Lung Macrophages: Pivotal Immune Effector Cells Orchestrating Acute and Chronic Lung Diseases

Abstract

Macrophages are key immune cells, where they play a pivotal role in host defense and tissue homeostasis. The lungs have two major subsets, alveolar macrophages (AMs) found in airspaces and interstitial macrophages (IMs) found in lung tissues. Lung macrophages (LM) are highly heterogeneous and have high levels of plasticity. A long-lasting population of LM with self-renewal ability populate the lung during embryogenesis and monocyte-derived macrophages recruited during infection, inflammation, or tissue repair, which are more short lived. AMs have been the main focus of research due in part to their abundance, accessibility, and ease of isolation compared with IMs. With advances in multichannel flow cytometry and single-cell sequencing, the importance of IMs has been recently appreciated. LM’s functions in the lungs include maintenance of homoeostasis, immune surveillance, removal of cellular debris, tissue repair, clearance of pathogens, and the resolution of inflammation. They also activate the adaptive immune response by functioning as antigen-presenting cells. LMs are pivotal in the pathogenesis of acute and chronic inflammatory lung conditions including lung cancer. This chapter will discuss the ontology, phenotypic heterogeneity, and functions of LM’s and how these characteristics orchestrate and impact common acute and chronic lung conditions.

1. Introduction

Macrophages are immune effector cells that are present in most organs and tissues, are highly phagocytic in nature and produce large amounts of a wide variety of mediators. They are either resident in tissues or are recruited and as part of the innate immune effector system, are activated to mount an appropriate immune response to neutralize harmful insults [1].

The lungs are continuously challenged by a variety of foreign inhaled substances which include allergens, microbial pathogens, chemicals, particulates matter and noxious gasses. These insults require an exquisite capacity to appropriately calibrate inflammatory responses in the airways and lung tissues to maintain physiologic homeostasis (e.g., gas exchange). The lung macrophages have all the properties to orchestrate and calibrate such inflammatory responses given their location in the lungs, their large abundance in tissue and their high degree of functional plasticity and ability to communicate with neighboring cells. Research over the past three decades has shed light on the origins of lung macrophages, their ability to adapt to the local microenvironment, their plasticity, and their functional responses to maintain tissue homeostasis. In addition, their pivotal role in orchestrating the innate immune response and activating adaptive immunity when airways are challenged with pathogenic insults has also been elucidated [1, 2].

1.1 Macrophage populations in the lung

Two well-studied populations of lung macrophages have been defined: (1) alveolar macrophages (AMs), which are predominantly located on alveolar epithelial surfaces and can be harvested from the lungs by bronchoalveolar lavage (BAL) and (2) interstitial macrophages (IMs), which are located within alveolar walls or interstitial lung tissue and can be harvested directly from lung tissue specimens through biopsy or surgical resections [3]. Two less well-defined populations of macrophages are airway macrophages, which are found on mucosal surfaces of the airways and intravascular macrophages, which reside on capillary blood vessel walls. Limited data suggest that airway macrophages are phenotypically and functionally very similar to AMs and may represent AMs that have migrated up the tracheobronchial tree [4]. Airway macrophages are usually grouped with AMs because of their phenotypic similarity and their ability to be captured by BAL. The intravascular macrophages are located on the inner side of capillaries, suggesting that they fight against blood-borne pathogens. Limited (older) data suggest that their function is comparable to that of AMs and that they may reflect an intermediate stage of differentiation between blood monocytes and AMs [5, 6].

It was previously thought that resident lung macrophages originate primarily from blood monocytes, which are produced and released from the bone marrow [7, 8]. The last decade this paradigm has been turned on its head as research has shown that most resident macrophages in different tissues throughout the body, including lung macrophages, arise predominantly from embryonic precursors. These macrophages are produced before birth, become colonized in the lungs in the prenatal period and are maintained throughout life by local proliferation [9, 10, 11]. Resident alveolar macrophages originate as erythromyeloid progenitors (EMPs) in the yolk sac on embryonic day (E) 8.5. EMPs colonize the fetal liver by E10.5, and then give rise to fetal monocytes, which migrate to the lungs by E12.5 [12]. The maturation of fetal monocytes to alveolar macrophages occurs in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and is fully completed by the third postnatal day (Figure 1).

Figure 1.

The origin of alveolar (AM) and interstitial (IM) macrophages in mice. Resident AMs, derived from the embryo (yolk sac and/orfetal liver), are capable of self-replicating during homeostasis and when challenge in lung. IMs are derived from bone marrow monocytes and there are three unique IMs in murine lung in homeostasis: IM1, IM2 and IM3 (Gibbings et al.). During steady state, the maintenance of AM pool does not need the contribution of bone marrow -derived monocytes, but in the circumstance of inflammation, monocytes are strongly recruited to areas of inflammatory alveoli and differentiated into recruited monocyte-derived AMs. IM3 are originated from BM-derived monocytes and play different roles in diseases such as pulmonary fibrosis (Chakarov et al.).

