Alveolar macrophages: plasticity in a tissue-specific context.

Nature Reviews Immunology | AOP, published online 21 January 2014; doi:10.1038/nri3600

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Alveolar macrophages: plasticity in a tissue-specific context Tracy Hussell and Thomas J. Bell

Abstract | Alveolar macrophages exist in a unique microenvironment and, despite historical evidence showing that they are in close contact with the respiratory epithelium, have until recently been investigated in isolation. The microenvironment of the airway lumen has a considerable influence on many aspects of alveolar macrophage phenotype, function and turnover. As the lungs adapt to environmental challenges, so too do alveolar macrophages adapt to accommodate the ever-changing needs of the tissue. In this Review, we discuss the unique characteristics of alveolar macrophages, the mechanisms that drive their adaptation and the direct and indirect influences of epithelial cells on them. We also highlight how airway luminal macrophages function as sentinels of a healthy state and how they do not respond in a pro-inflammatory manner to antigens that do not disrupt lung structure. The unique tissue location and function of alveolar macrophages distinguish them from other macrophage populations and suggest that it is important to classify macrophages according to the site that they occupy.

Manchester Collaborative Centre for Inflammation Research, University of Manchester, 2nd floor, Core Technology Facility, Oxford Road, Manchester M13 9PT, UK. Correspondence to T.H. e-mail: tracy.hussell@ manchester.ac.uk doi:10.1038/nri3600 Published online 21 January 2014

Macrophage function needs to be tailored to the requirements of the tissue in which the cells reside — an adaptation that is driven by tissue-derived factors and by the physiological environment. As such, Kupffer cells in the liver, osteoclasts in the bone and alveolar macro phages have very different roles and can be pheno typically differentiated from one another 1,2. Tissue macrophage diversity has led researchers to question the origins of these cells and the factors that promote their maintenance. Bone marrow haematopoietic stem cells (HSCs) give rise to circulating monocytes, which can differentiate in tissues into macrophages. However, a recent study showed that the mouse embryo yolk sac is a sufficient source of specific macrophage subtypes in the liver, skin and central nervous system (CNS) in the absence of HSCs3. Furthermore, initial colonization of the airways with alveolar macrophages occurs in the first few days after birth — a process that is wholly dependent on fetal monocytes4. In addition, models of transplantation5, radiation chimaeras6, parabiosis4 and strontium-mediated depletion of blood monocytes7 have shown that alveolar macrophages have a marked capacity for self-renewal and that this is the main means by which these cells are replenished throughout life8,9. The alveolar macrophage pool is at least partially depleted during influenza infection8,10; however, in situ proliferation of the remaining alveolar macrophages

seems to be capable of replenishing the population8. Only in the case of radiation-induced depletion of alveolar macrophages, when any remaining cells have a reduced capacity for proliferation, do HSC-derived circulating monocytes eventually contribute to alveolar macrophage repopulation4,8. In this Review, we describe the specialized micro environment of the airspaces and explain how this creates a unique and flexible population of alveolar macrophages. Furthermore, we contrast the functional and phenotypical properties of alveolar macrophages with other tissue-resident macrophages and discuss how the unique features of alveolar macrophages enable them to avoid mounting excessive and potentially pathological immune responses in the lungs.

The specific environment of the airspaces Alveolar macrophages are distinct from the macrophages that reside between the airway epithelium and the blood vessels, which implies that further specialization of macrophage populations occurs in the lungs. Alveolar macrophages reside in a tissue compartment that shows marked environmental fluctuations; for example, the partial O2 pressure (PO2) in healthy alveoli is 100–110 millimetres of mercury (mm Hg), but this alters with exposure to environmental antigens, acute infections and during chronic inflammatory diseases, such as cystic

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M1 macrophages M1, or classically activated, macrophages are induced by Toll-like receptor signalling and interferon‑γ. They have enhanced antimicrobial properties and secrete pro-inflammatory cytokines such as interleukin‑1 (IL‑1), IL‑6, IL‑12, IL‑23 and tumour necrosis factor (TNF). They can also express CXC-chemokine ligand 9 (CXCL9), CXCL10, CXCL11 and CC-chemokine ligand 5 (CCL5).

M2 macrophages M2, or alternatively activated, macrophages are generally induced by interleukin‑4 (IL‑4) and IL‑13, although various M2‑like subtypes have been described. These cells contribute to wound healing, are anti-inflammatory and typically express the mannose receptor (also known as CD206), the tyrosine protein kinase MER, growth arrest-specific protein 7 (GAS7), CD163, arginase and tumour necrosis factor-β (TGFβ).

fibrosis, in which localized bronchial mucosa PO2 levels as low as 2.5 mm Hg are observed11,12. Hypoxia is a fairly common occurrence, even in healthy airways, and can have marked effects on airway immune responses, as tissue hypoxia leads to the induction of hypoxia-inducible factor 12, nuclear factor-κB (NF‑κB)13 and cyclic AMP-responsive element-binding protein (CREB)14. The lower airways in mice and humans also contain a microbial flora that fluctuates with changes to the lung microenvironment15,16, the airway mucus (that functions as a physical and a biological barrier, but that also contains immune modulators) and a considerable number of epithelial cells that have been shown to modulate alveolar macrophage activity through both direct 17,18 and indirect 19 mechanisms (FIG. 1). The vast majority of studies that investigate macrophages in the airways, and the techniques used to collect them, do not distinguish between ‘alveolar’ macrophages from the alveoli and ‘airway’ macrophages found in the larger airspaces, and it is not clear whether these are in fact distinct populations. In this Review, we refer to the undefined mixture of macrophages found in the lumen of the healthy airways as alveolar macrophages. In healthy individuals, the airspaces are replete with mechanisms that prevent an inflammatory response from occurring. This in turn affects the function and the phenotype of the alveolar macrophages, which are one of the few cell populations to reside in the healthy airspaces, in addition to a small number of lymphocytes20,21. Alveolar macrophages from mice have been shown to be poor at presenting antigens to T cells22–24, although they are capable of transporting antigens to the lung-draining lymph nodes25. Human alveolar macrophages also induce T cell antigen-specific unresponsiveness as a result of poor antigen presentation and a lack of expression of co‑stimulatory molecules, such as CD86 (REF. 26); this promotes tolerance to innocuous antigens. In addition, alveolar macrophages show decreased phagocytic activity compared with lung interstitial macrophages27 and also have a reduced respiratory burst 28. Furthermore, they produce immunosuppressive prostaglandins and transforming growth factor‑β (TGFβ), which suppress T cell activation29. Alveolar macrophages may drive the development of forkhead box P3 (FOXP3)+ regulatory T cells by secreting TGFβ and retinoic acid30, although recent evidence suggests that tissue-resident macrophages in the lungs can also secrete these molecules31. Although interactions between alveolar macrophages and T cells have mostly been studied in vitro, there are several scenarios in which these interactions could also occur in vivo. First, there is a population of T cells present in the lumen of the healthy airways32 that could be influenced by the resident macrophages, as well as a large number of infiltrating T cells in the inflamed lungs. Second, breakdown of the epithelial barrier during inflammation, for example, as indicated by the presence of serum proteins such as albumin in the airways after influenza infection15, may also facilitate the diffusion of soluble mediators between luminal alveolar macrophages and lung T cells.

