nature reviews immunology - october 2013

The classification of macrophages and dendritic cells (DCs) has mainly been based on cell morphology, phenotype and/or select functional attributes. However, many of these markers are not unique to a specific cell type, which has resulted in much debate as to whether a given mononuclear phagocyte should be classified as a DC or a macrophage. Reporting in Cell, Reis e Sousa and colleagues show that the precursors of conventional DCs (cDCs) in mice are marked by dendritic cell natural killer lectin group receptor 1 (DNGR1; encoded by CLEC9A), and they describe a model in which these cells and their progeny are genetically labelled, which facilitates the identification of cDCs in mice on the basis of ontogeny rather than phenotype or function.

Previous studies have shown that plasmacytoid DCs (pDCs) and specific cDC subsets express DNGR1. Now the authors show that DNGR1 is also expressed by bone marrow progenitor cells that pheno-typically resemble

common DC precursors (CDPs). To test the differentiation potential of these cells, lineage-negative CD115+DNGR1+ bone marrow cells were transferred to congenic mice. In contrast with unfractionated bone marrow, which gave rise to a variety of lymphoid and myeloid lineages, lineage-negative CD115+DNGR1+ bone marrow cells almost exclusively generated CD11c+MHC class II+ cDCs, but not pDCs. This suggests that DNGR1 expression marks pre-cursor cells that have a cDC-restricted differentiation potential. Of note, on the basis of the data in the paper, the authors suggested that the acronym CDPs should therefore be defined as conventional DC precursors.

Next, the authors generated mice in which DNGR1-expressing cells and their progeny are indel-ibly marked with enhanced yellow fluorescent protein (YFP), termed Clec9a+/creRosa+/EYFP mice. As expected, the expression of YFP in these mice was restricted to CDPs, to resident CD8+ and CD11b+ cDC subsets in the spleen and skin-draining lymph nodes, and to migratory CD103+ cDCs in the skin. No YFP expression was observed in CD169+ metallophilic macrophages, in Langerhans cells, or in LY6Chi and LY6Clow monocytes. Of note, pDCs (SIGLEC-H+B220+) expressed only low levels of YFP, which, together with the results of the transfer study described above, suggests that these cells arise from a distinct pDC-specific precursor cell. However, CD8+CD205 cells, which have previously been reported to resemble pDCs, expressed high levels of YFP, which suggests that they arise from CDPs.

Further investigation of these mice showed that cDCs in non-lymphoid tissues, including the lungs, intestines and kidneys, can be specifically identified by their DNGR1 expression history. In the lungs, CD103+ cells, CD103CD11b cells and CD11b+ cells were labelled with YFP, even though the CD103CD11b and CD11b+ cell subsets lacked DNGR1 expression. By contrast, pDCs and CD64+ cells (which have been argued to represent monocyte progeny) were inefficiently labelled with YFP. Similarly, in the small intestine, CD11bCD103+ cells, CD11b+CD103+ cells and CD11b+CD103 cells (a cell subset which has previously been suggested to arise from monocytes) all expressed YFP, which indicates that they descend from CDPs. By contrast, monocyte-derived CD11clowCD64+ cells were poorly labelled with YFP. Interestingly, CD64+ cells specifically in the kidneys were also labelled with YFP, which suggests that the expression of CD64 does not dif-ferentiate between CDP-derived and monocyte-derived cells in this tissue site. Further analysis showed that CD64+CD11blowF4/80hi kidney cells had phenotypic and functional properties that are typical of cDCs.

Finally, Clec9a+/creRosa+/EYFPmice were shown to faithfully trace CDP-derived cells, but not monocyte-derived cells that resemble cDCs, during inflammation following infection with Listeria monocytogenes or following dextran sulphate sodium treatment to induce colitis.

So, Clec9a+/creRosa+/EYFP mice represent an in vivo model to identify cDCs on the basis of their onto-genetic descendence from a commit-ted precursor cell and have been used in this study to confirm that cDCs are an independent leukocyte lineage.

Olive Leavy

D E N D R I T I C C E L L S

Tracing the origins of cDCs

ORIGINAL RESEARCH PAPER Schraml, B. U. etal. Genetic tracing via DNGR-1 expression history defines dendritic cells as a hematopoietic lineage. Cell 154, 843858 (2013)

a model...which facilitates the identification of cDCs in mice on the basis of ontogeny rather than phenotype or function

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NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | OCTOBER 2013

2013 Macmillan Publishers Limited. All rights reserved

The molecular mechanisms that underlie allergic inflammatory diseases remain unclear and this is an area of active debate. Reporting in Science, Millien et al. now suggest that hyperactivation of an antifungal pathway involving fibrinogen cleav-age and Toll-like receptor 4 (TLR4) signalling contributes to allergic airway inflammation.

Wild-type mice that were intra-nasally challenged daily for 2 weeks with a fungal proteinase derived from Aspergillus oryzae (PAO) developed canonical features of asthma, includ-ing airway hyperresponsiveness, eosinophilia, enhanced mucin 5AC (Muc5ac) expression, airway goblet cell hyperplasia and production of

the T helper 2 (TH2) cell-associated cytokines interleukin-4 (IL-4), IL-5 and IL-13.

By contrast, Tlr4/ mice chal-lenged with PAO showed reduced or attenuated disease symptoms, but equivalent levels of IL-4 production compared with wild-type mice. Similarly, the induction of allergic lung inflammation, but not IL-4 pro-duction, in response to allergen chal-lenge with proteinase-free ovalbumin or with the conidia of Aspergillus niger was TLR4 dependent. These data indicate that TLR4 is required for the development of allergic airway inflammation, irrespective of allergen proteinase content, but not for IL-4 production in the lungs.

Alveolar macrophages from PAO-treated mice had increased TLR4-dependent expression of several genes that are associated with antifungal immunity. In addition, PAO-activated bone marrow-derived macrophages (BMDMs) from wild-type mice, but not from Tlr4/ mice, controlled fungal growth (known as fungistasis) when the conidia of A.niger were added to the cell cul-tures. Of note, this fungistatic activity only occurred in the presence of fetal bovine serum. These data suggest that PAO functions through both a serum factor and TLR4 to induce macrophage antifungal immunity.

Further investigations showed that stimulation of BMDMs with the endogenous proteinase thrombin, which converts the serum factor fibrinogen to fibrin, resulted in similar fungistatic activity to that observed after PAO stimulation. Furthermore, fibrinogen cleavage products (FCPs) generated by incubation of fibrinogen with PAO

or thrombin induced fungistasis when added to BMDM cultures with-out fetal bovine serum. Moreover, the proteinase inhibitor hirudin reduced the fungistatic activity of PAO-stimulated BMDMs in the presence of fetal bovine serum. Interestingly, FCPs also induced TLR4-dependent MUC5AC and IL-13 receptor-1 (IL-13R1) expression, as well as fungistatic activity, by ex vivo airway epithelial cells. These data suggest that exogenous and endogenous proteinases can cleave fibrinogen to generate TLR4 ligands that prime epithelial cells to respond to IL-13.

But does this pathway have a role in allergic airway disease? Mice chal-lenged with high-dose FCPs showed modest eosinophil recruitment and Muc5ac expression in the lungs but not airway hyperresponsiveness or IL-4 production. Furthermore, the inclusion of hirudin during allergen challenge greatly reduced PAO-induced airway hyperresponsiveness, eosinophilia, Muc5ac expression and IL-13 production. Thus, both FCPTLR4 signalling and cytokine signalling from TH2 cells are required for the full induction of allergic airway disease.

Taken together, these data sug-gest that overwhelming exposure to endogenous or exogenous pro-teinases may lead to hyperactivation of an antifungal pathway and may drive allergic airway inflammation through both fibrinogen and TLR4-dependent and TLR4-independent pathways.

Olive Leavy

A S T H M A A N D A L L E R GY

A fibrinogen root to airway inflammation

ORIGINAL RESEARCH PAPER Millien, V. O. et al. Cleavage of fibrinogen by proteinases elicits allergic responses through Toll-like receptor 4. Science 341, 792796 (2013)

exogenous and endogenous proteinases can cleave fibrinogen to generate TLR4 ligands that prime innate immune cells to respond to IL-13

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Nature Reviews Immunology | AOP, published online 16 September 2013; doi:10.1038/nri3538


Nucleic acids from pathogens are important activators of innate immune responses, but inappropriate responses to self nucleic acids can lead to autoimmune disease. This study now shows that oxidative damage to DNA potentiates its detection by the immune system, which has implications for both self and nonself recognition.

A series of experiments showed that ultraviolet irradiation of DNA has a dosedependent effect on type I interferon (IFN) production by immune and nonimmune cells exposed to this DNA in mice and

humans. The authors observed the generation of intracellular reactive oxygen species (ROS) in cells exposed to ultraviolet radiation, which resulted in a dosedependent increase in oxidative damage to DNA, as measured by 8hydroxyguanine (8OHG), which is the oxidation product of guanine. The 8OHG content of DNA correlated with its ability to stimulate IFN production.

The authors went on to investigate how ROS production by myeloid cells might affect the detection of ingested pathogen DNA. The type I IFN response of mouse dendritic cells that had been transfected with bacterial or viral genomic DNA was enhanced in a dosedependent manner when the DNA was first incubated with hydrogen peroxide. So, in addition to directly damaging pathogens, ROS production might also indirectly facilitate their immune detection.

The oxidative burst of neutrophils can be followed by the expulsion of self genomic DNA to form neutrophil extracellular traps (NETs). NET DNA had a higher 8OHG content than control neutrophil DNA and induced higher levels of type I IFN production when transfected into monocytes. At high concentrations, oxidized self DNA induced a typeI IFN response in monocytes even when no mechanism of intracellular delivery was used, which indicates that extracellular oxidized DNA that has been released from neutrophils might stimulate surrounding immune cells.

