RATIONALE:Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of ...aortic leukocytes is only poorly understood.
OBJECTIVE:We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to define an atlas of the immune cell landscape in atherosclerosis.
METHODS AND RESULTS:Employing scRNAseq of aortic leukocytes from chow (CD) and western diet (WD) fed Apoe and Ldlr mice, we detected 11 principal leukocyte clusters with distinct phenotypical and spatial characteristics, while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on a single cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe and Ldlr mice. We confirmed the phenotypic diversity of these clusters with a novel CyTOF 35-marker panel with metal-labelled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of scRNAseq, CyTOF, and FACS, we detected three principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Finally, we used leukocyte cluster gene signatures to enumerate leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.
CONCLUSIONS:The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.
Although mouse models exist for many immune-based diseases, the clinical translation remains challenging. Most basic and translational studies utilize only a single inbred mouse strain. However, ...basal and diseased immune states in humans show vast inter-individual variability. Here, focusing on macrophage responses to lipopolysaccharide (LPS), we use the hybrid mouse diversity panel (HMDP) of 83 inbred strains as a surrogate for human natural immune variation. Since conventional bioinformatics fail to analyse a population spectrum, we highlight how gene signatures for LPS responsiveness can be derived based on an Interleukin-12β and arginase expression ratio. Compared to published signatures, these gene markers are more robust to identify susceptibility or resilience to several macrophage-related disorders in humans, including survival prediction across many tumours. This study highlights natural activation diversity as a disease-relevant dimension in macrophage biology, and suggests the HMDP as a viable tool to increase translatability of mouse data to clinical settings.
OBJECTIVE—Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other ...pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature.
APPROACH AND RESULTS—To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet–fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36 mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein.
CONCLUSIONS—Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.
Persistent inflammation contributes to the development of cardiovascular disease (CVD) as an HIV-associated comorbidity. Innate immune cells such as monocytes are major drivers of inflammation in men ...and women with HIV. The study objectives are to examine the contribution of circulating non-classical monocytes (NCM, CD14dimCD16+) and intermediate monocytes (IM, CD14+CD16+) to the host response to long-term HIV infection and HIV-associated CVD. Women with and without chronic HIV infection (H) were studied. Subclinical CVD (C) was detected as plaques imaged by B-mode carotid artery ultrasound. The study included H-C-, H+C-, H-C+, and H+C+ participants (23 of each, matched on race/ethnicity, age and smoking status), selected from among enrollees in the Women's Interagency HIV Study. We assessed transcriptomic features associated with HIV or CVD alone or comorbid HIV/CVD comparing to healthy (H-C-) participants in IM and NCM isolated from peripheral blood mononuclear cells. IM gene expression was little affected by HIV alone or CVD alone. In IM, coexisting HIV and CVD produced a measurable gene transcription signature, which was abolished by lipid-lowering treatment. In NCM, versus non-HIV controls, women with HIV had altered gene expression, irrespective of whether or not they had comorbid CVD. The largest set of differentially expressed genes was found in NCM among women with both HIV and CVD. Genes upregulated in association with HIV included several potential targets of drug therapies, including LAG3 (CD223). In conclusion, circulating monocytes from patients with well controlled HIV infection demonstrate an extensive gene expression signature which may be consistent with the ability of these cells to serve as potential viral reservoirs. Gene transcriptional changes in HIV patients were further magnified in the presence of subclinical CVD.
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In this issue of Cell Chemical Biology, Lane et al.1 introduce a transgene-based system to express fusion proteins that recruit transcription factors to E3 ligases. This approach expands the target ...repertoire for engineered cell therapies as exemplified by the targeting of SMAD proteins to overcome a TGFβ-mediated suppressive mechanism that weakens anti-tumor responses.
In this issue of Cell Chemical Biology, Lane et al. introduce a transgene-based system to express fusion proteins that recruit transcription factors to E3 ligases. This approach expands the target repertoire for engineered cell therapies as exemplified by the targeting of SMAD proteins to overcome a TGFβ-mediated suppressive mechanism that weakens anti-tumor responses.
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite the best available treatment, prognosis remains poor. Current standard therapy consists of surgical removal of ...the tumor followed by radiotherapy and chemotherapy with the alkylating agent temozolomide (TMZ). Experimental studies suggest that antisecretory factor (AF), an endogenous protein with proposed antisecretory and anti-inflammatory properties, may potentiate the effect of TMZ and alleviate cerebral edema. Salovum is an egg yolk powder enriched for AF and is classified as a medical food in the European Union. In this pilot study, we evaluate the safety and feasibility of add-on Salovum in GBM patients.