Alveolar macrophages are long-lived, with a turnover rate of only approximately 40% in 1 year 33,34. By contrast, substantial turnover of both lung tissue and peritoneal macrophages occurs within a period of 21 days8,34. Studies investigating human alveolar macrophages in situ in healthy tissue are limited or may be confounded by the condition, such as cancer, that initially led to the lung resection (providing the tissue from which they could be studied); for example, alveolar macrophages taken from unaffected tissue in patients with lung cancer seem to be more pro-inflammatory in nature than macrophages in the interstitial tissue35. However, it is unclear how the distal cancer affects cells in unaffected sections of the same tissue. For this reason, it is difficult to validate ideas that have been developed using healthy alveolar macrophages from mice with those available in humans, as they are invariably taken from patients with underlying respiratory conditions.

Alveolar macrophages in health In addition to general tissue-specific influences, macro phages are also conditioned by the requirements of the tissue during normal (and pathological) processes. The roles of macrophages in clearing apoptotic cells and cellular debris in health and disease are equally as important as the participation of these cells in immuno logical responses. However, each function requires plasticity within the resident macrophage population so that pro-inflammatory responses to tissue debris or to innocuous antigens are inhibited, but effective immune responses to pathogenic microorganisms are not compromised. The ability of tissue macrophages to adapt and to carry out such disparate functions led to their broad classification as either classically activated M1 macrophages or alternatively activated M2 macrophages36. Since their initial description, the functional and phenotypical characteristics of macro phages within the M1 phenotype have remained mostly unaltered, but the M2 macrophage category has been expanded to accommodate a broad range of macro phage functions in wound healing and in immune regulation37–39. Studies using healthy alveolar macro phages in mice and humans are few or do not directly address the question and imply that these cells do not neatly fit into any current macrophage classification. Attempts to subdivide alveolar macrophages into subtypes have mostly been done on the basis of experiments that examined these cells during inflammation of the lungs or after the cells were driven to a particular subtype using combinations of recombinant cytokines in vitro. Notably, this has led to the use of a variety of descriptions that encompass a broad range of alveolar macrophage phenotypes. A transcriptional analysis of human alveolar macro phages that were polarized ex vivo using interferon‑γ (IFNγ), or with interleukin‑4 (IL‑4) and IL‑13, highlighted 41 and 33 genes that were associated with M1 macrophages and M2 macrophages, respectively. Genes associated with macrophages that were polarized using IFNγ included those encoding CD69, Tolllike receptor 2 (TLR2), TLR4, CXC-chemokine ligand 9

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Type II alveolar epithelial cell

Type I alveolar epithelial cell Bacterium T cell Blood vessel

Alveolus Alveolar macrophage

b

Large airway (bronchus) Goblet cell

Prostaglandin Activation

Small airway (bronchiole)

TGFβ FOXP3

Alveolar macrophage

Mucus DC Interstitial macrophage

Retinoic acid

T cell

Interstitial space

Clara cell

a Alveolar macrophage CD200R IL-10R

TGFβR

IL-10

αvβ6

CD200

Figure 1 | Leukocyte interactions in the healthy lungs. Alveolar macrophages reside in the airspaces juxtaposed Immunology with type I alveolar epithelial cells (which account for as much as 98% of the total surface Nature area of Reviews the lungs| 157 ) or with type II alveolar epithelial cells . Macrophages found in the larger airways (also referred to in this Review as alveolar macrophages) reside within the mucous layer. Mucus-producing goblet cells are present in both large and small airways, and secretory non-ciliated Clara cells are more common in the bronchioles158. Macrophages are also found in the interstitial space between the alveoli and the blood vessels where T cells, dendritic cells (DCs) and a sparse population of B cells also reside. Commensal (and pathogenic) bacteria reside within the airway mucosa and in the alveoli. a | Alveolar macrophages are regulated by the airway epithelium through their interactions with CD200, which is expressed by type II alveolar cells, with transforming growth factor‑β (TGFβ), which is tethered to the epithelial cell surface by αvβ6 integrin, and with secreted interleukin‑10 (IL‑10). These interactions can also take place in the larger airways, where CD200 and αvβ6 integrin are also expressed by the bronchial epithelium. b | The secretion of TGFβ and retinoic acid by alveolar macrophages can induce forkhead box P3 (FOXP3) expression in both naive and activated CD4+ T cells that are present in the lumen of the airways. In addition, TGFβ and prostaglandins suppress T cell activation. CD200R, CD200 receptor; IL‑10R, IL‑10 receptor; TGFβR, TGFβ receptor.