These resident lung macrophages are the primary “janitors” of the lung, protecting the lungs from inhaled environmental insults. In contrast to resident alveolar macrophages, recruited monocytes, which enter airspaces 24-72 hrs after the onset of an inflammatory stimulus in the lung, differentiate into macrophages in the tissues where they initially take on a pro-inflammatory phenotype (by promoting inflammation) and later assumes a regulatory role by suppressing the inflammatory process. Classically, the initial phenotype has been described as M1 macrophages and the regulatory phenotype as M2 macrophages.

While M1 macrophages promote inflammation, M2 macrophages induce efferocytosis (phagocytosis of apoptotic and dead cells) to enable inflammatory clearance, and secrete anti-inflammatory cytokines such as IL-10 and soluble (decoy) IL-1R [13].

Studies in animal models have shown that resident AMs can be replaced by IMs or be derived from circulating monocytes [8, 14, 15], underlining the adaptability of macrophage kinetics and function. Schneider and co-workers have shown that the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-𝛾) is essential for the differentiation of AMs and that PPAR-𝛾 deletion in the myeloid lineage cells prevents the formation of mature AMs, resulting in impaired microbial clearance and development of a clinical condition called pulmonary alveolar proteinosis, which is characterized by excess surfactant accumulation in lungs [16]. PPAR-𝛾 expression requires GM-CSF, which is produced largely by type II alveolar epithelial cells. A lack of GM-CSF leads to a loss of mature AMs, accumulation of surfactant and ultimately alveolar proteinosis [9]. Recently the interesting concept of innate immune memory resulting from resetting of the cell’s epigenetic program and functional state after infection, leading to enhanced protection against a secondary infection in the ensuing weeks to months [17]. These “memory AMs” have been shown to originate from resident AMs following mild infection and are characterized by a high level of MHCII molecules and host defense-ready genes, and enhanced production of neutrophil attracting chemokines and interferon-gamma (IFN-γ) derived from effector T cells, which are primed to respond during infection [18].

Interstitial macrophages are less well defined mostly because access to pure populations are difficult in humans. Studies in laboratory animals suggest that there are at least 3 different populations of IM: IM1, IM2 and IM3. Expression of CD11c and MHCII on IMs at a steady state classifies these population into three subsets, IM1 (CD11c^loMHCII^lo), IM2 (CD11c^loMHCII^hi), and IM3 (CD11c^hiMHCII^hi) in mice [19]. At least two of these three populations have been identified in humans [20, 21]. Whether these IMs are resident, long lived cells possessing self-renewal properties similar to resident AMs, or represent derived cells from blood monocytes is still unclear.

Macrophages in the lungs have a range of function, including maintenance of homeostasis, immune surveillance, repair, removal of cellular debris and surfactant clearance, and elimination of microbes, allergens and particulate matter, and resolution of inflammation. These distinct functions are linked to the different subsets of macrophages, ontogeny, and location of the macrophages and influence of the local microenvironment.

2. Macrophage phenotypes

Similar to T helper 1/2 (Th1/Th2) cells, macrophages are categorized as either classically activated M1 macrophages or alternatively activated M2 macrophages. The classical or M1 macrophages are activated by microbial products and/or IFN-γ. MI cells are pro-inflammatory and possess anti-microbial functions, and ability destroy tumor cells [22]. Signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor (IRF)3, IRF5, and nuclear factor-κβ (NF-κβ) are key molecules that become activated in M1 macrophages to generate pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), Interleukin-1 (IL-1), IL- 12 and IL- 23, [23], nitric oxide (NO), and reactive oxygen intermediates (ROI). They also promote increased expression of major histocompatibility complex (MHC) molecules (Figure 2). Over the inflammatory period, the microenvironment changes and Th2 cytokines, including IL- 4 and IL- 13, stimulate monocytes or macrophages to transform into a M2 phenotype. M2 macrophages promote resolution of the inflammatory process and stimulate wound healing, favoring a milieu of angiogenesis and tissue remodeling. IL- 10, glucocorticoid hormones and IL-1R may also induce M2 macrophage polarization.

Figure 2.

Paradigm of macrophage phenotypes. Naïve macrophages are activated to either a M1 phenotype (classical activation) or to M2 phenotype (alternatively activated) dependent on their microenvironment milieau. Macrophages are polarized to an M1 (or T-helper type 1 cell [Th1]) phenotype in response to IFN-g, granulocyte–macrophage colony–stimulating factor (GM-CSF), and bacterial products (e.g., LPS). They are characterized by a high production of the proinflammatory cytokines IL-12 and IL-23, and with high expression of major histocompatibility complex class II (MHCII) molecules and coactivation proteins (CD80 and CD86) are considered excellent antigen-presenting cells that can activate an adaptive immune response and fight foreign insults through the production of toxic intermediates such as reactive oxygen intermediates [ROI]). Macrophages are polarized to an M2 (or Th2) phenotype in response to macrophage colony–stimulating factor (M-CSF), IL-3 and IL-10 and antiinflammatoryfactors. They produce a variety of antiinflammatorycytokines, immunosuppressive mediators, and matrix-degrading enzymes, and promote angiogenesis and are involved in tissue remodeling and repair.