The immunostimulatory capacity of oxidized self DNA could have implications for systemic lupus erythematous (SLE), which

involves antibody responses against self nucleic acids. MRLlpr mice (which are a model of SLE) produced higher levels of typeI IFN in response to intravenous injection of ultravioletdamaged self DNA compared with control mice, presumably as a result of increased antibodymediated uptake of DNA. This mechanism could account for the ultraviolet photosensitivity of patients with SLE. Indeed, injection of ultravioletdamaged DNA into the ears of MRLlpr mice resulted in skin lesions similar to those found in patients with SLE.

Finally, the authors showed that the recognition of oxidized DNA involves signalling through the cytosolic cyclic GAMP synthase (cGAS)STING (stimulator of interferon genes protein) pathway. 8OHGcontaining DNA was degraded more slowly by the cytosolic exonuclease TREX1 than unmodified DNA, which resulted in the accumulation of oxidized DNA and therefore increased signalling through the cGASSTING pathway.

Together these results show that not only foreign DNA but also self DNA induces a potentiated immune response after oxidation, which shows that it is a bona fide damageassociated molecular pattern.

Kirsty Minton

PAT T E R N R E C O G N I T I O N R E C E P TO R S

DNA damage drives detection

ORIGINAL RESEARCH PAPER Gehrke, N. et al. Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing. Immunity http://dx.doi.org/10.1016/j.immuni. 2013.08.004 (2013)FURTHER READING Broz, P. & Monack, D. M. Newly described pattern recognition receptors team up against intracellular pathogens. Nature Rev. Immunol. 13, 551565 (2013)

The 8-OHG content of DNA correlated with its ability to stimulate IFN production

Watercolour image courtesy of Christiane Ahlemeyer, Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Germany.





Effector and memory T cells show an increased propensity to traffic back to the tissue sites in which they were originally activated. Dendritic cells (DCs) have been shown to drive the selective trafficking of T cells to the skin and small intestine by inducing T cell expression of tissue-specific homing molecules. Two studies now describe the mechanisms by which lung DCs can promote T cell migra-tion to both the lungs and the small intestine.

Luster and colleagues found that CD4+ T cells homed more efficiently to the lungs if they were activated by lung DCs than if they were activated by DCs from various other tissue sites. They showed that lung DCs promote homing to the lungs

partly through the induction of CC-chemokine receptor 4 (CCR4) expression by T cells, which enables T cell recruitment to the lungs in response to CC-chemokine ligand17 (CCL17) and CCL22.

The physiological relevance of this process was shown by transfer-ring antigen-specific wild-type or CCR4-deficient CD4+ T cells that had been activated by DCs from different tissue sites into mice infected with influenza virus. Mice that received wild-type Tcells activated by lung DCs showed reduced weight loss and cleared their infection more rapidly than mice that received CCR4-deficient Tcells activated by lung DCs or than mice that received wild-type Tcells activated by DCs not from the lungs. Thus, DC-mediated imprinting of T cell homing to the lungs is important to drive more effective immune responses to pathogens in the airways.

Mehandru and colleagues compared the ability of DCs from different tissues to imprint gut-homing molecules on CD4+ Tcells and found that DCs from the lungs, but not from the spleen or skin-draining lymph nodes, could induce T cell expression of the gut-homing molecules CCR9 and 47 integrin as efficiently as DCs from the mesenteric lymph nodes (MLNs). Similarly to what has been shown for DCs from the MLNs, the induc-tion of 47 integrin expression on Tcells by lung DCs was dependent on retinoic acid and transforming growth factor--mediated signal-ling. However, although imprinting with intestinal homing molecules has been reported to be exclusively driven by CD103+ intestinal DCs, both CD103+ and CD103 DCs from the lungs induced CCR9 and 47 integrin expression on T cells.

In addition, the authors showed that T cells activated in an antigen-specific manner by lung DCs could migrate to the intestinal lamina propria. Furthermore, intranasal immunization with antigens expressed by the gastrointestinal pathogen Salmonella enterica subsp. enterica serovar Typhimurium was more effective than subcutaneous immunization in protecting mice against subsequent enteric infection with this pathogen.

Taken together, these studies show that lung DCs can promote immunity to mucosal pathogens by driving T cell homing to the lungs and intestines. They also add further support to the much older concept of a common mucosal immune system.

Yvonne Bordon

M U C O S A L I M M U N O LO GY

Air miles for T cells

ORIGINAL RESEARCH PAPERS Mikhak, Z., Strassner, J. P. & Luster, A. D. Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4. J. Exp. Med. 210, 18551869 (2013) | Ruane, D. et al. Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract. J.Exp. Med. 210, 18711888 (2013)

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lung DCs can promote immunity to mucosal pathogens by driving Tcell homing to the lungs and intestines





Although it is not completely under-stood, the pain that is associated with acute bacterial infections is thought to be secondary to activation of the immune system. Clifford Woolf and colleagues now show that bacteria directly activate pain responses by triggering nociceptor neurons. Furthermore, activation of these sensory neurons by bacteria leads to the release of neuropeptides that can suppress immune responses to the infection.

The authors aimed to inves-tigate the mechanisms by which Staphylococcus aureus (which is a common cause of wound infections) induces pain. Pain thresholds were assessed by infecting mice with S.aureus and then measuring their sensitivity to mechanical, heat- or cold-associated stress. Mice showed signs of hyperalgesia within 1 hour of S. aureus infection, with the pain response peaking 6 hours after infec-tion and beginning to decrease after 24 hours. Surprisingly, the kinetics of the pain response did not correlate with the kinetics of tissue swelling or with the kinetics of immune cell recruitment. By contrast, bacterial loads in the tissue closely correlated with pain hypersensitivity, which suggests that bacteria may directly

interact with sensory neurons. In keeping with this idea, pain percep-tion during S.aureus infection was not decreased in mice deficient for Toll-like receptor signalling compo-nents, or in mice lacking neutrophils, monocytes or lymphocytes. Indeed, antibody-mediated depletion of neu-trophils or monocytes led to higher bacterial loads and to increased pain hypersensitivity.

To test whether bacteria directly induce pain, the authors applied heat-killed bacteria to dorsal root ganglia (DRG) sensory neurons. Various strains of heat-killed bac-teria, including S. aureus, induced robust calcium fluxes in DRG neurons. Additional experiments suggested that bacterial N-formylated peptides trigger mechanical (but not heat) hyperalgesia by activating nociceptors. Furthermore, they showed that -haemolysin, which is a pore-forming toxin produced by S.aureus, binds to and activates a subset of nociceptor neurons and directly induces mechanical, heat and cold hypersensitivity.

The authors proceeded to examine how the activation of nociceptors by bacteria can modulate immune responses. Conditional ablation of nociceptors abolished pain responses

during S. aureus infection and this was associated with increased local inflammation, despite there being similar bacterial tissue loads in noci-ceptor-deficient and control mice. Microarray analyses showed that receptors for the neuropeptides calci-tonin gene-related peptide (CGRP), galanin and somatostatin are highly expressed by neutrophils, monocytes and macrophages. Furthermore, the treatment of macrophages with these neuropeptides suppressed their production of tumour necrosis factor in response to S. aureus. Finally, the exposure of DRG neurons to super-natant from S. aureus cultures or to -haemolysin promoted the release of CGRP in a dose-dependent manner.

This study shows that bacte-rial products can directly activate nociceptors to induce pain and the release of immunosuppressive neuropeptides. The authors suggest that pathogenic bacterial strains have evolved to trigger nociceptors in order to suppress the host immune system and increase their own spread in infected tissues.

Yvonne Bordon

N E U R O I M M U N O LO GY

Pain blame it on the bug, eh?

ORIGINAL RESEARCH PAPER Chiu, I. M. et al. Bacteria activate sensory neurons that modulate pain and inflammation. Nature http://dx.doi.org/ 10.1038/nature12479 (2013)

bacterial products can directly activate nociceptors to induce pain and the release of immuno-suppressive neuropeptides

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NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | OCTOBER 2013 | 1



Following injury, the regeneration of skeletal muscle is supported by muscle-associated macrophages. These macrophages initially show pro-inflammatory characteristics and stimulate myoblast prolif-eration, but later they switch to an anti-inflammatory phenotype that supports the terminal differentia-tion of myoblasts and the growth of new muscle fibres. Mounier et al. now report that the metabolic regulator AMP-activated protein kinase (AMPK) is crucial for such macrophage skewing during muscle regeneration.

AMPK senses cellular energy levels by monitoring ADP:ATP and AMP:ATP ratios, and it regulates

many metabolic processes that are involved in cellular energy homeostasis. Previous studies have associated increased AMPK activity with decreased inflamma-tory responses in macrophages, so Mounier et al. reasoned that AMPK could be involved in the macrophage phenotype-switching that occurs during muscle repair. As AMPK1 is the only catalytic subunit of AMPK that is expressed in macrophages, they examined muscle regeneration in AMPK1-deficient mice.

Histological analysis showed that skeletal muscle repair was delayed in AMPK1-deficient mice and that this was associated with higher numbers of necrotic myofibres, a decrease in the size of new myofibres and lower overall muscle mass. Closer compari-son of the reparatory process in wild-type and AMPK1-deficient animals showed that AMPK1 deficiency does not alter muscle cell homeosta-sis or the fusion of muscle cells into myofibres. Furthermore, mice in which AMPK1 was deleted under the control of a myeloid-specific pro-moter also showed impaired skeletal muscle regeneration. Thus, AMPK1 expression by macrophages, but not by other cells in the tissue, is needed for skeletal muscle repair.