REVIEWS Table 1 | The specific phenotype of mouse macrophages from different sites Surface marker

Peritoneal macrophage

Interstitial macrophage Alveolar macrophage

Refs

CD11b

Intermediate expression


Not expressed

45,47

CD11c

Not expressed

Not expressed

High expression

45,47

CD14



Low expression

50,159

CD200R

Low expression*


High expression

17

DEC205

Not expressed

Expression unknown


F4/80


Low expression

Low expression

50,160

Mannose receptor (also known as CD206)

Low expression


High expression

50,161

MHC class II



Low expression

47,50

SIGLEC-F

Not expressed

Not expressed

High expression

50,162

47

CD200R, CD200 receptor; SIGLEC-F, sialic acid-binding immunoglobulin-like lectin F. *Expression shown on splenic macrophages.

(CXCL9), CXCL10, CXCL11 and CC-chemokine ligand 5 (CCL5), whereas genes associated with alveolar macrophages that were polarized using IL‑4 and IL‑13 included those encoding the mannose receptor (also known as CD206), matrix metalloproteinase 2 (MMP2), MMP7, MMP9, the tyrosine protein kinase MER, growth arrest-specific protein 7 (GAS7), CD163, stabilin 1 (STAB1), arginase and the adenosine A3 receptor 40. In humans, there is no general consensus about whether alveolar macrophages in the healthy state are M1- or M2‑like in nature. Only a minor population of IL‑13‑producing M2‑like macrophages are observed in the airspaces and, although this population increases as a result of exposure to cigarette smoke, this would imply that macrophages in the healthy lungs predominantly have an M1‑like phenotype40,41. In contrast to this observation, other studies have shown that between 8% and 20% of macrophages in human bronchoalveolar lavage fluid express CD206 or STAB1 (which are associated with M2 macrophages)42 or that 50% of human alveolar macrophages are CD206+ and, therefore, presumably they are M2 macrophages43. These conflicting reports highlight the fact that alveolar macrophages in healthy individuals do not neatly fit into either a strict M1 or M2 classification, and it may not be the case that a resting tissue-resident macrophage would necessarily be driven to become one or the other cell subtype. An increase in M2 macrophage characteristics in alveolar macrophages seems to be a feature of many inflammatory lung diseases in both humans and rodents; for example, one study showed that when alveolar macro phages were subdivided into M1 macrophages (according to their expression of IFN-regulatory factor 5 (IRF5)), M2 macrophages (according to their expression of YM1 (also known as chitinase 3‑like protein 3)) or M2‑like macrophages (according to their expression of IL‑10), M2 macrophage numbers were shown to be increased in the allergen-exposed mouse lungs in a manner that was dependent on the allergen dose44. In mice, alveolar macrophages are easily distinguished from interstitial macrophages by their unusual phenotype (TABLE 1), which to a certain extent reflects their unique function. Alveolar macrophages express

low levels of CD11b, but have high levels of expression of CD11c (also known as integrin α X)45, which combines with CD18 to form an integrin that mediates phagocytosis of inactivated complement component C3b (iC3b)‑opsonized particles46. Interestingly, CD11c is also expressed by macrophages in the gut mucosa but is not expressed by non-mucosal macrophages. Alveolar macrophages also express DEC205 (also known as LY75 and CD205)47, which, similarly to CD11c, is usually expressed at different levels on dendritic cells (DCs), B cells, T cells and thymic epithelial cells48 and is reported to recognize and to facilitate the uptake of the TLR9 agonist CpG49. We are unaware of any reports of DEC205 expression on macrophages from other sites. This unique phenotype is not simply a misinterpretation of cell origin by flow cytometry, as bone marrow-derived or peritoneal macrophages that are intranasally transferred to other recipients upregulate CD11c expression in the airspaces47. Mouse alveolar macrophages also express sialic acidbinding immunoglobulin-like lectin F (SIGLEC-F)50 — the functional analogue of human SIGLEC‑8 — which is predominantly expressed by eosinophils. Crosslinking of SIGLEC-F induces eosinophil apoptosis51,52, and antibodies that bind to SIGLEC-F reduce the eosinophil-associated inflammation that is observed in mouse models of allergic airway disease53. The function of SIGLEC-F on alveolar macrophages is currently unknown.

Regulatory effects of the lung microenvironment Alveolar macrophage activation is tightly controlled through several cell–cell and soluble mediator interactions; this creates a regulatory environment that is highly malleable in order to limit unwanted inflammatory responses. The regulatory receptors described below are of particular interest when considering the regulation of alveolar macrophages because of their increased expression on macrophages in the airways compared with those in other sites, because of the availability of their ligands and other immune modulators in the airway microenvironment and/or because of the induction of their expression on alveolar macrophages following inflammatory events.



REVIEWS Airspace MD2 TLR2

Mannose receptor

TLR4

Alveolar macrophage

Induction of expression of inflammatory cytokines Induction of expression of miR-146b