The M2 macrophage phenotype is not homogenous and consists of several subtypes including M2a, M2b, M2c and M2d (Figure 3). The M2a macrophages are produced by exposure to cytokine such as IL-4 or IL-13. They can also be induced by fungal and helminth infections. They are characterized by expression of high levels of the mannose receptor (CD 206), CD209, IL-4R and FcεR, secreting transforming growth factor β1 (TGF-β1) and insulin-like growth factor. Functionally M2a macrophages contribute towards wound healing and tissue repair [24]. In contrast, M2b macrophages are induced by immune complexes, IL-1β and lipopolysaccharide (LPS) exposure, and secrete abundance of IL-10, IL-1β, IL-6 and TNF-α, that have predominantly anti-inflammatory properties [25]. The M2c macrophages are induced cytokines such as IL-10, TGF-β and glucocorticoids and have increased expression of receptor for advance glycation and end products (RAGE), CD163 and CD206. M2c macrophages are thought to be involved in immunosuppression, tissue repair and matrix remodeling [26, 27]. Lastly, M2d macrophages, are activated by leukocyte inhibitory factor, toll like receptor (TLR) ligands and adenosine. They express low levels of CD206, and produce large amounts of IL-10, TGF-β and vascular endothelial growth factor (VEGF) which facilitate immunosuppression and angiogenesis [28].

Figure 3.

Different M2 macrophage phenotypes are generated by different microenvironments and stimuli. The functional responses of the four different M2 sub-phenotypes are determine by them producing different mediators.

A unique characteristic of macrophages is their plasticity and their ability to differentiate into several phenotypes and also to de-differentiate. For example, M2 macrophages may revert to M1 macrophages under certain conditions. This switch in polarization is dynamic and induced predominantly by their microenvironment. The distinct M1 and M2 subtypes is an overly simplification of macrophage polarization, for example, 5% of the macrophages in lung cancer specimens express both M1 and M2 markers [29], and mixed macrophage polarization have been described in other lung conditions [29, 30]. With the advent of genetic analysis and specifically single cell RNA sequencing technology, the basic macrophage characterization of M1 and M2 phenotypes has been challenged and may change in the near future [31].

3. Macrophages in lung infections

Airspace or resident AMs represent the first line of defense and play a central role in protecting the lungs against a range of respiratory pathogens. Yet, pulmonary immune responses need to be contained and refined to avoid excessive tissue damage and safeguard gas exchange. Resident AMs (ResAM) are less responsive than recruited monocyte-derived macrophages in the context of infection in the lung [32]. As resident AMs are predominantly involved in lung tissue homeostasis, resident AMs are generally “anti-inflammatory”, which in part is linked to their production of type I interferons (IFNs) [33]. Notably Type I IFNs negatively regulate IL-1 production and positively regulate IL-10 production in monocyte-derived macrophages [34]. AMs have low expression of complement-associated genes such as C1qa, C1qb, C1qc, C2, C4b and C3ar1 [19], in contrast to macrophages isolated from other tissues. Epigenetic profiling of AMs during inflammation has shown that while the C1q locus is inaccessible in ResAMs, complement genes are highly accessible in inflammatory AMs (InfResAMs) that arise in the AM pool during influenza infection [35]. With human SARS-CoV-2 infection, recruited macrophages were more C1q^hi compared with the ResAMs, which were more C1q^lo, suggesting that repression of the complement-associated genes in ResAMs is conserved in humans [36]. Importantly, the overt activation of complements has been linked to coronavirus 2019 (COVID-19) severity [37, 38]. It is reasonable to suggest that the sharp drop in ResAMs and their replacement by recruited macrophages expressing high levels of the complement-associated genes during SARS-CoV-2 infection may fuel the complement cascade and actively participate in the COVID-19 pathogenesis. Furthermore, it could be that repression of these complement genes in ResAMs is the result of evolutionary pressure to protect the lungs from complement-mediated collateral damage during infections.