The authors next investigated the differentiation of bone marrow-derived macrophages from AMPK1-deficient mice in vitro. Notably, although AMPK1-deficient macro-phages showed normal acquisition of a pro-inflammatory M1phenotype, they showed impaired acquisition of an anti-inflammatory M2 phenotype, as determined by lower levels of transforming growth factor-, CD163 and CD206 and higher expression of

inducible nitric oxide synthase. High oxygen consumption rates have been associated with the M2 macrophage phenotype but, whereas wild-type macrophages showed a marked increase in oxygen consumption rates under M2-polarizing condi-tions, this increase was not seen in AMPK1-deficient macrophages.

Further experiments in vivo confirmed that AMPK1-deficient macrophages do not switch from an M1 to an M2 phenotype during muscle repair. Although wild-type macrophages showed transition to an anti-inflammatory and pro- reparatory phenotype following phagocytosis of apoptotic and necrotic myoblasts, AMPK1-deficient mac-rophages showed impaired phagocytic responses and a failure to undergo this phenotypic switching. Finally, wild-type macrophages treated with an inhibitor of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2), which is an upstream activator of AMPK, also failed to switch to an M2phenotype follow-ing the phagocytosis of apoptotic myoblasts.

These data suggest that the phago-cytosis of cellular debris by inflam-matory macrophages in an injured tissue is associated with activation of the metabolic regulator AMPK. This enzyme then functions as a molecular switch to promote the acquisition of the pro-reparatory macrophage phenotype that is needed for tissue regeneration.

Yvonne Bordon

M AC R O P H AG E S

Metabolic master prompts a change of tack

ORIGINAL RESEARCH PAPER Mounier, R. et. al. AMPK1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration. Cell Metab. 18, 251264 (2013)

AMPK1 expression by macrophagesis needed for skeletal muscle repair

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S I G N A L L I N G

NF-B signalosomes on the ERThis study shows that stimulation of a range of innate and adaptive immune receptors results in the accumulation of ubiquitylated components of the nuclear factor-B (NF-B) signalling cascade on the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane. ER membrane fractions from stimulated cells could activate inhibitor of NF-B kinase (IKK) in a cell-free system, which indicates that the ER membrane anchors a signalosome that is sufficient to propagate NF-B signalling. The ER-resident protein metadherin was shown to associate with ubiquitylated NF-B signalling components, and knockdown of metadherin in both B and T cells inhibited the accumulation of ubiquitylated signalling components on the ER and selectively decreased NF-B activation downstream of various immune receptors. The results support a role for the ER in outside-in signalling.ORIGINAL RESEARCH PAPER Alexia, C. et al. The endoplasmic reticulum acts as a platform for ubiquitylated components of nuclear factor B signaling. Sci. Signal. 291, ra79 (2013)

R E P R O D U C T I V E I M M U N O LO GY

How NK cells affect pregnancy outcomeInteractions between killer cell immunoglobulin-like receptors (KIRs) expressed by maternal decidual natural killer (NK) cells and HLA-C molecules expressed by fetal trophoblast cells affect the extent of trophoblast invasion of the maternal blood supply by unknown mechanisms. This study reports that decidual NK cells expressing the activating receptor KIR2DS1 produce greater amounts of granulocytemacrophage colony-stimulating factor (GM-CSF) in response to HLA-C2 than NK cells expressing the inhibitory receptor KIR2DL1 or those expressing both KIR2DS1 and KIR2DL1. Trophoblast cells were shown to express GM-CSF receptor-, and stimulation with GM-CSF increased their migration through fibronectin-coated transwells. The authors suggest that women expressing KIR2DL1, with or without KIR2DS2, who carry a HLA-C2+ fetus will have decreased GM-CSF production in the decidua and hence decreased trophoblast invasion, which correlates with pregnancy disorders such as pre-eclampsia and fetal growth restriction.ORIGINAL RESEARCH PAPER Xiong, S. et al. Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation. J. Clin. Invest. http://dx.doi.org/10.1172/JCI68991 (2013)

I M M U N E R E G U L AT I O N

IL-27 induces immunosuppressive DCsThis study shows that, instead of directly affecting T cells as was previously thought, interleukin-27 (IL-27) modulates dendritic cells (DCs) to suppress T cells. Pretreatment of DCs with IL-27 decreased their ability to promote the differentiation of Thelper1 (T

H1) and T

H17 cells and increased

their ability to generate regulatory T cells. Consistent with the increased induction of pathogenic T

H cell subsets, chimeric

mice containing IL-27 receptor -chain (IL-27RA)-deficient DCs developed faster onset and more severe experimental autoimmune encephalomyelitis (EAE) than control mice. Microarray analysis of IL-27-treated DCs showed upregulation of expression of CD39, which reduced extracellular concentrations of ATP and suppressed nucleotide-dependent activation of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3). Finally, vaccination with IL-27-conditioned DCs suppressed EAE and reduced epitope spreading.ORIGINAL RESEARCH PAPER Mascanfroni, I. D. et al. IL-27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the immunoregulatory molecule CD39. Nature Immunol. http://dx.doi.org/10.1038/ni.2695 (2013)

IN BRIEF




The textbook tenet is that B cells arise in the bone marrow. But FredAlt, Duane Wesemann and their col-leagues now show that B cells can also develop in the mouse gut for a short time period after birth.

The authors used recombination activating gene 2 (Rag2)-reporter mice, in which the Rag2 gene is fused to the gene that encodes green fluorescent protein (GFP), to mark immature B cells undergoing RAG2-mediated generation of B cell receptor repertoires. Analysis of these mice showed that approximately 3% of total CD19+ B cells in the small

intestinal lamina propria expressed RAG2GFP. The RAG2+ B lineage cells were mainly located near to the bases of the villi, whereas mature Bcells were distributed throughout the lamina propria but were not found in the mesenteric lymph nodes or among intraepithelial lymphocytes and only in very low frequencies in the large intestinal lamina propria. Interestingly, the numbers of lamina propria RAG2+ Blineage cells gradu-ally increased after birth, peaking at about 1823 days, before decreasing to undetectable levels by postnatal day 35.

The RAG+ B lineage cell populations in the bone marrow comprise pro-Bcells (IgIg), pre-B cells (Ig+Ig) and immature B cells undergoing receptor editing (Ig+Ig+). Similar relative levels of these three subsets were found in the gut and the bone marrow. Further investigation of repertoire diversity indicated that the immunoglobulin heavy chain (IgH) repertoires of the gut and the bone marrow cells were indistinguishable, but the immuno-globulin light chain (IgL) repertoires were distinctive. The authors proposed that the lamina propria IgL repertoires were generated by receptor editing in RAG2+ immature B cells in response to commensal micro-organisms. In support of this idea, colonization of germ-free mice with commensal microorganisms led to increases in RAG1 and RAG2 expres-sion and increased the percentages of pro-Bcells relative to total Bcells in the gut and the bone marrow. Moreover, there was a commensal bacteria-dependent increase in Ig usage a marker of receptor editing specifically in the lamina propria.

So, B cell development and diver-sification can occur in the intestinal mucosa in response to colonization of the intestinal microbiota at weaning. Whether this process enhances overall antibody diversity or whether it helps to eliminate antibody reactivity to commensal bacteria and self antigens will require further study.

Lucy Bird

B C E L L D E V E LO P M E N T

A window of opportunity

ORIGINAL RESEARCH PAPER Wesemann, D. R. et al. Microbial colonization influences early B-lineage development in the gut lamina propria. Nature 501, 112115 (2013)FURTHER READING Schlissel, M. B cell development in the gut. Nature 501, 4243 (2013)

B cell development and diversification can occur in the intestinal mucosa in response to colonization of the intestinal microbiota

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Atherosclerosis results from a maladaptive inflamma-tory response that is initiated by the intramural reten-tion of cholesterol-rich, apolipoprotein B-containing lipoproteins in susceptible areas of the arterial vascu-lature1. Lipoproteins that are sequestered in the arte-rial wall are susceptible to various modifications (such as oxidation, enzymatic and non-enzymatic cleavage, and aggregation), which render these particles pro-inflammatory and which induce the activation of the overlying endothelium. The ensuing immune response is mediated by the recruitment of monocyte-derived cells into the subendothelial space, where these cells dif-ferentiate into mononuclear phagocytes that ingest the accumulated normal and modified lipoproteins, which transforms them into the cholesterol-laden foam cells. Foam cells, typically classified as a type of macrophage, persist in plaques, which promotes disease progression. Although macrophage clearance of lipoproteins is likely to be beneficial at the outset of this immune response, there is little negative feedback following uptake and thus these cells become grossly engorged with lipids. The resulting dysregulation of lipid metabolism alters the macrophage phenotype and compromises crucial immune functions.

Notably, macrophages that accumulate in athero-sclerotic plaques seem to have a diminished capacity to migrate, which contributes to their failure to resolve inflammation and to the progression of these lesions to more advanced, complex plaques in which other immune cell subsets and vascular smooth muscle cells participate in the inflammatory process2. In these advanced plaques, macrophages continue to be major

contributors to the inflammatory response through their secretion of pro-inflammatory mediators (includ-ing chemokines, cytokines and reactive oxygen and nitrogen species) and matrix-degrading proteases, and through their eventual death by necrosis or apoptosis. Dying macrophages release their lipid contents and tissue factors, which leads to the formation of a pro-thrombotic necrotic core. This necrotic core is a key component of unstable plaques and contributes to their rupture and the ensuing intravascular blood clot that underlies myocardial infarction andstroke.