RHOA

STAT3 JAK1

SHP1

p38

TLR4

Phagocytosis SMADs

ERK

CD14

TREM2

Induction of expression of inflammatory cytokines SOCS3

TLR6

NF-κB

SIRPα

TGFβR TGFβ

JNK

SPA and SPD

αvβ6 integrin

RASA2 DOK2

IL-10R

CD200R

IL-10 CD200

Tissue

Type I alveolar epithelial cell


Figure 2 | Negative regulators of alveolar macrophage activation. Alveolar macrophagesNature are restricted soluble Reviewsby| Immunology mediators in the lumen of the airways and by cell–cell interactions, for example, with bronchial and alveolar epithelial cells. Interleukin‑10 (IL‑10) is abundant in the lungs and restricts inflammation by triggering the Janus kinase 1 (JAK1)–signal transducer and activator of transcription 3 (STAT3) pathway to induce the expression of negative regulators such as suppressor of cytokine signalling 3 (SOCS3) and the microRNA miR‑146b. SOCS3 blocks the expression of pro-inflammatory cytokines, whereas miR‑146b directly inhibits Toll-like receptor 4 (TLR4) expression and signalling. Transforming growth factor‑β (TGFβ) regulates inflammation through both SMAD-dependent and SMAD-independent signalling pathways. The αvβ6 integrin is mainly expressed on bronchial epithelial cells, but is also expressed on inflamed alveolar epithelial cells. Binding of latent TGFβ by αvβ6 integrin induces a conformational change in TGFβ that facilitates access of the TGFβ receptor (TGFβR) to the αvβ6 integrin‑bound TGFβ. Triggering receptor expressed by myeloid cells 2 (TREM2), via the adaptor molecule DNAX-activation protein 12 (DAP12), negatively restricts inflammation in macrophages by binding to a currently unknown ligand or ligands. The mannose receptor blocks the recognition of TLR4 ligands and restricts phagocytosis of pathogens such as Pseudomonas aeruginosa. The CD200 receptor (CD200R) interacts with CD200 on the respiratory epithelium, recruiting docking protein 2 (DOK2) and RAS GTPase-activating protein RASA2 (also known as RASGAP), which blocks the extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and JUN N-terminal kinase (JNK) inflammatory pathways. Pulmonary surfactant-associated protein A (SPA) and SPD are abundant in the airways and block TLR2 and TLR4 interactions with their respective ligands, as well as their interactions with the TLR co‑receptors MD2 and CD14, which prevents the activation of nuclear factor-κB (NF‑κB) and the initiation of the inflammatory response. Binding of surfactant proteins to signal-regulatory protein‑α (SIRPα) recruits SH2 domain-containing protein tyrosine phosphatase 1 (SHP1) and activates RHOA, which inhibits phagocytosis. IL‑10R, IL‑10 receptor.

The receptors expressed by alveolar macrophages that are described in the section below are only physiologically relevant if their respective ligands are also present in the airspaces. In the non-challenged airways there are only a few cells that could provide such ligands, for example, T cells; however, by far the most predominant cell populations at this location are bronchial and alveolar epithelial cells. Research in the past

few years has highlighted the complex crosstalk that occurs between the airway epithelium and the alveolar macrophages via cell surface-expressed receptors and secreted products (FIG. 2). For example, culture supernatants from the human BEAS‑2B bronchial epithelial cell line (that constitutively secretes TGFβ) regulate multiple properties of macrophages, monocytes, DCs and T cells in vitro54. These epithelial cells restrict the in vitro



REVIEWS inflammatory responses of alveolar macrophages through undefined soluble factor-mediated and cell contact-mediated mechanisms54. In addition, spontaneous cytokine production by alveolar macrophages during macrophage–epithelial cell co‑culture only occurs when the CD200 receptor (CD200R) is present 17. These and other interactions discussed below highlight the regulatory potential of the respiratory epithelium and reinforce the idea that an intact respiratory epithelium determines how immediately responsive alveolar macrophages are in their environment. Although they are well characterized in mice, many of these important regulatory pathways between alveolar macrophages and primary epithelial cells have not been investigated in humans because of sampling difficulties.

CD200 receptor (CD200R). The CD200R limits inflammatory macrophage responses by activating RAS GTPase-activating protein (RASGAP), which inhibits the extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and JUN N-terminal kinase (JNK) signalling pathways. CD200, the ligand for the CD200R, is found on bronchial and type II alveolar epithelium and on some T cells.

Type II alveolar epithelial cells Unlike their type I structural counterparts, type II alveolar epithelial cells are involved in airway innate immunity; they secrete pulmonary surfactantassociated proteins and cytokines, and recognize pathogens through Toll-like receptors. They also express ligands, such as CD200, for macrophage regulatory receptors.

Type I alveolar epithelial cells Type I alveolar epithelial cells make up as much as 98% of the total surface area of the lungs. They have a large surface area and are very thin to facilitate gas exchange between the alveoli and the underlying capillaries.

Negative regulation of alveolar macrophages CD200R. The CD200R is of considerable interest in the airways as its ligand, CD200, is expressed on the luminal aspect of respiratory epithelial cells17. Compared to interstitial lung macrophages and those from other tissues, mouse alveolar macrophages express higher levels of CD200R17, which is a type 1 transmembrane glycoprotein of the immunoglobulin superfamily that is present on most leukocytes, especially on cells of the myeloid lineage55. CD200R expression is observed on human monocyte-derived macrophages that are polarized to an M2a macrophage phenotype in vitro by the addition of IL‑4 and IL‑13 (REF. 56), but the expression of CD200R on human alveolar macrophages has not yet been thoroughly investigated. CD200R engagement by the widely expressed amino‑terminal V‑type domain of CD200 (REF. 57) inhibits the activation of myeloid cells and T cells. CD200 expression has been described on the luminal aspect of the airway epithelium in mice and rats, where it is expressed on bronchial epithelial cells and on type II alveolar epithelial cells, but not on type I alveolar epithelial cells17,58, and on apoptotic leukocytes in the inflamed airways of mice15. Binding of CD200 phosphorylates a tyrosine residue in an NPXY (where X represents any amino acid residue) motif in the cytoplasmic tail of CD200R59. Docking protein 2 (DOK2) is then phosphorylated and recruits RAS GTPaseactivating protein (RASGAP), which inhibits activation of the pro-inflammatory extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways in myeloid cells 59,60. DOK1 is also phosphorylated by CD200R and recruits SH2 domain-containing inositol‑5‑phosphatase (SHIP1; also known as phosphatidylinositol 3,4,5‑trisphosphate 5‑phosphatase 1); however, this is not required for CD200R‑mediated inhibition in human myeloid cells60. Instead, DOK1 seems to be a negative regulator of DOK2‑induced CD200R signalling 61. Although this DOK1‑mediated negative feedback loop limits the amount of RASGAP that is recruited by DOK2 (REF. 61), the overall outcome of CD200R signalling is inhibitory. An absence of CD200 results in a twofold increase in alveolar macro phage numbers and in their spontaneous upregulation of activation markers17.