Kinetic studies have shown that bone marrow derived monocytes are recruited into alveolar space within 24 hrs following a focal instillation of Streptococcus pneumoniae into the lung [15]. These monocyte derived macrophages become the dominant immune cells in the airspaces during the resolution phase of pneumonia. Bacteria have developed strategies to take advantage of hypo-responsive ResAMs by enhancing their survival and promoting spread [39]. An example of this concept is the relocation of Mycobacterium tuberculosis-infected AMs into pulmonary tissues, which is a pivotal preparatory step for bacterial dissemination in the host [40]. Furthermore, following viral infection such as influenza, the resident AM population are depleted and subsequently restored by monocyte recruitment from the bloodstream which differentiate into long-lived InfResAMs and partially replace the resident AM [41, 42]. These newly recruited monocytes replace the resident macrophages and the extend of this replacement is linked to the dose and/or virulence of the pathogen. The reduction of resident macrophages by viral infections renders the host more susceptible to other microbial infections until the resident macrophages recover their numbers via self-renewal [43]. InfResAMs that develop during a viral infection have been shown to differ functionally from the ResAMs that were present before the infection [16, 42]. For example, InfResAMs that developed during influenza infection are more responsive to TLR ligands and subsequent S. pneumoniae infection compared with ResAMs. The InfResAMs acquired innate memory qualities via enhanced IL-6 production that provides protection against subsequent bacterial infections such as S. pneumoniae infection. Comparing the epigenetic signature of these ResAMs and InfResAMs shows that monocytes recruited by the virus infection have an epigenetic profile similar to tissue residency, but addition also acquire a more inflammatory signature induced by the viral infection. For example, the IL-6 enhancer regions are more accessible in InfResAMs that are recruited during an influenza lung infection compared with ResAMs. This altered epigenetic signature is still evident 1 month after the viral infection but started to alter at 2 months post-infection (Figure 4).

Figure 4.

Inflammation and/or infection in the lung and subsequent resolution reshape the composition of the pulmonary macrophage pool. A) An infectious episode in the lung results in the resident macrophages (ResMac) send signals for recruitment of monocytes from the blood. B) Monocytes from the blood are recruited into alveolar spaces to help dealing with the infection. C) these monocytes change intotransitional inflammatory macrophages that help clearing the infection. D) some of these transitional macrophages change intoinflammatory resident macrophages (InfResMac), the rest disappear. The InfResMacremain in the airspaces for months and ResMacproliferate to slowly replace them over time.

Long-term memory cells, which are characterized by altered gene expression, metabolism and antimicrobial responsiveness, have been proposed as a subset of ResAMs [44]. These cells have reduced phagocytic capacity by over-expressing signal regulatory protein α [45]. Similarly, InfResAMs that develop during an infection can acquire innate memory, thus altering their responses to subsequent infections. However, these InfResMacs show increased epigenetic plasticity. Much less is known about the impact of viral infections on resident IMs and their plasticity after an infectious insult. These cells have a significantly shorter half-life compared with ResAMs and robust fate-mapping systems have not been developed for IM.

Our ability to separate lung ResMacs from InfResMacs has shown that InfResMacs are more strongly imprinted across a range of infectious stimuli. This imprinting has the capacity to generate long-lasting innate immune memory cells that alter macrophage function during subsequent challenges. Multiple lung infectious challenges may therefore allow the engraftment of many waves of InfResMacs into lungs. However, over time and in a steady state, InfResMac may lose their unique genetic or epigenetic signature, leading to a loss in memory. Recurrent infections or inflammatory stimuli, on the other hand, could revive the memory of these InfResMacs. Innate memory in InfResMacs may be beneficial in some cases and may confer resistance to subsequent infections, but could also exacerbate tissue inflammation and injury during subsequent infections [46]. This has been proposed as a potential reason for the difference in severity of Covid-19 infection in older versus younger subjects and the generally mild inflammatory response seen in children. Therefore, the duration of residence in a homeostatic environment such as the lung, becomes a key factor determining lung macrophage biology [47, 48].

4. Macrophages and COPD

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory lung disease caused by the long-term exposure to toxic particles and gasses. Worldwide, tobacco cigarette smoke is the main culprit, though biomass exposure may be a more important cause of COPD in some parts of the developing world [49]. These exposures elicit a persistent innate and eventually an adaptive immune response in the airways and lung tissues, which is characterized by overproduction of mucus in the central airways, fibrosis and obstruction of small airways and eventual destruction of the lung parenchyma leading to emphysema [50]. There is also evidence for impaired tissue repair responses and altered tissue remodeling that contribute to a progressive disease phenotype [51].