Although many cell types, including endothelial cells, monocytes, dendritic cells (DCs), lymphocytes, eosino-phils, mast cells and smooth muscle cells, contribute to the formation of atherosclerotic plaques, foam cells are so central to the pathophysiology of atherosclerosis that emphasis has long been placed on understanding the mechanisms of monocyte recruitment into plaques and on identifying strategies to reduce monocyte influx to retard plaque progression. However, it has become apparent that the recruitment of monocytes and other leukocytes into the artery may also be crucial to promote atherosclerosis regression and inflammation resolution3. In addition, studies in some models of atherosclerosis regression have shown that macrophage retention can be reversed46, which led to the identification of path-ways that promote macrophage accumulation in, or egress from, the inflamed plaque. These advances have shown that both the quantity and the phenotype of mac-rophages influence the inflammatory state of the plaque, and have potentially identified new targets for plaque intervention. In this Review, we discuss the key roles of

Department of Medicine, Leon H.Charney Division of Cardiology, Marc and RutiBell Vascular Biology and Disease Program, NewYork University School of Medicine, New York, New York 10016, USA. Correspondence to K.J.M.and E.A.F. e-mails: [email protected]; [email protected]:10.1038/nri3520Published online 2 September 2013

Foam cellsMacrophages in the arterial wall that ingest oxidized low-density lipoprotein and assume a foamy appearance. These cells secrete various substances that are involved in plaque growth.

Myocardial infarctionAn episode of acute cardiac ischaemia that leads to death of heart muscle cells. It is usually caused by a thrombotic atherosclerotic plaque.

Macrophages in atherosclerosis: a dynamic balanceKathryn J.Moore, Frederick J.Sheedy and Edward A.Fisher

Abstract | Atherosclerosis is a chronic inflammatory disease that arises from an imbalance in lipid metabolism and a maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Through the analysis of the progression and regression of atherosclerosis in animal models, there is a growing understanding that the balance of macrophages in the plaque is dynamic and that both macrophage numbers and the inflammatory phenotype influence plaque fate. In this Review, we summarize recently identified pro- and anti-inflammatory pathways that link lipid and inflammation biology with the retention of macrophages in plaques, as well as factors that have the potential to promote their egress from these sites.

REVIEWS



Atherosclerosis regressionA decrease in atherosclerotic plaque size that is typically accompanied by a reduction in lipid levels, immune cells and inflammatory gene expression.

macrophages in the initiation, progression and resolu-tion of atherosclerotic inflammation, with a focus on how the dynamics of macrophage recruitment, egress and death alter the fate of theplaque.

Circulating monocytes and their recruitmentHypercholesterolaemia and monocytosis. Hyper-cholesterolaemia is associated with increased numbers of circulating monocytes in mice, pigs and rabbits7,8. In apolipoprotein E-deficient (Apoe/) mice, the numbers of circulating monocytes are ~50% higher than in wild-type mice9,10. How does hypercholesterolaemia cause mono-cytosis? Studies using mice have shown that cholesterol enrichment of haematopoietic stem and progenitor cells (HSPCs; precursors of monocytes and neutrophils) increases their expression of the common -subunit of the interleukin-3 (IL-3) and the granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor and thus, HSPC proliferation11. Notably, the expression of factors that promote cholesterol efflux (high-density lipoprotein (HDL) and APOE) in hypercholesterolemic mouse models corrected HSPC proliferation.

Circulating monocytes in mice consist of two major subsets, LY6Chi and LY6Clow monocytes (BOX1). Interestingly, the monocytosis in hypercholesterolemic mice primarily derives from an increase in the more inflammatory LY6Chi subset, which constitutes the majority of cells recruited to progressing atherosclerotic plaques and which is thought to be the source of the M1 macrophages (also known as classically activated macrophages) that are found in the plaques911. The basis for this cell bias has been postulated to be due to a hypercholesterolaemia-induced impairment of a pro-cess in which LY6Chi cells are converted to LY6Clow cells9; however, this remains an area of active investigation.

Recruitment of monocytes into athero-prone arterial sites. The early steps of atherogenesis have been the subject of numerous reviews (for example, REFS1214) and will only be briefly covered here. Atherosclerotic plaques are not randomly distributed, but tend to form at the inner curvatures and branch points of arteries, where laminar flow is either disturbed or insufficient to maintain the normal, quiescent state of the endothelium.

Box 1 | Characteristics of monocyte and macrophage subsets

LY6Chi monocytesExpresshighlevelsofCC-chemokinereceptor2

Thoughttobepro-inflammatorybecauseoftheirrecruitmenttositesofinflammation,includingtoatheroscleroticplaques

Normallyrepresent50%ofmonocytesinmice,buttheirfrequencyisincreasedinhyperlipidaemia

ThoughttocorrespondtotheCD14+CD16monocytesubsetinhumans,whichrepresent95%ofmonocytesinhumans

ProposedtobeprecursorsofM1macrophages

LY6Clow monocytesExpresshighlevelsofCX

3C-chemokinereceptor1

Thoughttopatrolthevasculatureinahomeostaticfunction

ThoughttocorrespondtotheCD14lowCD16+monocytesubsetinhumans

ProposedtobeprecursorsofM2macrophages

M1 macrophagesClassicalactivationbylipopolysaccharide(orbyotherToll-likereceptorligands)andinterferon-Enrichedinprogressingplaques

Secretepro-inflammatorycytokines,suchasinterleukin-1(IL-1),IL-12andtumournecrosisfactorProducehighlevelsofinduciblenitricoxidesynthaseandnitricoxide

Expressthepro-inflammatorytranscriptionfactorsnuclearfactor-B,activatorprotein1andhypoxia-induciblefactor1ExpressMHCclassIImoleculesandtheco-stimulatorymoleculesCD80andCD86

M2 macrophagesAlternativeactivationbyIL-4andIL-13

Enrichedinregressingplaques

Highendocyticactivity

Takeupandoxidizefattyacids

Secreteanti-inflammatorycytokines,suchasIL-1receptorantagonistandIL-10

Expresshighlevelsofarginase1andhaveincreasedsecretionofcollagen,whichpromotestissuerepair

ExpressthetranscriptionfactorsKrppel-likefactor4,peroxisomeproliferatoractivatedreceptor-andsignaltransducerandactivatoroftranscription6(STAT6)

ExpressCD163,mannosereceptor1(alsoknownasCD206)andFIZZ1

Mox macrophagesInducedbyoxidizedphospholipidsandnitrosylatedfattyacids

Enrichedinprogressingplaques

Expresshighlevelsofreactiveoxygenspeciesandhaemoxygenase1,andthetranscriptionfactornuclearerythroid2-relatedfactor2(NRF2)

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710 | OCTOBER 2013 | VOLUME 13 www.nature.com/reviews/immunol


Retentionmolecules Netrin 1 Semaphorin 3E

Cytokines IL-1 IL-6 TNF

Chemokines CCL2 CCL5 CXCL1

Nature Reviews | Immunology

T cell

DC

OxidizedLDL

LDL

SR-A1

Foam cellMacrophage

CCR1

CX3CR1

CX3CL1CXCL1and CCL5

P-selectin

PSGL1

Crawling(patrolling)

Capture Rolling Arrest

LDL

Endothelialcell

Extravasation

ICAM1

LFA1

LFA1VCAM1 ICAM1

VLA4

20%

PSGL1lowLFA1hi

GR1LY6Clow

PSGL1hiLFA1low

80%

Media

Lumen

Intima

CCR5

CD36

GR1+LY6Chi monocyteGR1LY6Clow monocyte

Leukocyte adhesion cascadeThe key steps that are involved in leukocyte adhesion to the endothelium. These include rolling (which is mediated by selectins), activation (which is mediated by chemokines) and arrest (which is mediated by integrins). Recent additional steps have been defined that include capture (also known as tethering), slow rolling, adhesion strengthening and spreading, intravascular crawling, and paracellular and transcellular transmigration.

Firm adhesionThe interactions of rolling leukocytes with chemokines or lipid mediators, such as leukotriene B4, at the endothelial surface leads to the activation of leukocyte integrins another family of adhesion molecules. After they are activated, integrins mediate the high-affinity adhesive interactions between leukocytes and endothelial cells, which results in the arrest and firm adhesion of rolling leukocytes.

This activation of the endothelium leads to increased permeability to lipoproteins and an accumulation of extracellular matrix proteins that cause a poorly under-stood diffuse intimal thickening and the retention of the atherogenic APOB lipoproteins. The arterial intima at these athero-prone sites contains an increased number of myeloid cells that have features ofDCs15.

The activation of the endothelium also promotes the recruitment of circulating monocytes (FIG.1). In addi-tion to the bone marrow origin of these monocytes, it has recently been recognized that splenic HSPCs can be an extramedullary myelopoietic source of monocytes, which are mobilized to inflammatory sites, including to atherosclerotic plaques16. The steps that regulate mono-cyte entry into the arterial intima are apparently inde-pendent of the source of the cells and depend on the upregulation on activated endothelial cells of molecules that mediate the arrest of circulating monocytes by the leukocyte adhesion cascade17. The capture and rolling

phases of this cascade depend on the immobilization of chemokines, particularly CC-chemokine ligand 5 (CCL5) and CXC-chemokine ligand 1 (CXCL1), on endothelial cell glycosaminoglycans, and on P-selectin, which is expressed on the luminal side of endothelial cells. Very recent results have shown that the arrest of LY6Chi monocytes through CCL5 depends not only on its interaction with CC-chemokine receptor 5 (CCR5) but also on its interaction with CCR1 (REF.18). Vascular cell adhesion molecule 1 (VCAM1) and intercellu-lar adhesion molecule 1 (ICAM1), which bind to the integrins very late antigen 4 (VLA4; also known as 41 integrin) and lymphocyte function-associated anti-gen1 (LFA1; also known as L2 integrin), respectively, are important for the firm adhesion of monocytes to the luminal surface of the endothelium. Comparatively more LFA1 is expressed by LY6Clow cells than by LY6Chi cells, which may underlie the greater tendency of LY6Clow cells to adhere to, but not to enter, the vasculature19.