SIRPα. Mouse alveolar macrophages express signalregulatory protein‑α (SIRPα; also known as SHPS1 and CD172a)62, which is also expressed on early haemato poietic progenitor cells, on myeloid cells such as macro phages and granulocytes, and on DCs and neurons. Following the phosphorylation of tyrosine residues in its cytoplasmic tail, SIRPα can recruit and activate either SH2 domain-containing protein tyrosine phosphatase 1 (SHP1; also known as PTPN6) or SHP2, depending on the cell type that expresses SIRPα. In haematopoietic cells, SHP1 has a crucial role in the negative regulation of various immunological pathways such as macrophage cytokine-receptor signalling and natural killer (NK) cell inhibitory signals63. SHP1‑deficient mice develop severe autoimmunity and succumb to fatal pneumonia a few weeks after birth owing to excess accumulation and outgrowth of macrophages and neutrophils in the lungs63. Ligands of SIRPα include CD47 and the surfactant proteins pulmonary surfactant-associated protein A (SPA) and SPD64. The extracellular immunoglobulin domain of CD47, which is expressed by many different cell types, binds to the N‑terminal IgV domain of SIRPα. In mice and humans the CD47–SIRPα interaction prevents the development of inflammation following the phagocytosis of erythrocytes65,66, promotes better tolerance following engraftment of human lymphocytes in mouse models67,68 and decreases tumour necrosis factor (TNF) production, but increases nitric oxide generation69,70. Apoptotic or aged cells have redistributed or reduced expression of CD47, which prevents macrophage inhibition by SIRPα, facilitating their timely clearance71,72. Binding of surfactant proteins to human and mouse SIRPα suppresses alveolar macrophage-mediated phagocytosis through the recruitment of SHP1 and the GTP exchange factor RHOA, as well as by inhibiting the activation of p38 MAPK73. Mannose receptor and MARCO. The mannose receptor is a carbohydrate-binding C‑type lectin that is expressed by DCs and macrophages, including by alveolar macrophages. The mannose receptor interacts with glycoproteins and glycolipids that are found on the surface of pathogens. Recognition of unopsonized bacteria results in the suppression of alveolar macrophages in humans, rats, mice and rabbits74,75 — an effect that is dependent on the expression of mannose receptors by these cells76. This suggests that, unless other receptors are co‑ligated or the bacteria are ‘flagged’ for immune recognition in some way, signalling through the mannose receptor prevents alveolar macrophages from initiating proinflammatory responses against microorganisms. This may be of benefit in preventing inflammatory responses against commensal bacteria in the airways. Macrophage receptor with collagenous structure (MARCO) is also immunoregulatory in certain settings. This scavenger receptor mediates the clearance of apoptotic cells, lung pathogens, Streptococcus spp., unopsonized particulate matter and ozone-generated oxidized lipid intermediates. However, in the steady state, MARCO also seems to limit inflammation as MARCO-deficient mice show an early enhanced development of inflammation in response to influenza infection77.



REVIEWS TREM family. Another class of immunoregulatory receptors that is expressed by macrophages is the triggering receptor expressed on myeloid cells (TREM) family. In mice and humans, this family consists of at least one inhibitory and two activating receptors of the immunoglobulin superfamily. TREM1 is expressed by human and mouse macrophages and neutrophils, and promotes their pro-inflammatory functions. Conversely, TREM2 regulates the development and the functions of DCs, microglia and osteoclasts, and has been reported to inhibit TLR-induced macrophage inflammation. Silencing of Trem2 causes an enhanced alveolar macrophage response to TLR4 agonists, which further emphasizes its role as a negative regulator of macrophage function78. Alveolar macrophages upregulate TREM1 expression in response to the recognition of TLR ligands79, whereas TREM2 is expressed by mouse alveolar macrophages following inflammation78,80. Research investigating the TREM family with respect to normal and pathological responses in the lungs is gaining momentum. Pulmonary surfactant-associated proteins. SPA and SPD are abundant in the fluid that lines the epithelium and are produced in the lower airways by type II alveolar epithelial cells81. In addition to binding to SIRPα, SPA promotes an anti-inflammatory state in the lungs by inhibiting the formation of the intact C1 complex that is required to activate complement 82 and blocks the binding of TLR ligands to TLR2, TLR4 and the TLR co‑receptors MD2 (also known as LY96) and CD14 (REFS 83,84). SPD has a key regulatory role in the airways, as SPD‑deficient mice show constitutive activation of alveolar macrophages85. IL‑10 and TGFβ. Although they are widely expressed in other sites, TGFβ and IL‑10 are worthy of mention in this section as they are important components of the healthy lungs and they regulate the activities of alveolar macrophages86. The TGFβ receptor (TGFβR) is composed of a heterodimer of two transmembrane serine/ threonine kinase subunits; TGFβR1 and TGFβR2 (REF. 87). Activation of the TGFβR2 subunit occurs when the receptor binds to TGFβ, which leads to the phosphorylation of the TGFβR1 kinase domain and initiates a broad range of SMAD-dependent and SMAD-independent signalling pathways. SMAD-mediated signalling involves phosphorylation of the receptor-regulated SMADs (RSMADS; including SMAD2 and SMAD3) by TGFβR1, which then form a complex with SMAD4 (REF. 88). These phosphorylated RSMAD–SMAD4 complexes translocate into the nucleus and function as either a positive or a negative transcriptional regulator of several genes by interacting with transcription factors, co‑activators, repressors and CREB-binding protein (CBP) or p300 transactivators88. The SMAD-independent pathway can induce several signalling cascades, including p38 MAPK, RHOA, ERK and JNK, which regulate a broad range of cellular functions88. The cellular effect of TGFβ signalling and the activation of either SMAD or non-SMAD pathways is partly determined by the expression level of the TGFβR2 subunit of the TGFβR89.