Eight patients with newly diagnosed, histologically confirmed GBM were prescribed Salovum during concomitant radiochemotherapy. Safety was determined by the number of treatment-related adverse events. Feasibility was determined by the number of patients who completed the full prescribed Salovum treatment.
No serious treatment-related adverse events were observed. Out of 8 included patients, 2 did not complete the full treatment. Only one of the dropouts was due to issues directly related to Salovum, which were nausea and loss of appetite. Median survival was 23 months.
We conclude that Salovum is safe to use as an add-on treatment for GBM. In terms of feasibility, adherence to the treatment regimen requires a determined and independent patient as the large doses prescribed may cause nausea and loss of appetite.
ClinicalTrials.gov NCT04116138. Registered on 04/10/2019.
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Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). ...Interrupting the interaction between this ligand–receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist.
In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet.
CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion.
These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis.
•Long-acting FKN-Fc reduced atherosclerosis in LDLR KO mice in a prevention model.•FKN-Fc administration accelerated diet-induced regression of established atherosclerosis.•Monocyte adhesion in vitro and in vivo is reduced in the presence of FKN-Fc.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Atherosclerosis in the single-cell era Winkels, Holger; Ehinger, Erik; Ghosheh, Yanal ...
Current opinion in lipidology,
10/2018, Volume:
29, Issue:
5
Journal Article
Peer reviewed
Open access
The immune system plays a critical role in the development and modulation of atherosclerosis. New high-parameter technologies, including mass cytometry (CyTOF) and single-cell RNA sequencing ...(scRNAseq), allow for an encompassing analysis of immune cells. Unexplored marker combinations and transcriptomes can define new immune cell subsets and suggest their functions. Here, we review recent advances describing the immune cells in the artery wall of mice with and without atherosclerosis. We compare technologies and discuss limitations and advantages.
Both CyTOF and scRNAseq on leukocytes from digested aortae show 10-30 immune cell subsets. Myeloid, T, B and natural killer cells were confirmed. Although cellular functions can be inferred from RNA-Seq data, some subsets cannot be identified based on current knowledge, suggesting they may be new cell types. CyTOF and scRNAseq each identified four B-cell subsets and three macrophage subsets in the atherosclerotic aorta. Limitations include cell death caused by enzymatic digestion and the limited depth of the scRNAseq transcriptomes.
High-parameter methods are powerful tools for uncovering leukocyte diversity. CyTOF is currently more powerful at discerning leukocyte subsets in the atherosclerotic aorta, whereas scRNAseq provides more insight into their likely functions.
Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, ...unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.
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•High-dimensional analysis of human bone marrow reveals neutrophil progenitor (NeP)•NeP is unipotent and produces only neutrophils in vivo•NeP suppresses T cell activation and promotes tumor growth in mouse models in vivo•NeP expands in blood of humans with melanoma and in tumor-bearing mice
Zhu et al. discover an early unipotent neutrophil progenitor (NeP) in mouse and human bone marrow using high-dimensional profiling. NeP expand in cancer, suppress T cells, and promote tumor growth in vivo. NeP is found in the blood of human melanoma patients, suggesting that NeP could be a new cancer biomarker.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
RATIONALE:Non-classical mouse monocyte (CX3CR1, Ly-6C) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Due to technical challenges it is ...currently not established how patrolling occurs in large arteries.
OBJECTIVE:This study was undertaken to elucidate the molecular, migratory and functional phenotype of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic and atherosclerotic conditions.
METHODS AND RESULTS:Applying a new method for stable, long-term two-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP mice, we show that non-classical monocytes patrol inside healthy carotid arteries at a velocity of 36 μm/min, three times faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wildtype mice a western diet or by applying topical TLR7/8 agonists. A similar increase is seen in CX3CR1/apoE mice on chow diet, with a further 2-3-fold increase on western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 and α4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1 apoE mice leads to pronounced endothelial apoptosis.
CONCLUSIONS:Arterial patrolling is a prominent new feature of non-classical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in an CX3CR1-independent fashion, and plays a potential role in endothelial protection.Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.
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