2-mediated inflammation leading to atopy. Our previous studies demonstrated that allergen-specific IgG immune complexes (ICs) and house dust mite (HDM) extract both induced dendritic cells (DCs) to drive TH2-mediated inflammation, but the mechanism by which these diverse stimuli produce similar responses is unknown. OBJECTIVE: We sought to identify the DC signaling pathways used by TH2 stimuli to promote TH2-mediated inflammation. METHODS: C57BL/6, FcγRIII(-/-), FcRγ(-/-), and ST2(-/-) mice were sensitized and challenged with HDM, and inflammation was assessed based on results of flow cytometry and histology and cytokine production. Bone marrow-derived DCs from these strains were used in signaling and adoptive transfer experiments. RESULTS: Our findings indicate that 2 distinct TH2 stimuli, ICs and HDM, use the FcRγ-associated receptors FcγRIII and Dectin-2, respectively, to promote TH2-mediated lung inflammation. In this study we demonstrate that both ICs and HDM induce expression of IL-33, a critical mediator in asthma pathogenesis and the differentiation of TH2 cells, in DCs. Upregulation of IL-33 in DCs is dependent on FcRγ, Toll-like receptor 4, and phosphoinositide 3-kinase. Exogenous IL-33 is sufficient to restore the development of TH2 responses in FcRγ-deficient mice. Finally, adoptive transfer of allergen-pulsed FcRγ(+/-) bone-marrow derived DCs restores the development of TH2-type inflammation in FcRγ-deficient mice, demonstrating the necessity of this signaling pathway in DCs for allergen-induced inflammation. CONCLUSION: These data identify a mechanism whereby TH2 stimuli signal through FcRγ-associated receptors on DCs to upregulate IL-33 production and induce TH2-mediated allergic airway inflammation.