TGFβ is widely produced by cells in the normal human and mouse lungs90, and regulation of macro phage function by TGFβ may be autocrine (for example, activated rat alveolar macrophages secrete TGFβ, which is activated from its latent form by the protease plasmin and the interaction between thrombospondin 1 and CD36 (also known as platelet glycoprotein 4)91) or paracrine (for example, αvβ6 integrin expressed on the bronchial epithelium and at low levels on the alveolar epithelium activates latent TGFβ in a cell– cell contact-dependent manner 18). Mice lacking αvβ6 integrin‑activated TGFβ develop spontaneous pulmonary emphysema because of an upregulation of MMP12 expression by alveolar macrophages, as well as developing general lung inflammation, both of which can be rescued by the administration of exogenous active TGFβ1 or by the targeted expression of functional αvβ6 integrin in the lung epithelium86. In addition, alveolar macrophages in mice and humans express the IL‑10 receptor (IL‑10R)92. Binding of soluble IL‑10 by its receptor sends an inhibitory signal via Janus kinase 1 (JAK1), which in turn phosphorylates two tyrosine residues in the IL‑10R. These phosphorylated tyrosine residues are the docking site for the signal transducer and activator of transcription 3 (STAT3) SRC homology 2 (SH2) domains. STAT3‑induced genes, including suppressor of cytokine signalling 3 (SOCS3), in turn suppress the expression of pro-inflammatory cytokines 93,94. The IL‑10R also induces expression of the microRNA miR‑146b, which directly inhibits the TLR4 signalling pathway by preventing the transcription of TLR4, myeloid differentiation primary-response protein 88 (MYD88) and TNF receptor-associated factor 6 (TRAF6) mRNA95. The epithelium in mice is clearly a source of IL‑10; however, in humans there are mixed reports regarding IL‑10 and IL‑10R expression by airway epithelial cells17,96. GM-CSF. The respiratory epithelium is also a major source of granulocyte–macrophage colony-stimulating factor (GM‑CSF)97–99. Bone marrow-derived macro phages cultured with either GM‑CSF or SPD upregulate their CD11c expression 47 and, in fact, GM‑CSF is required for the differentiation of fetal monocytes into alveolar macrophages4. Macrophages found in the airways of GM‑CSF-deficient mice are not fully differentiated alveolar macrophages4 and are functionally limited; their functional limitations include reduced responsiveness to the bacterial TLR4 agonist lipopolysaccharide (LPS) and impaired phagocytosis 100. The absence of GM‑CSF also results in an accumulation of pulmonary surfactant-associated proteins in the airways, as the remaining macrophages are unable to catabolize surfactant lipids and proteins100. PPARγ. Compared with other macrophage populations, alveolar macrophages differentially express genes that are involved in lipid metabolism101. One such gene product that seems to be important for alveolar macro phage function is peroxisome proliferator-activated



REVIEWS Efferocytosis The cell-mediated engulfment and clearance of apoptotic cells, which is similar to phagocytosis. This process is mediated by bridging molecules and cell surface receptors such as the TAM (TYRO3, AXL and MER) receptor family. Efferocytosis typically induces anti-inflammatory signalling pathways within the engulfing phagocyte.

TAM receptor family Made up of TYRO3, AXL and MER receptor tyrosine kinases. These receptors promote efferocytosis by recognizing externalized phosphatidylserine expressed on the surface of apoptotic cells via the bridging molecules growth arrest-specific protein 6 (GAS6) and protein S.

receptor‑γ (PPARγ). PPARγ is expressed by both human and mouse alveolar macrophages, but its expression is lost in GM‑CSF-deficient mice and in patients with pulmonary alveolar proteinosis 102. Alveolar macrophages from these patients and mice show excessive lipid accumulation, but overexpression of PPARγ in macrophages in the airways of GM‑CSFdeficient mice restores the ability of these animals to clear lipids102. Notably, mice lacking PPARγ expression in macrophages develop spontaneous inflammation in the lungs103, which highlights the important role of lipid metabolism by alveolar macrophages in the maintenance of airway homeostasis.

Alveolar macrophages in the inflamed lungs Alveolar macrophages are the masters of contradictory function. They are essential for steady-state ‘hoovering’ of daily cellular debris but are also ideally placed to initiate a strong inflammatory response to something more pathogenic. How do alveolar macrophages so rapidly distinguish between these two functions? Mannose receptor

IL-1R IFNGR

IL-10R SIRPα

TNFR

CD14

TGFβR TLR2 Activating signal

Inhibitory signal TREM2

TLR6

TLR4 CD200R MD2 Alveolar macrophage

Figure 3 | The balancing act of macrophage activation. Alveolar macrophage activation and the initiation of inflammation involves a complex between Naturebalancing Reviews | act Immunology activating and repressing signals. On the one hand, Toll-like receptors (TLRs), along with their co‑receptors such as MD2 and CD14, recognize pathogen-associated molecular patterns and receptors for inflammatory cytokines, such as tumour necrosis factor (TNF), interleukin‑1β (IL‑1β) and interferon-γ, which perpetuate inflammation. On the other hand, mediators such as IL‑10 and soluble or αvβ6 integrin‑tethered transforming growth factor‑β (TGFβ) block pathways that lead to inflammation. Cell–cell interactions with bronchial or alveolar epithelial cells also deliver inhibitory signals to alveolar macrophages, for example, through CD200 receptor (CD200R), triggering receptor expressed by myeloid cells 2 (TREM2) or signal-regulatory protein‑α (SIRPα). Loss of the ligands for the negative regulators, for example, following epithelial cell loss during inflammation, will tip the balance towards alveolar macrophage activation. Conversely, increased expression of the negative regulators and inhibition of TLR signalling pathways, for example, in the resolution of inflammation, tips the balance towards the repression of alveolar macrophages. IFNGR, interferon-γ receptor, IL‑1R, IL‑1 receptor; TGFβR, TGFβ receptor; TNFR, TNF receptor.