Figure 1 | Mechanisms regulating monocyte recruitment and accumulation in plaques. Hyperlipidaemia increases the number of GR1+LY6Chi monocytes, which constitute 80% of the monocytes recruited to mouse atherosclerotic plaques, with the remainder being the GR1LY6Clow patrolling monocytes. These monocyte subsets use different chemokinechemokine receptor pairs to infiltrate the intima, which is facilitated by endothelial adhesion molecules, including selectins, intercellular adhesion molecule 1 (ICAM1) and vascular adhesion molecule 1 (VCAM1). The recruited monocytes differentiate into macrophages or dendritic cells (DCs) in the intima, where they interact with atherogenic lipoproteins. Macrophages avidly take up native and modified (for example, oxidized) low-density lipoprotein (LDL) via macropinocytosis or scavenger receptor-mediated pathways (including via scavenger receptor A1 (SR-A1) and CD36), which results in the formation of the foam cells that are a hallmark of the atherosclerotic plaque. These foam cells secrete pro-inflammatory cytokines (including interleukin-1 (IL-1), IL-6, and tumour necrosis factor (TNF)) and chemokines (such as CC-chemokine ligand 2 (CCL2), CCL5 and CXC-chemokine ligand 1 (CXCL1)), as well as macrophage retention factors (such as netrin 1 and semaphorin 3E) that amplify the inflammatory response. CX

3CL1,

CX3C-chemokine ligand 1; CX

3CR1, CX

3C-chemokine receptor 1; LFA1, lymphocyte function-associated antigen 1;

PSGL1, P-selectin glycoprotein ligand 1; VLA4, very late antigen 4.

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Pattern recognition receptors(PRRs). Host receptors (such as Toll-like receptors) that can sense pathogen-associated or damage-associated molecular patterns and that can initiate signalling cascades (which involve activation of nuclear factor-B) that lead to an innate immune response.

The transmigration of monocytes across the endothe-lium into plaques is mediated by chemokines that are secreted by endothelial cells, intimal macrophages and smooth muscle cells. Although several chemokines have been implicated in atherosclerosis20, the three major chemokine receptorchemokine pairs that are thought to be involved in monocyte transmigra-tion are CCR2CCL2, CX3C-chemokine receptor1 (CX3CR1)CX3C-chemokine ligand 1 (CX3CL1) and CCR5CCL5 (REF.10). Indeed, the elimination of these three chemokine axes led to a ~90% reduc-tion in atherosclerosis in Apoe/ mice21. In addition to these chemokines, CD31 (also known as PECAM1; an endothelial cell surface immunoglobulin-like adhe-sion molecule) and VCAM1 may also have a role in monocyte transmigration into atherosclerotic plaques. It should also be noted that CCR2 and CX3CR1, in addi-tion to their effects on transmigration, indirectly influ-ence the number of monocytes that enter the plaques: in particular, CCR2 is required for the extravasation of LY6Chi cells from the bone marrow and CX3CR1 promotes their survival by inhibiting apoptosis22,23.

In addition to the factors described above, emerg-ing evidence suggests that neuronal guidance cues are involved in monocyte recruitment in atherosclerosis. We recently reported that members of the netrin, semaphorin and ephrin families are expressed by arte-rial endothelial cells and that they are differentially regulated under conditions that promote or protect from atherosclerosis24; for example, the expression of ephrinB2 is upregulated under pro-atherosclerotic conditions and is a chemoattractant, which increases leukocyte recruitment to athero-prone arterial sites in the absence of additional chemokines24. By con-trast, the expression of netrin 1 and semaphorin 3A, which inhibit the chemokine-directed migration of human and murine monocytes invitro, are decreased in athero-prone regions, and the inhibition of these molecules by blocking peptides in wild-type mice increased leukocyte adhesion to the endothelium24. Although further studies in hyperlipidemic mouse models are needed, the data suggest that the coor-dinated regulation of positive and negative guidance cues facilitates leukocyte infiltration of the endothe-lium. Notably, these neuronal guidance cues have additional roles in atherosclerosis as they regulate the chemostasis of plaque macrophages25,26 (see below).

Therefore, overall, the recruitment of circulating monocytes into plaques requires the integration of at least three discrete processes, namely, their capture, roll-ing and transmigration, and each step is regulated by multiple, and sometimes overlapping, molecular factors. The fates of these recruited monocytes in the plaques are addressed in the sectionsbelow.

Foam cell formation in atherosclerosisLipoprotein uptake. Lipoprotein uptake by monocyte-derived macrophages is thought to be one of the earliest pathogenic events in the nascent plaque and results in the development of foam cells (FIG.2). The mechanisms of foam cell formation have been intensely studied

(reviewed in REF.27). Although macrophages can clear APOB-containing lipoproteins through the low-density lipoprotein (LDL) receptor, the expression of this recep-tor is downregulated early during foam cell formation by the increased cellular cholesterol levels. These observa-tions led to the early hypothesis that lipoproteins must become modified in the artery wall and that they must be taken up by other mechanisms. Multiple means of LDL modification have now been identified that facili-tate cholesterol loading of macrophages invitro (FIG.2); however, the physiologically relevant pathways of foam cell formation invivo remain an area ofdebate.

A prevailing paradigm has been that increased oxidative stress in the artery wall promotes modifica-tions of LDL, which generates damage signals that are recognized by pattern recognition receptors (PRRs) on cells of the innate immune system. This hypothesis is supported by the presence of oxidized LDL in both human and mouse atheromas, and of natural antibodies (predominantly IgM) that recognize oxidation-specific epitopes of LDL28. A variety of mechanisms mediated by enzymes (such as 12/15-lipoxygenase and myeloperoxi-dase) and by free radicals (such as superoxide, hydro-gen peroxide and nitric oxide) have been identified that could promote LDL oxidation in the arterywall28, and invitro preparations of such modified LDLs are avidly endocytosed by macrophages29,30.

Scavenger receptors, which are a type of PRR expressed by macrophages, have an important role in atherosclerosis and were originally characterized by their ability to recognize and process modified LDL27. Numerous scavenger receptor family members including scavenger receptor A1 (SR-A1; encoded by MSR1), macrophage receptor with collagenous struc-ture (MARCO; also known as SR-A2), CD36 (also known as platelet glycoprotein 4), scavenger receptorB1 (SR-B1), lectin-like oxidized LDL receptor 1 (LOX1), scavenger receptor expressed by endothelial cells 1 (SREC1) and scavenger receptor for phosphatidylserine and oxidized LDL (SR-PSOX; also known as CXCL16) can bind to oxidized LDL and can promote foam cell formation31. SR-A1 and CD36 mediate 7590% of the degradation of LDL that has been modified by acety-lation or oxidation by macrophages invitro29. These receptors internalize the lipoproteins and, in the late endolysosomal compartment, the cholesteryl esters of the lipoproteins are hydrolysed to free cholesterol and fatty acids. Free cholesterol in the endolysosomal compartment is then trafficked to the endoplasmic reticulum (ER), where it undergoes re-esterification by acetyl-coenzymeA:cholesterol acetyltransferase 1 (ACAT1) to cholesteryl fatty acid esters that provide the foam of the foam cells32.

Combined deficiency of SR-A1 and CD36 reduced foam cell formation in Apoe/ mice; however, this effect was incomplete, which suggests that there are addi-tional mechanisms of macrophage cholesterol uptake invivo33,34. Despite this redundancy in cholesterol uptake mechanisms, plaques in mice that are deficient in both CD36 and APOE (Cd36/Apoe/ mice) and in mice that are deficient in SR-A1, CD36 and APOE

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ER stress

Apoptosis

Cytokines andchemokines

NLRP3inammasomeactivation


Lipid eux

Pro-inammatorysignalling

Lipoprotein uptake

Macro-pinocytosis

Phagocytosisaggregated LDL

SR-A1 LOX1LDL SR-B1CD36 Oxidized LDL

Cholesterol-rich lipid raft

VLDL

ABCA1

ABCG1

Nascent HDL

Lipid-poorAPOA1

Mature HDL

Acid lipolysis

Lipophagy

Phagophore

AutophagosomeLipolysis

Lipiddroplets

NCEH1

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LXRRXR

ER

Nucleus

Lysosome

Endosome

Freecholesterol

Cholesterolcrystals

Lysosomaldysfunction

CD36

TLR4TLR6

TLR4

NF-B

(Msr/Cd36/Apoe/ mice) have reduced signs of inflammation, macrophage apoptosis and secondary necrosis, which suggests that these scavenger receptors have roles beyond lipid uptake33,34. Nevertheless, the invivo relevance of oxidative processes in atheroscle-rosis remains speculative. Several well-powered human clinical trials of antioxidant vitamins, such as vitaminE and vitaminC, have failed to show a reduction of cardio-vascular events35, which encourages the field to consider alternative mechanisms for foam cell formation.