Lung macrophages are key immune effector cells in the pathogenesis of COPD. Airspace macrophages are directly exposed to inhaled antigens, pathogens and noxious particles and gasses, and several studies have shown an increase in their numbers in subjects with COPD compared to controls [52, 53, 54]. Cigarette smoking is still the major cause of COPD in developed countries, but biomass exposure is a more important cause of COPD in the developing world [49]. For both of these exposure types, lung macrophages play a pivotal role in processing and clearing these particles from the lungs. Due to the chronicity of these exposures in COPD, functional responses of lung macrophages to these exposures are thought to participate in the development of COPD. There is a significant increase in macrophages in induced sputum and BAL fluid (BALF) samples in COPD patients [55], supporting this notion [54]. In COPD, lung macrophages also secrete large amounts of potential tissue damaging enzymes such as elastase, matrix metalloproteinases (MMPs) MMP-2, MMP-9, MMP-12 and cathepsin S in response to exposure to cigarette smoke, ambient particulate matter or micro-organisms [55, 56]. In addition, continuous exposure to cigarette smoke or biomass markedly depletes intracellular anti-oxidants such as glutathione, causing excessive oxidative stress, which suppresses macrophage bacterial phagocytosis and efferocytosis [57]. Therefore, macrophages in COPD generate a more pro-inflammatory milieu that promotes tissue injury. They also demonstrate defective immune surveillance and protective (phagocytic) functions that collectively contribute to the progression of COPD. The majority of acute exacerbations of COPD are triggered by either viral or bacterial respiratory infections that could alter the airway microbiome and cause frequent exacerbations [58], a clinical phenotype that is associated with a poor long-term outcome [51].

In COPD, M1 macrophages demonstrate enhanced pro-inflammatory capacity producing more TNF-α and MMPs [59], leading to increased extracellular matrix (ECM) deposition, elastin breakdown and excessive accumulation of collagen in the lung parenchyma. In contrast, Stout and co-workers [60] showed that M2 macrophages have a lower pro-inflammatory capacity (TNF-α, IL-1β, and IL-6), when stimulated. Morphological studies have shown that macrophages accumulate in areas of persistent inflammation in lung tissues of COPD including airway walls that lead to airway narrowing and obstruction and destructive changes in lung parenchyma [50, 61]. Several studies have shown phenotypic shifts in lung macrophages in COPD airways. Dewhurst and co-workers showed that the total number of macrophages in the airspaces of COPD subjects increased and morphologically became predominantly larger macrophages, which produced fewer pro-inflammatory cytokines and demonstrated reduced phagocytic ability [62]. Berenson and co-workers showed that macrophages from patients with COPD have impaired phagocytosis of respiratory pathogens which strongly correlated with COPD severity (FEV1% predicted) [63]. Studies from our laboratory recently showed that the majority of airspace macrophages in COPD do not express either M1 or M2 markers and that these “non-polarized” macrophages have significantly reduced phagocytic capacity compared to polarized (M1 or M2) macrophages [30]. In this study, airspace macrophages could be divided into 4 distinct groups using surface markers, as either M1, M2, dual positive for M1 & M2 (double polarized) or negative for both M1 & M2 markers (non-polarized). Using the phagocytosis of opsonized Staphylococcus aureus as a readout, we showed that the double polarized macrophages had the best phagocytic function while the non-polarized macrophages had the worst (Figure 5). These data highlight the importance of macrophage micro-environment that impacts polarization and ultimately function.

Figure 5.

Phagocytic activity of airspace macrophages, harvested from bronchial alveolar lavage, in subjects with COPD. CD40 was used to label M1 and CD163 to label M2 macrophages. The phagocytosis of flourescentlabelled StapholococcusAureas was measured using flow cytometry. Phagocytosis was reduced in COPD subjects compared to control subjects in all the different macrophage populations but was particular lowin the most abundant non-labeled or double negative cells.

The inability of macrophages to polarize may render the airways in COPD more vulnerable for colonization with pathogens and subsequent infection resulting in COPD exacerbation. The presence of large numbers of non-polarized macrophages in COPD may collectively reduce phagocytosis of pathogens and noxious particles, and in certain cases promote a pro-inflammatory milieu that contributes to airway and lung tissue injury and remodeling.

The air spaces have their own unique microbiome shown by next-generation sequencing technologies, such as 16 s RNA gene measurement, and studies in COPD cohorts have shown alterations in this microbiome that vary with the severity of COPD, during and after an acute COPD exacerbations, and with the use of inhaled steroids and/or antimicrobial treatment [64]. Alterations in the lung microbiome may contribute to the pathogenesis of COPD by impacting inflammatory and/or immune processes in the lungs. Lung macrophages play a central role in clearing harmful bacteria such as Haemophilus influenzae, Moraxella catarrhalis and S. pneumoniae, from the lungs, and this macrophage function deteriorates as the disease progresses [63] leading to colonization of airspaces and exacerbations of COPD [65]. Therefore, the defective phagocytic function of macrophages in COPD could contribute to the colonization of the airways with various bacteria, specifically those known to cause acute exacerbations and pneumonia during COPD.

One of the key functions of lung macrophages is to remove and clear cellular debris as well as dead or damage cells following an inflammatory insult to the lungs. This process, which is termed efferocytosis, is defective in subjects with COPD [66]. In most subjects with COPD, there are an access of neutrophils in the airspaces (as measured by bronchial alveolar lavage), which further increase during acute COPD exacerbations. Defective clearance of these recruited neutrophils results in the accumulation of necrotic neutrophils that indiscriminately release toxic granule proteins containing neutrophil elastase and proteases that has been associated with tissue damage and COPD progression [67]. Since LMs are the primary “janitors” of the lungs, dysfunctional processing and clearance of apoptotic and necrotic cells and cellular debris could contribute to ongoing lung tissue inflammation in subjects with COPD, even long after they stop smoking [68].