Studies examining how alveolar macrophages are altered following inflammation and the resolution of lung inflammation suggest that the destruction of the airway epithelium and the loss of exposure to the regulatory ligands that are expressed by the epithelium may lead alveolar macrophages to respond to airway antigens in a pro-inflammatory, as opposed to a regulatory, manner (FIG. 3). Whether the cells that are cleared by alveolar macrophages are apoptotic or necrotic is another important determinant of whether alveolar macrophages will respond in an anti-inflammatory or in a proinflammatory manner. As in other body sites, apoptotic cell efferocytosis requires alveolar macrophages to adopt an anti-inflammatory state in order to prevent inflammatory responses to self proteins (for a review see REF. 104). Recognition of externalized phosphatidylserine on apop totic cells by the TAM receptor family induces SOCS1 and SOCS3 expression by alveolar macrophages, which inhibits cytokine receptor and TLR signalling105. Apoptotic cells also show increased expression of the CD200R ligand CD200 and, as discussed above, this can also transmit inhibitory signals to the phagocytic cell17. Conversely, necrosis (and secondary necrosis, which occurs if apoptotic cells are not cleared) liberates pro-inflammatory damage-associated cellular constituents.

It takes two to tango Another factor that enables macrophages to distinguish between situations that require a tolerogenic response and those that require an inflammatory response is the need for multi-receptor signalling in innate immune cells. For example, tonic TLR or pattern recognition receptor (PRR) signalling occurs at all mucosal sites via the interaction of innate cells with commensal microorganisms or with the by‑products of cellular and extracellular matrix turnover. However, more pathogenic encounters disrupt structural cells (causing a loss of regulation) and also upregulate TLR co‑receptors such as CD14 and TREM1 (REF. 106). Therefore, the initiation of inflammation requires a combination of events that override the inhibitory mechanisms that regulate alveolar macrophages. Once initiated, the outcome of their activation is determined by pathogen-specific properties and by the host immune response to them. The exposure of alveolar macrophages to oxidative stress enhances exocytosis and hence the surface expression of TLR4, which can be reversed with antioxidants107. Stimulation of alveolar macrophages through TLR2, TLR4 or TLR9 inhibits IL‑10R signal transduction and releases these cells from the suppression that is usually mediated by epithelial cell-produced IL‑10 (REF. 92). Signalling via one TLR in alveolar macrophages markedly changes the pattern of expression of other TLRs on the same cells108,109. The inhibition of T cell responses by alveolar macrophages is overcome by the addition of pro-inflammatory cytokines, such as GM‑CSF and TNF110. Once activated, alveolar macrophages generally have a greater phagocytic capacity 111, with a higher oxidative burst and higher pro-inflammatory cytokine production112. Therefore, alveolar macrophages carry out an important gate-keeping role in the defence of the respiratory tract.



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Myeloid-derived suppressor cells (MDSCs). A group of immature CD11b+GR1+ cells that include precursors of macrophages, granulocytes, dendritic cells and myeloid cells. These cells are produced in response to various tumour-derived cytokines and have been shown to inhibit tumour-specific immune responses.

Alveolar macrophages in airway disease It is a matter of debate as to the relative contributions to the perpetuation and the resolution of inflammatory responses of tissue-resident macrophages, the macrophages that are recruited to the airways and become alveolar macrophages, and the original alveolar macrophages that persist after inflammation has resolved. Indeed, the conclusion reached seems to be highly dependent on the model of inflammation that is used to study the functions of these macrophage populations. Although space limits an in‑depth discussion on this topic, the recent use of radiation chimaeras, microparticle uptake and dye labelling has enabled the distinction to be made between newly recruited and original tissue-resident alveolar macrophages34,113. The fate of the original alveolar macrophages is interesting, as many seem to be lost through apoptosis or necrosis during inflammation. Whether the airways are repopulated by recruited replacements from the periphery or from the lung tissue, or by the division of remaining alveolar macrophages in situ, all airway macrophages will be influenced by the airway microenvironment that is dominant at that time, regardless of their site of origin. Alveolar macrophages certainly contribute to the development of severe inflammation (for examples, see REFS 114–116) but they also have a crucial role in limiting the excess inflammation that is caused by infection; for example, an influenza virus infection in the absence of alveolar macrophages results in reduced viral clearance and therefore more inflammation and pathology 117–119. Site-specific suppression of alveolar macrophages may itself contribute to disease severity by enabling pathogens to replicate to a higher load before the negative immune regulators are overcome. Reducing this negative regulation, for example, by the deletion of the genes encoding CD200R 17, MARCO77 or the ubiquitin-editing protein A20 (also known as TNFAIP3) 120 before influenza infection, results in faster viral clearance and a better outcome. Recruited macrophages may also phenotypically differentiate into alveolar macrophages in the steady state — a process that is accelerated by previous inflammation33. Alveolar macrophages that have an M2‑like phenotype have a role in the resolution of many different lung inflammatory conditions (for a review see REF. 121), but also contribute to fibrotic pathology 122 via increased production of TGFβ, CCL18, resistin-like secreted protein, found in inflammatory zone (FIZZ1; also known as resistin-like-α) and chitinase-like secretory lectin YM1 (FIZZ1 and YM1 are involved in extracellular matrix dynamics). Furthermore, increased l‑arginine metabolism generates polyamines and proline, which are precursors for collagen synthesis 123; patients with Hermansky– Pudlak syndrome type 1, who have defective biogenesis of lysosome-related organelles, develop progressive fibrotic lung disease that is associated with increased alveolar macrophage activity 124; alveolar macrophages from patients with asthma produce higher levels of IL‑13 compared with cells from healthy volunteers125

and have impaired IL‑12 production126; and CD163, which is a scavenger receptor for the haemoglobin– haptoglobin complex, is expressed at higher levels by M2‑like human macrophages and shows enhanced expression on alveolar macrophages from patients with idiopathic pulmonary fibrosis 127. In addition, alveolar macrophages from patients with emphysema produce excess MMP1 and MMP12, which contribute to structural changes in the lungs128. Therefore, alveolar macrophages contribute to health and disease, but are these roles transient or are they maintained?