Modification by various proteases and lipases that are present in the intima can also mediate LDL modi-fications, particularly the aggregation of LDL. The extracellular matrix glycoproteins contribute to this process by retaining the lipoproteins and by modulat-ing the activity of various enzymes, including group IIA secretory phospholipase A2 (PLA2G2A), PLA2G5 and PLA2G10, as well as secretory sphingomyelin27. These lipolytic enzymes produce modified forms of LDL that are taken up via pathways that are independent of

Figure 2 | Mechanisms controlling macrophage lipoprotein uptake and efflux. Macrophages internalize native low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) as well as oxidized lipoproteins in the plaque via macropinocytosis, phagocytosis of aggregated LDL and scavenger receptor-mediated uptake (including by scavenger receptor A1 (SR-A1), lectin-like oxidized LDL receptor 1 (LOX1), SR-B1 and CD36). The internalized lipoproteins and their associated lipids are digested in the lysosome, which results in the release of free cholesterol that can travel to the plasma membrane and be effluxed from the cell or to the endoplasmic reticulum (ER) membrane. In the ER, it can then be esterified by acetyl-coenzyme A:cholesterol acetyltransferase 1 (ACAT1) and is ultimately stored in this form in cytosolic lipid droplets. These stored lipids can be mobilized for efflux either via lipolysis by neutral cholesterol ester hydrolase 1 (NCEH1) or via lipophagy, which is a form of autophagy, resulting in the delivery of lipid droplets to lysosomes. The accumulation of cellular cholesterol activates the liver X receptor (LXR)retinoid X receptor (RXR) heterodimeric transcription factor that upregulates expression of the ATP-binding cassette subfamily A member 1 (ABCA1) and ABCG1. This mediates the transfer of free cholesterol to lipid-poor apolipoprotein A1 (APOA1) to form nascent high-density lipoprotein (HDL) or more lipidated HDL particles in which free cholesterol has been esterified and stored in the core of the particle (known as mature HDL). Excessive free cholesterol accumulation can induce cholesterol crystal formation in the lysosome to activate the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome, and may also interfere with the function of the ER (inducing ER stress), which, if prolonged, results in cell death by apoptosis. In addition, lipid rafts are enriched in sphingomyelin, which forms a complex with the free cholesterol. As the cholesterol content of lipid rafts increases, pro-inflammatory Toll-like receptor 4 (TLR4) signalling is promoted, which can also be induced by oxidized LDL, through a heterotrimeric complex composed of CD36TLR4TLR6. This signalling results in the activation of nuclear factor-B (NF-B) and in the production of pro-inflammatory cytokines and chemokines.

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PinocytosisAlso known as fluid-phase endocytosis. A process of engulfment of extracellular fluid and its solutes. It can be mediated by an actin-dependent mechanism that results in the engulfment of large volumes (macropinocytosis) or by other mechanisms that result in the engulfment of smaller volumes (micropinocytosis).

EfferocytosisThe process of macrophage clearance of apoptotic cells.

ATP-binding cassette subfamily A member 1(ABCA1). A member of a superfamily of proteins that transport various molecules across extracellular and intracellular membranes using the energy of ATP hydrolysis. Eukaryotic ABC genes are classified in seven families, from ABCA to ABCG, on the basis of gene organization and primary sequence homology. Functional characterization can be partly made by differential sensitivity to inhibitory drugs.

AutophagyAn evolutionarily conserved process in which acidic double-membrane vacuoles sequester intracellular contents (such as damaged organelles and macromolecules) and target them for degradation, through fusion to secondary lysosomes.

NLRP3 inflammasomeA molecular complex containing NLRP3 (NOD-, LRR- and pyrin domain-containing 3) and the adaptor molecule ASC that controls the activity of caspase 1. Formation of this complex results in the cleavage of the highly pro-inflammatory cytokines pro-interleukin-1 (IL-1) and pro-IL-18, thereby producing active IL-1 and IL-18.

scavenger receptors36. Evidence from mouse models sup-ports a role for PLA2 family members in atherosclerosis progression37, and circulating PLA2 levels in humans correlate with coronary artery disease risk38,39, which identifies it as a promising therapeutic target, although further validation is required.

Finally, although a role for native LDL in foam cell formation was initially discounted, recent studies have shown that, in the arterial intima, LDL under-goes pinocytosis by macrophages when it is at concen-trations similar to those that occur in hyperlipidemic conditions, which results in foam cell formation40. This receptor-independent endocytic pathway also delivers cholesterol to the endolysosomal compartment and stimulates cholesterol esterification. Thus, rather than the originally envisioned single modification model in which LDL and other APOB-containing lipoproteins would be rendered atherogenic it is probable that multiple, simultaneous pathways contribute to foam cell formation invivo.

Defective cholesterol trafficking. Macrophage choles-terol metabolism can become overwhelmed during excessive cholesterol uptake, which results in patho-logical processes. When stored in the cell as cholesteryl ester, cholesterol is fairly inert; however, free cholesterol can be toxic to cells. Enrichment of ER membranes with free cholesterol can result in defective cholesterol esterification by ACAT1 in macrophages, which pro-motes the further accumulation of free cholesterol. In addition, free cholesterol enrichment of cell membranes can enhance inflammatory signalling from lipid rafts, particularly Toll-like receptor (TLR) signalling and acti-vation of nuclear factor-B (NF-B)4143. Furthermore, trafficking of free cholesterol out of lysosomes may also become defective in these macrophages, which consti-tutes a barrier to cholesterol efflux and further amplifies inflammation44. Such dysregulation in lipid metabolism contributes to ER stress in macrophages, which, if pro-longed and combined with other insults, can ultimately result in apoptotic cell death45. Efficient clearance of apoptotic cells by surrounding macrophages (the process of efferocytosis) requires intact lipid metabo-lism pathways (such as cholesterol esterification and efflux) in the engulfing cell to deal with the ingested lipids from the apoptotic bodies. Thus, as macrophage lipid metabolism becomes dysregulated, the increase in macro phage apoptosis combined with defective effero-cytosis results in secondary necrosis and in the release of cellular components and lipids that form the necrotic core46. This feature of advanced atherosclerotic plaques, along with the thinning of the fibrous cap, may increase the vulnerability of plaques to rupture.

Lipid efflux. Cells respond to excessive lipid accumula-tion by increasing the expression of pathways that pro-mote the removal of cholesterol and other lipids from the cell. In foam cells several macrophage transporters facilitate the efflux of lipids including ATP-binding cassette subfamily A member1 (ABCA1), ABCG1 and SR-B1 (FIG.2) although passive diffusion from the

plasma membrane also occurs47. ABCA1 promotes cho-lesterol efflux to lipid-poor APOA1, which is the building block of HDL, whereas ABCG1 promotes efflux to mature HDL particles. The genes encoding ABCA1 and ABCG1 are transcriptionally upregulated in response to elevated cellular cholesterol levels by liver X receptors (LXRs), which are ligand-activated nuclear receptors that function as sterol sensors; for example, LXR activation by choles-terol derivatives (such as oxysterols) or by desmosterol (which is a molecule similar to cholesterol)48, promotes macrophage cholesterol efflux via ABCA1 and ABCG1 and also has anti-inflammatory effects49. Thus, synthetic LXR agonists have been actively investigated for the treatment of atherosclerosis.

In addition, autophagy has a crucial role in mac-rophage cholesterol efflux: lipid droplets in macrophages and other cell types are targeted to and hydrolysed by the autophagy machinery in a process known as lipophagy50. Fusion of the autophagosome with the lysosome degrades cholesteryl esters and makes free and modified cholesterol available for efflux through an ABCA1-dependent pathway51 (FIG.2). The protective role of autophagy has been shown in studies in Apoe/ mice in which the deletion of components of the autophagy machinery enhanced atherosclerosis52,53. Furthermore, autophagy regulates innate and adaptive immune responses (discussed below), including inflammasome activation, antigen presentation and Tcell activation5355. Thus, pathways that stimulate the efflux of cholesterol from the macrophage have two atheroprotective func-tions: they promote lipid homeostasis and they protect against inflammation.

Innate immune activationEvidence supports the idea that innate immune activa-tion is a central process in the pathogenesis of atheroscle-rosis. As reviewed above, dysregulated lipid metabolism contributes to the development of foam cells. Such aber-rations and the resulting endogenous danger ligands that accumulate in atherosclerotic plaques trigger PRRs that are expressed by macrophages, including NOD-like receptors (NLRs), scavenger receptors and TLRs, thereby activating the inflammatory response.

NLRs and inflammasome activation. Cholesterol crys-tals are present in atherosclerotic plaques and are found in both extracellular spaces and within plaque macro-phages. Although previously thought to be a feature of advanced plaques, a recent study using combined confocal-reflection microscopy showed the presence of cholesterol crystals in early lesions in Apoe/ mice56,57 and showed that macrophage engulfment of choles-terol crystals induces the NLRP3 inflammasome (FIG.2). Uptake of pre-formed crystals by human and mouse macrophages induces lysosomal destabilization as well as the release of proteases and/or reactive oxygen species into the cytosol that activate NLRP3 (NOD-, LRR- and pyrin domain-containing 3), which leads to the pro-cessing and secretion of the cytokine IL-156,5861. The potential importance of this pathway in atherogenesis was shown using LDL receptor (Ldlr)/ mice, in which

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M1 macrophagesMacrophages that are activated by Toll-like receptor ligands (such as lipopolysaccharide) and interferon- and that express, among others, inducible nitric oxide synthase and nitric oxide.