Lung macrophages are primary responsible for processing and removing of inhaled irritants and particulate matter from the lungs. In this process they release proinflammatory mediators that could also inflict damage to lung tissues, promoting a dysregulated inflammatory response, which may lead to dysfunctional tissue repair and a persistent state of chronic low-grade lung inflammation, a hallmark of COPD. Studies that unravel the mechanisms promoting macrophage anti-inflammatory and reparative functions could contribute to the development of more targeted therapeutic interventions to reduce the destructive inflammatory response induced by cigarette smoke and environmental exposures that eventually lead to COPD.

5. Macrophages and lung cancer

The majority of lung cancers (~80%) are diagnosed at an advanced stage with >50% in older subjects who are ineligible for surgery [69, 70] leaving chemotherapy as their primary treatment modality. A better understanding of tumor immunology and our body’s natural immune response to combat cancer over the last two decades have highlighted the key role macrophages play in containing the progression and metastasis of tumor cells. The tumor microenvironment (TME), characterized by low levels of nutrients, hypoxia and acidity, promotes tumor growth, invasion and metastasis [71]. The most abundant immune cells in or surrounding lung tumors are “tumor-associated macrophages” (TAMs), and the functions of these macrophages are determined by the TME [72].

The tumor microenvironment recruits both innate and adaptive immune cells to the tumor site, with macrophages abundant at all stages of tumorigenesis. Evidence suggests that TAMs originate predominantly from blood monocytes, and are recruited to tumor sites by tumor-derived chemotactic signals, including monocyte chemo-attractant protein-1 (MCP-1), which is also known as CCL 2 [73]. Initially these macrophages have an M1-like phenotype, activated by interferon-γ (IFN-γ), demonstrating pro-inflammatory functions with the capacity to facilitate tumor cell destruction. They are also characterized by a high production of nitric oxide (NO) and reactive oxygen intermediates (ROI), and pro-inflammatory cytokines, including TNF-α, IL- 1, IL- 12 and IL- 23 and MHC molecules [74]. These mediators recruit cytotoxic CD8+ T and NK cells that destroy the tumor cells [72] (Figure 6).

Figure 6.

The effects of tumor associated macrophages (TAMs) on tumor growth, progression and metastasis. The immune response elicited by lung cancer cells included recruitment of monocytes from the blood (via CCL2/CCR2 interaction) that convert to macrophages and are recruited tothe tumor niche where they become either M1 type TAMs under the influence of mediators such as GM-CSF and INF-λand inhibit tumor growth via secreting mediators such as TNF-α, IL-1β, IL-12 and 23. Mediators such as IL-10, IL-4 and M-CSF will change the macrophages to a more M2 type TAMs that promote tumor growth via immuno-suppressive properties that include blocking NK-cell and other T-cells tumorcidaleffects. The M2-TAMsenvironment will also promote angiogenesis via mediators such as VEGF and PDGF and tumor invasion and metastasis via mediators such as MMPs and TGF-B. other mediators in the tumor environment produced by M2 macropahagessuch as IL-6, TGF-βand MMPs have also been shown to elicit chemotherapy resistance of tumors.

With tumor progression the TME changes to a milieu that converts macrophages to a more M2-like phenotype macrophages, which suppress anti-tumor immune responses. This in turn promotes cell proliferation, angiogenesis and ultimately metastasis. Damage-associated molecular patterns (DAMPs) from dead or dying cells in the tumor microenvironment promote polarization of macrophages to immunosuppressive TAM [75]. These M2-like TAM have a similar phenotype as LPS-tolerant macrophages and is thought to contribute to the immunosuppression in the tumor microenvironments. They express a variety of mediators that inhibit the host anti-tumor immune responses. These include cell surface receptors, cytokines, chemokines and a variety of enzymes. This anti-tumor immune response is via inhibition of direct cell-to-cell contact between TAM receptors and their ligand counterpart death/inhibitory receptors expressed by the target immune effector cells. For example, TAMs express the ligand receptors for PD-1 and CTLA-4 that upon activation suppress cytotoxic functions of T- cell and NK cells. They also express the ligand for the death receptors FAS and TRAIL that triggers T-cells and induce caspase dependent apoptosis of tumor cells. The TAMs produce TGF-β that impedes the cytotoxicity of NK cells, and promotes expression of PD-L1 that impedes the anti-tumor activity of T cells [76]. They also secrete cytokines IL-10 that inhibit T cells effector functions and chemokines such as CCL5, CCL20, CCL22 that recruit Treg cells that are immunosuppressive.