Long-term alteration of alveolar macrophages The resolution of airway inflammation and tissue repair lead to the development of an altered alveolar macrophage population. Following viral infection, mouse alveolar macrophages express higher levels of CD200R17 and express lower levels of bacterial scavenger receptors such as MARCO129. They are also blunted in their responsiveness to the bacterial TLR agonists lipoteichoic acid (LTA; a TLR2 ligand), LPS (a TLR4 ligand) and flagellin (a TLR5 ligand); in the case of flagellin, this is associated with reduced nuclear translocation of the p65 subunit of NF-κB130. IL‑10 expression is also markedly increased in the lungs following the resolution of viral infections131. In addition, both CD200R and TREM2 expression are increased following the establishment of inflammation in a mouse model of allergic airway disease80 (FIG. 4). In humans, emphysema enhances TLR2 and TLR4 expression by alveolar macrophages and leads to increased inflammation in response to infection with Streptococcus pneumoniae132. Conversely, cigarette smoke and chronic obstructive pulmonary disease (COPD) lead to decreased TLR2 expression by alveolar macrophages133,134, and morphine reduces the TLR9‑mediated triggering of NF‑κB signalling in alveolar macrophages and impairs the clearance of S. pneumoniae135. This implies that M1‑like macrophage function is reduced and/or M2‑like characteristics are increased. These phenotypical changes that occur in both the original population of resident alveolar macrophages and in any recruited macrophages that have differentiated into alveolar macrophages and that remain in the tissue following the resolution of the inflammatory response may be a consequence of several factors, such as efferocytosis of apoptotic cells and altered regulation of macrophages by the recently repaired epithelium, by the environmental factors that mediate lung tissue repair and/or by autocrine influences of the macrophages themselves21. A similar increase in M2 characteristics (although not proven) probably occurs in the alveolar macrophages of patients with lung neoplastic malignancies. In addition to regulatory T cells and myeloid-derived suppressor cells (MDSCs), tumourassociated macrophages facilitate tumour growth by suppressing antitumour effector cells136. Similarly to the processes that repair the lung architecture following non-malignant inflammation, tumorigenesis is associated with the deposition of extracellular matrix and hence the development of M2 macrophages.



REVIEWS Resting airways

Post-inflammation TLR2 MARCO

Decreased expression of MARCO

TLR4

TLR5

Resolution of inflammation

NF-κB

NF-κB

Pro-inflammatory genes

Impaired NF-κB translocation Increased levels of IL-10

IL-10R

Type I alveolar epithelial cell

Increased CD200R expression

CD200R CD200

IL-10

Increased TREM2 expression TREM2


Figure 4 | Altered macrophage regulation after inflammation. Following the resolution of inflammation, alveolar macrophages have an increased regulatory level compared to naive macrophages. CD200 receptor (CD200R) and Nature Reviews | Immunology triggering receptor expressed by myeloid cells 2 (TREM2) expression is markedly increased following influenza or house dust mite-induced allergic airway disease in mice, and there is more soluble interleukin‑10 (IL‑10) present in the lungs. Conversely, the expression of macrophage receptor with collagenous structure (MARCO), which is a scavenger receptor involved in the clearance of bacteria, is reduced after inflammation. In addition, several months after the resolution of an influenza infection, mouse airway macrophages are restricted in their responsiveness to the Toll-like receptor (TLR) agonists lipopolysaccharide (LPS; in the case of TLR4), lipoteichoic acid (LTA; in the case of TLR2) and flagellin (in the case of TLR5), and they have impaired nuclear factor‑κB (NF‑κB) translocation to the nucleus after TLR5 activation (dashed arrows). IL‑10R, IL‑10 receptor.

Consequences of heightened regulation A major complication that is associated with a functionally altered alveolar macrophage population is a heightened susceptibility to bacterial infection. This is a feature of many acute and chronic lung inflammatory disorders and usually occurs during the resolution phase of inflammation when antibacterial defences are at their lowest 15,80,137,138. Infection with different bacterial strains, including S. pneumoniae, Haemophilus influenzae and Staphylococcus aureus, is reported to have exacerbated the inflammatory lung disease that occurred during the 1918 and 2009 influenza pandemics. Such bacterial infections also exacerbate lung inflammation in patients with asthma or COPD, and in patients infected with respiratory syncytial virus (RSV) or rhinovirus. The literature detailing these complications is extensive and is reviewed in REFS 139–141. However, in some contexts — for example, when excessive inflammation exacerbates the disease pathology — a blunted innate immune response can be beneficial142. Perspective It is important that the inflammatory ‘tone’ of alveolar macrophages is correctly set from birth and appropriately re‑set following acute or during chronic inflammation; however, in some groups of patients this does not happen.

Much information about how alveolar macrophages promote tolerance has been provided by examining the lungs in the healthy state. An under-researched area is the study of alveolar macrophages soon after birth, when the level of tolerance and the pathways required must be initiated and presumably tailored to the various environments that are present across the world. Interestingly, exposure to microbial extracts enables neonatal mice to control a subsequent S. pneumoniae infection, which is attributed to an increased production of SPA and SPD and an increase in alveolar macrophage scavenging activity143. Furthermore, unlike in adult mice, alveolar macrophages from neonatal mice fail to activate NF‑κB in response to bacterial infection — a defect that decreases with age144. As a result of the unique position of alveolar macrophages in the airspaces, factors that promote an accurate assessment of the environment are crucial. Reduced tolerance would probably result in the development of inflammation in response to innocuous allergens, whereas over-regulation would enable pathogens to escape immediate recognition; indeed, both scenarios are observed in animal models145–148. In addition, there is mounting evidence to suggest that pathological macrophage dysregulation occurs in both paediatric149–153 and adult 154–156 patients, in whom the regulatory mechanisms discussed in this Review may have a role that is currently unknown.



REVIEWS Alveolar macrophages have an important role in maintaining airway immune homeostasis and are directly accessible, even to large molecules. Knowledge of their site-specific regulation will provide strategies to improve the efficacy of inhaled vaccines and to revert the long-term alterations that occur in alveolar macro phages in severe and chronic inflammatory diseases. Whether alveolar macrophages are recruited from the periphery or derived from lung tissue-resident

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Acknowledgements

The authors would like to thank M. Exley for his critical reading of the manuscript.

Competing interests statement

The authors declare no competing interests.


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