M2 macrophagesMacrophages that are stimulated by interleukin-4 (IL-4) or IL-13 and that express arginase 1, the mannose receptor 1 (also known as CD206) and the IL-4 receptor -chain.

transplantation with bone marrow cells deficient in IL-1 or in components of the NLRP3 inflammasome complex led to reduced plaque formation56. A subse-quent study using Apoe/ mice with somatic deficiency of Nlrp3 failed to show protection from atherosclerosis62. Although the reasons for this discrepancy will need to be investigated, a potential confounding factor may have been the different cholesterol contents of the Western diet used in the two studies (0.3% versus 1.25%).

In addition to pre-formed cholesterol crystals, recent work indicates that loading of macrophages with cho-lesterol can lead to the denovo formation of intracel-lular cholesterol crystals that trigger NLRP3 (REF.63). CD36 has a crucial role in the accrual and the nuclea-tion of cholesterol crystals within macrophages that have been treated with oxidized LDL, as well as in the ensuing lysosomal disruption and NLRP3 inflamma-some activation63. Consequently, macrophages lacking CD36 failed to induce IL-1 production in response to oxidized LDL, and targeting CD36 in atherosclerotic mice decreased serum IL-1 levels and plaque choles-terol crystal accumulation. Notably, the CD36-mediated uptake of amyloid-forming peptides that are implicated in Alzheimers disease and type2 diabetes also activates NLRP3. This suggests that there is a common pathway of lysosomal-mediated NLRP3 activation that occurs in the cell after the new aggregation and transformation of these soluble ligands into their pathogenic forms63. Although not yet investigated, other crystalline or amyloid substances in atherosclerotic plaques, such as calcium phosphate crystals or serum amyloid A64, may also represent damage-associated molecular patterns (DAMPs) that could trigger the inflammasome and IL-1 secretion.

TLR signalling. The participation of TLR signalling pathways in the promotion of atherosclerosis is sup-ported by mouse studies in which the whole body deletion of Tlr2 orTlr4 (REFS6568) or of the adaptor proteins used by these TLRs, including IL-1 recep-tor-associated kinase 4 (IRAK4)69,70, TNF receptor-associated factor 6 (TRAF6)71, TIR-domain-containing adaptor protein inducing IFN (TRIF; also known as TICAM1)72 and myeloid differentiation primary-response protein 88 (MYD88)65,73, confers protection from atherosclerosis. This finding has initiated inves-tigations of the endogenous ligands that accumulate during hypercholesterolaemia and in plaques that may trigger these microbial-sensing pathways in mac-rophages. Among the candidates proposed, oxidized LDL species have been extensively studied as ligands for both the scavenger receptors and the TLRs, and the extent of oxidation influences their recognition by these receptors (FIG.2); for example, minimally oxidized LDL is recognized by CD14TLR4MD2 and initiates cytoskeletal rearrangements, as well as tumour necrosis factor (TNF), IL-6 and IL-10 production74. Moderately oxidized LDL that is recognized by CD36 signals via a heterodimer of TLR4 and TLR6, which results in NF-B activation and in the expression of chemokines that promote monocyte recruitment to atherosclerotic

lesions67. Finally, oxidized phospholipids and saturated fatty acids induce cooperative signalling of CD36 and TLR2 that promotes apoptosis in macrophages under-going prolonged ER stress75. However, in addition to these ligand-initiated signalling pathways, the enrich-ment of macrophage plasma membranes with free cholesterol can also lead to the sustained activation of various TLRs, including TLR3 and TLR4 (REFS43,76). Thus, numerous pathways may contribute to the initia-tion and the maintenance of TLR-induced macrophage inflammation in atherosclerotic plaques.

Macrophage polarization and plasticityOne consequence of the TLR-dependent activation of monocyte-derived cells entering the plaque might be their polarization to M1 macrophages. These inflamma-tory macrophages secrete pro-atherosclerotic cytokines (such as IL-6 and IL-12), as well as reactive oxygen and nitrogen species that would exacerbate oxidative stress in the plaque77 (BOX1). Histological analysis of human plaques showed M1 macrophages to be enriched in lipids and localized to areas that are distinct from those in which the less inflammatory M2 macrophages (also known as alternatively activated macrophages) are localized78. Studies of M1 and M2 macrophages that have been polarized invitro and in mouse mod-els of atherosclerosis have led to a simplified view that M1macrophages promote plaque inflammation and M2 macrophages resolve plaque inflammation. However, the phenotypic range of macrophages invivo is likely to be complex, as macrophages encounter a microenvironment of diverse, and even opposing, sig-nals; for example, in addition to inducing TLR signal-ling that can lead to M1 polarization, oxidized LDL has also been reported to induce the expression of the M2 macrophage phenotypic marker arginase 1 via the acti-vation of peroxisome proliferator activated receptor- (PPAR)79. In addition, oxidized phospholipids present in oxidized LDL induce a macrophage phenotype that is distinct from M1 or M2 macrophage phenotypes and that has been termed Mox; these macrophages are characterized by the increased expression of nuclear factor erythroid 2-related factor 2 (NRF2; also known as NFE2L2)-dependent genes and reactive oxygen spe-cies80. It is probable that T helper 1 (TH1) and TH2 cells in plaques secrete macrophage-polarizing factors81 that also contribute to the balance of M1 and M2 macro-phages. Nonetheless, the factors in the plaque micro-environment that promote the polarization of these cells invivo remain incompletely defined.

The recent identification of transcriptional pro-grammes that regulate macrophage polarization has provided some insights into the effects of M1 and M2 macrophages on atherogenesis. Whole body or bone marrow-specific deletion of the transcription factor NR4A1 (also known as NUR77), which has been sug-gested to control the LY6Clow patrolling monocyte phe-notype and to favour M2 macrophage differentiation82, resulted in increased polarization of macrophages to an M1 macrophage phenotype and an acceleration of atherosclerosis in Apoe/ and Ldlr/ mice83,84, although

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this result has been inconsistent85. Similarly, the targeted deletion of the transcription factor Krppel-like factor4 (KLF4), which promotes M2 macrophage polarization and inhibits M1 macrophage polarization86, enhanced both pro-inflammatory M1 macrophage activation and foam cell formation, and accelerated atherosclerosis in Apoe/ mice87. Notably, the expression of KLF4 in macro phages is reduced by pro-inflammatory cytokines and oxidized phospholipids found in plaques87, which suggests that the KLF4-driven M2 macrophage phe-notype may be repressed during atherogenesis and that this contributes to disease progression when such signals predominate. Indeed, the administration of the M2-polarizing cytokine IL-13 to Ldlr/ mice was shown to drive plaque macrophages to M2-like cells and to inhibit atherosclerosis progression88. Moreover, an enrichment of M2 macrophages has been shown to occur in plaques in which the regression of atheroscle-rosis in mice (TABLE1) is induced by aggressive lowering of lipids or raising of HDL levels4,5,89 (discussed further below). Collectively, these studies suggest that path-ways that promote the M2 polarization of macrophages protect against atherosclerosis.

The origin of M1 and M2 macrophages in plaques remains an area of debate. Although it has been sug-gested that LY6Chi monocytes are the precursors of M1 macrophages, studies using Apoe/ mice have shown that M2 macrophages populate early lesions (also known as fatty streaks), which are present at the stage in which LY6Chi monocytes are thought to be the predominant monocyte subset recruited into plaques. However, as plaques progress to more complex inflammatory lesions, the M1 macrophage phenotype becomes more frequent90. Further studies are needed to address the origins of M1 and M2 mac-rophages in atherosclerosis, particularly whether the recruitment of LY6Clow monocytes thought to pref-erentially become M2 macrophages predominate in the earliest lesions, whether there is interconversion

between M1 and M2 macrophage phenotypes invivo, or whether M2 macrophages are derived from the proliferation of a small population of tissue-resident M2 macrophages, as has recently described in other disease models91,92. A better understanding of the regulation of macrophage polarization is likely to offer insights into pathways that could be used for the potential manipulation of macrophage behaviour towards an atheroprotectivestate.

Plaque macrophage retention and emigrationThe number of macrophages in a plaque is kinetically determined by monocyte recruitment and local prolifera-tion, and is counterbalanced by the emigration and death of macrophages. The factors that determine macrophage recruitment to plaques were discussed above. With regard to the local proliferation of monocyte-derived macrophages, this probably occurs in the plaque, as has been suggested by the assessment of proliferation mark-ers in lesional macrophages and DCs93. Nevertheless, the quantitative importance of macrophage prolifera-tion in atherosclerosis progression remains to be deter-mined. Of note, on the basis of a recent report showing a lower percentage of proliferating macrophages in early plaques compared with advanced plaques, it is likely to be variable in different stages of the disease117.

Macrophage emigration has been shown to occur in early atherosclerotic plaques, but the rate of macrophage egress has been reported to decrease with atherosclero-sis progression94 (FIG.3). It is probable that plaque mac-rophages are subject to both retention and emigration signals, and that the balance of these forces contributes to the net accumulation of plaque macrophages. These sig-nals are only beginning to be defined. Cholesterol loading of macrophages has been shown to increase the expres-sion of the neuro-immune guidance cues netrin 1 and semaphorin 3E, which both function to induce macro-phage chemostasis invitro25,26. Macrophage expression of these migration-inhibitory molecules is also induced

Table 1 | Selected mouse models of atherosclerosis progression and regression

Mouse model Important Features Lipoprotein profile Refs

Progression

Apoe/ mice Spontaneous development of complex plaques when mice are fed on a chow diet; and acceleration of plaque formation when mice are fed on a Western diet

Intestinally derived remnant lipoprotein particles

114,115

Ldlr/ mice Development of plaques following feeding mice a cholesterol and fat-enriched diet; and lipoprotein profile similar to that of humans

VLDL and LDL 116

Regression

Aortic transplant mice Rapid regression of atherosclerosis; but requires surgical procedure, for example, the transplantation of aortas from Apoe/ mice to wild-type mice

Lipid levels revert to the levels in wild-type mice

103

Reversa mice An Ldlr/ mouse-based platform that shows inducible reversal of hyperlipidaemia after conditional inactivation of Mttp

Lipid levels revert to nearly the levels that are observed in wild-type mice

4

Reconstitution of Apoe/ mice with APOE

The inducible regression of atherosclerosis by adenoviral delivery of Apoe to the liver or by correcting a hypomorphic allele of the Apoe gene

Lipid levels revert to nearly the levels that are observed in wild-type mice

100,102

Apoe, apolipoprotein E; Ldlr, low-density lipoprotein receptor; Mttp, microsomal triglyceride transfer protein large subunit; VLDL, very low-density lipoprotein.