The density of macrophages, in particular M2 phenotypes, has been associated with a poor prognosis in almost all human cancer types including lung cancers in clinical trials [77]. The CD68⁺CD163⁺ or CD68⁺CD206⁺ markers on TAM are used to identify M2-like macrophages and these macrophages are associated with more dense peritumoral lymphatic microvessels, a pathological feature that relate to poor patients’ prognoses in subjects with lung cancer [78]. Furthermore, an increased density of CD68⁺CD163⁺ macrophages in tumor nests and stroma was associated with lymph node metastases [78] and Cao et al showed expression levels of CD 68⁺CD163⁺on M2 macrophages were inversely correlated with overall survival, and disease free survival in non-small cell lung cancer (NSCLC) [77].

The overwhelming evidence that TAMs and especially M2 macrophages promote tumorigenesis has made TAMs a target for a novel anti-tumor strategy in lung cancer. Several strategies that have been explored include blockade of the CCL2-CCR 2 interaction and the CSF1-CSF1R recruitment of monocyte pathways that decrease TAM infiltration, thereby reversing their immunosuppressive effects [73]. Mu and co-workers have suggested that reprogramming TAM macrophages can be a promising approach to address immunosuppressive failure in the cancer environment [79]. Re-educating TAMs to a M1 phenotype or switching M2 to M1 macrophages with several drugs has also shown promise including the use of BTH1677 (a yeast β-glucan immunomodulator), hydroxychloroquine, and celecoxib [80, 81]. Another approach is to block the levels of critical TAM-secreted cytokines involved in tumor biology such as CCL18, CCL 22, and MIP-3α, which are mainly produced by M2-type macrophages, and promote malignant behavior of tumors [82, 83]. Furthermore, nanoparticles or nanoparticle-based drug delivery are more reliable and effective in regulating the macrophage phenotype by ensuring that the drug reaches the cancer site without off-target activities [84, 85]. In addition, materials used in nanoparticle production, including TiO2 and Ag, may preferentially polarize TAMs towards an M1 phenotype [86, 87].

6. Macrophages and their role in asthma

Asthma is characterized by chronically inflamed airways, leading to remodeling and constriction in response to a wide variety of stimuli. One of the prototypical traits of asthma is airway hyperresponsiveness. Typical triggers of bronchoconstriction are aero-allergens and viral pathogens but non-specific stimuli such as irritating chemicals, cold air, and exercise (increase flow) can also trigger this response. Airway inflammation is characterized by increased mucus secretion, thickening of all the components of the airway wall and luminal narrowing, leading to symptoms of shortness of breath, chest tightness, wheezing, and cough [88]. The inflammatory response in the airways is typically type 2 (Th2) in which allergens are detected by pattern recognition receptors (PRRs) on epithelial cells, which, upon activation, secrete alarmins such as interleukin (IL)-33, IL-25 and thymic stromal lymphopoietin (TSLP) and cytokines such as GM-CSF. These mediators induce type 2 inflammation by activating dendritic cells (DCs) and type 2 innate lymphoid cells (ILC2s) and differentiating naïve T cells into T helper (Th) 2 cells, which produce IL-4, IL-5, and IL-13 [88, 89]. These type 2 cytokines are involved in producing IgE and recruiting eosinophils into the airways. A classic type 2 inflammatory response can be suppressed by treatment with corticosteroids. In adults a substantial subset of patients with non-atopic asthma may be driven by a Th1 response, which is characterized by infiltration of neutrophils. Th1 inflammation is more difficult to treat, as it is often resistant to corticosteroids [90].

Macrophages are the most abundant leukocytes found in alveoli, and in small as well as conducting airways, suggesting that they have an important role in providing protection against foreign inhaled particulate matter including allergens, pathogens and noxious gasses. Links between lung macrophages and airway inflammation, including eosinophilic inflammation and airway remodeling are well documented in asthma [91, 92, 93, 94, 95]. However, it is not still unclear whether macrophages have a predominant pro-inflammatory or regulatory role in asthma.