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Circulatingmonocyte

Collagen

Lumen

Intima

Media

Necrotic core

Progressing plaque

Chemokinegradient

Foam cell

Macrophage

AdhesionReversetransmigration

Chemostasis

UNC5B

To adventitial lymphatics

CCR7ABCA1

Regressing plaque

SR-A1

OxidizedLDL

CD36

LOX1

Migration

Proliferation

Lipidunloading

LDL

Netrin 1Semaphorin 3ECadherins

ER stress

Apoptosis Defective

eerocytosis

Macrophage emigration

Eective eerocytosis

during hypoxia, which is intimately linked to athero-sclerosis26,95; this occurs in mice96 and has become rec-ognized as a primary trigger of plaque inflammation. Studies using Ldlr/ mice with a bone marrow deficiency of netrin 1 showed that they had reduced atherosclero-sis progression and increased macrophage emigration from lesions, which suggests that netrin 1 may function to retain macrophages in plaques25. Similar experiments using mice that lack semaphorin 3E in macrophages will be needed to extend these findings and are in progress. Other factors that inhibit cell movement (such as adhe-sion molecules97) or the resolution of inflammation are also likely to contribute to the retention of macrophages in the plaque, and studies comparing mouse models of atherosclerosis progression and regression are beginning to uncover these signals (see below).

The signals that guide macrophages to exit plaques, either by reverse transmigration through the endothelium to the lumen or by migrating through the media to the adventitial lymphatics, remain poorly defined. In stud-ies in which macrophage emigration from plaques was induced by normalizing the hyperlipidemic plasma profile of mice in an aortic transplant model, the cells that emi-grated expressed several markers that are characteristic of both macrophages and DCs98; for example, the expression of CCR7, which is the receptor for the chemokines CCL19 and CCL21 that regulate DC homing to the lymph nodes, was upregulated in the emigrating CD68 (also known as macrosialin)-expressing cells. Furthermore, blocking this pathway led to substantial retention of these cells in the plaque98. Further studies are needed to define other fac-tors in this and other models of atherosclerosis regression.

Figure 3 | Pathways regulating macrophage retention and emigration in plaques. Imbalances in macrophage lipid metabolism in the progressing plaque lead to the retention of macrophages and to chronic inflammation. The accumulating lipid-laden macrophages express retention molecules (such as netrin 1 and its receptor UNC5B, semaphorin3E and cadherins) that promote macrophage chemostasis. In this inflammatory milieu, these accumulating macrophages experience endoplasmic reticulum (ER) stress, which, if prolonged, results in apoptosis. This cell death, coupled with defective efferocytosis, results in the formation of the necrotic core that is characteristic of advanced plaques. The mechanisms that promote lipid unloading of the foam cell, including the factors that upregulate ATP-binding cassette subfamily A member 1 (ABCA1) expression on plaque macrophages and cholesterol efflux, reverse the accumulation of these foam cells. This plaque regression is characterized by an upregulation of CC-chemokine receptor 7 (CCR7) on myeloid-derived cells and a decrease in the expression of retention factors. The accumulating evidence summarized in this Review supports the idea that the regulation of these macrophage migration factors contributes to macrophage emigration from the plaque through reverse transmigration to the lumen or through trafficking to the adventitial lymphatics. LDL, low-density lipoprotein; LOX1, lectin-like oxidized LDL receptor 1; SR-A1, scavenger receptor A1.

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In addition, the continued presence of macrophage foam cells in the inflammatory lipid-rich environ-ment of the plaque can eventually lead to cytotoxic-ity from ER and oxidative stress1. Activation of ER stress responses occurs as a result of free cholesterol accumulation in macrophages and by saturated fatty acid signalling via SR-A1, TLR2 and TLR4 (REF.75). Prolonged ER stress leads to macrophage apoptosis, which is observed in 2 to 4% of cells in mouse plaques, with the highest levels in advanced plaques. In these late-stage plaques, the ability of macrophages to clear their dying counterparts through such receptors as tyrosine protein kinase MER (MERTK) and LDLR-related protein 1 (LRP1) becomes compromised, and this has been partly attributed to cholesterol accumula-tion in the engulfing cells99. This defective efferocytosis contributes to secondary necrosis and to the forma-tion and expansion of the lipid cores, which, in turn, contribute to plaque vulnerability and to rupture46. Therefore, it is possible that apoptosis, especially in the context of efficient efferocytosis, also contributes to net changes in macrophage or foam cell content, as has been suggested in a recent study of atherosclero-sis regression100; however, a mathematical analysis of those data suggest that a rate tenfold higher than usual would be required for the changes observed (S. Russell and E.A.F., unpublished observations). In summary, monocyte recruitment and cell retention, emigration and death are all potential kinetic contributors to the net plaque contents of macrophages and foam cells. The quantitative effect of each of these processes will probably vary during the different stages of the disease and in different models of progression and regression, as well as in co-morbid states, such as insulin resistance or diabetes, and chronic kidney disease.

Lessons from models of plaque regressionThe historical focus on atherosclerosis in both human and animal studies has been on its progression, with the prevailing view that, except for early lesions which are dominated by foam cells, atherosclerosis was essentially irreversible, although the mechanisms by which even an immature plaque regressed remained undefined. More recent discoveries, including finding that mac-rophages can emigrate from plaques in some animal models and that tissue-remodelling M2 macrophages are present in human and animal plaques, suggest that there is cause for optimism that clinical atherosclero-sis regression could be achieved. Nevertheless, under-standing the biology of atherosclerosis regression, and the discovery of therapeutic targets to achieve it, requires robust preclinical models. Therefore, several mouse models of atherosclerosis, such as Apoe/ mice and Ldlr/ mice, have been adapted for studies of ath-erosclerosis regression (TABLE 1). Common to all mod-els has been the finding that in the regressing plaque there is a decline in the number of macrophages and, in some, a change in their phenotypic characteristics, with an enrichment of M2 macrophage characteris-tics46,89,101104, which suggests that this is a common signature of regressing plaques.

Transcriptomic profiling of macrophages that have been isolated by laser capture microdissection98 of pro-gressing and regressing plaques in an aortic transplanta-tion mouse model showed there to be >700 differentially regulated genes97, including the recently described mac-rophage retention factors semaphorin 3E and netrin1. Other genes that are downregulated in macrophages in regressing plaques include adhesion molecules, such as members of the cadherin family97. By contrast, cellular motility factors were upregulated. In addition, CCR7 was expressed at low levels in plaque macrophages and was probably suppressed by hypercholesterolaemia as a result of a serum-response element in its promoter105. Notably, the transcription of Ccr7 was upregulated in macrophages when plaques were placed in a regression environment, thereby increasing the migratory capacity of the cells. Taken together, the transcriptomic data from the aortic transplantation model indicate that the emigra-tion of macrophages from plaques is a highly regulated process, and reflect coordinated changes in macrophage retention and movement. Transcriptome analyses from other models of atherosclerosis regression will be needed to determine how conserved these changesare.

Therapeutic targeting of plaque macrophagesTherapies that alter macrophage content by reducing macrophage recruitment to atherosclerotic plaques or by promoting macrophage apoptosis, efferocytosis or emi-gration have been proposed to have beneficial effects on disease. However, the quantitative effect of each of these processes on disease progression probably depends on the stage of disease; for example, macrophage recruitment dominates compared with emigration in disease progres-sion, whereas macrophage emigration is increased in sev-eral models of atherosclerosis regression. Furthermore, the low level of macrophage apoptosis that is seen in early atherosclerosis (typically ~24% of cells) increases as plaques become more complex, with secondary necrosis also becoming prominent as the efferocytosis of apoptotic cells falters1. In addition, as seen in models of progression and regression5,90,97, the inflammatory phenotype of the macrophages (using the simplified scheme of M1 versus M2 macrophages) is not constant, which probably reflects the well-known plasticity of monocyte-derived cells in response to microenvironmental changes. Therapies that alter macrophage inflammation by increasing polarization to an M2 macrophage phenotype, by increasing efferocyto-sis or by increasing macrophage emigration would be pre-dicted to be beneficial on the basis of preclinicalmodels.

The fact that new clinical targets are needed is obvious from the failure of conventional risk factor management to effectively eliminate the risk of cardiovascular disease, with more than half of patients in controlled clinical trials having heart attacks or strokes despite aggressive treat-ments. A recent example of the discovery of a potential target from mouse studies is our finding that neuronal guidance molecules function as macrophage retention factors in plaque progression and that their expression in macrophages decreases in regressing plaques25,26,97. Thus, it may be desirable to selectively deliver small interfering RNAs (siRNAs), or other therapeutics directed against

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MicroRNAA single-stranded RNA molecule of approximately 2123 nucleotides in length that regulates the expression of other genes.

these and other factors that facilitate the emigration of macrophages. There is considerable optimism that it is possible to specifically target agents to m

nature reviews immunology - october 2013

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