As discussed previously, macrophages have the ability to adapt to their microenvironment (plasticity). In asthmatic lung inflammation this plasticity of macrophage function is most likely responsible for their apparent dual or contrasting roles as pro-inflammatory versus immunosuppressive effector immune cells. Zaslona and co-workers showed that during allergic inflammation, resident alveolar macrophages proliferate locally and exert a protective effect on allergic inflammation, whereas recruited monocytes/macrophages aggravate allergic inflammation [96]. These recruited monocytes are also involved in the characteristic chronic remodeling of airways [97]. When circulating monocytes were depleted by intravenous injection of clodronate, there was significant attenuation of allergic inflammation in the airways and when clodronate was administered via the intratracheal route to deplete resident airspace macrophages, eosinophilic inflammation was enhanced [97]. Collectively, these data suggest that resident macrophages serve to maintain lung homeostasis by suppressing inflammatory responses, while recruited monocytes primarily promote allergic inflammation. The picture that emerges is one of rapid recruitment of monocytes to fight the perceived dangers of the allergen by mounting an inflammatory response and then subsequently expanding the pool of suppressive AMs in an attempt to restore homeostasis (Figure 7). The dominant macrophages in allergic asthma are alternatively activated AMs, which respond to IL-4/IL-13. Although the presence of these macrophages correlates well with the severity of airway inflammation, it is not clear whether these macrophages significantly contribute to the allergic inflammatory response or are the downstream consequence of the allergic inflammation [98, 99, 100]. This issue has been addressed in several experimental studies using transgenic murine models of asthma [101, 102, 103]. Together, these studies have shown distinct differences between resident and recruited macrophages in contributing to asthmatic inflammation. However, as there may be significant differences between mice and humans, these findings should be interpreted cautiously. Recent studies have shown that there is a mixed population of macrophage phenotypes in both human and mouse models of allergic inflammation [101, 102, 103] with functional studies showing that IFNγ-stimulated macrophages (M1) prevent the development of allergic inflammation in mice by suppressing DC maturation [104]. However in human studies of subjects with established asthma, there is a higher number of macrophages expressing the IFNγ-activated transcription factor, interferon regulatory factor 5 (IRF5), in airways which in turn correlates with the severity of airflow obstruction [102]. These studies highlight the potential dual role of classically activated macrophages (M1) in asthma.

Figure 7.

After allergen exposure, there is rapid recruitment of monocytes from the blood that become inflammatory macrophages that predominantly promote acute inflammatory responses in the airspace. Resident macrophages (ResMac), which are largely self-replicating, act to suppress the acute inflammation in an attempt torestore homeostasis. It is as yetunclear whether ResMaccan arise directly from recruited monocytes or from IMs as intermediate progenitors. These newly recruited monocyte-derived cells appear to oppose the suppressive actions of resident AMs. b inflammation becomes chronic after repeated exposures to allergen with the increased recruitment of immune cells and consequent elevated levels of cytokines such as IL-4, IL-13, and IFNγ. In response to these signals, ResMaccan polarize across a continuum of activation phenotypes, losing their suppressive functions and gaining pathogenic functions. IL-4/IL-13-induced AMs promote type 2/eosinophilic inflammation, and IFNγ-induced AMs are associated with type I/neutrophilic inflammation. It is as yetunclear whether these activated phenotypes can arise from recruited monocytes directly or from IMs as an intermediate progenitor.

The presence of type 1 cytokines, such as IL-6, would be expected during host responses to viruses and to a variety of exogenous and endogenous ligands that trigger asthma exacerbations [88, 105]. Infections with common respiratory viruses such as rhinovirus are a major trigger of asthma exacerbations and in these virus-induced exacerbations, there is direct interaction of the rhinovirus with airspace macrophages [105]. Asthma models in mice, support the role of macrophages in viral induced exacerbations, but interestingly, the pathogenic macrophage phenotype involved varies with the underlying inflammatory milieu. In mice with a predominant type 2 inflammatory response, viral infection induces activation of alternative AMs, which amplifies eosinophil recruitment into the airways. However, mice with predominantly type 1 inflammation demonstrate mostly classical activation of AMs, skewing the phenotype to a more neutrophilic inflammation upon virus infection [106].

The role of lung macrophages in allergic asthma is still evolving. Current paradigms suggest that macrophages of different origins or phenotypes have the potential to be either protective and/or harmful in different stages of allergic airway disease. In asthma, macrophages may have a dual role: with induction of the allergic inflammatory response they may be predominantly regulatory to resolve the inflammation but when persistently activated, they may contribute to chronic inflammation and further damage of the airways (Figure 7). The role of resident and recruited macrophages as well as the macrophage phenotypes in the pathogenesis of allergic airways disease requires additional studies.

7. Macrophages in interstitial lung disease

Chronic and aberrant lung repair responses that lead to irreversible scarring and remodeling of the airways and lung parenchyma are hallmarks of pulmonary fibrotic diseases. These diseases are characterized by excessive deposition of ECM leading to fibrotic remodeling of lungs and irreversible lung dysfunction [107]. Alveolar macrophages have been shown to be involved in ECM processing by secreting matrix metalloproteinases (MMPs) such as MMP9, a type IV collagenase known to degrade extracellular matrix, and numerous non-matrix protein, which have been demonstrated in a murine model of lung fibrosis induced by bleomycin [108, 109] These macrophages also endocytose collagen and produce soluble mediators required for collagen-degradation, and enhance the activity of fibroblast-specific protein-1 (FSP-1) that increases the proliferation and production of ECM by lung fibroblasts [110]. Recent animal studies have shown that the AMs involved in the bleomycin murine lung fibrosis model are monocyte derived and not resident AMs. Depletion of resident AMs by intratracheal instillation of liposomal clodronate before bleomycin administration does not alter the fibrotic response, indicating that resident AMs are dispensable for the development of fibrosis [111]. To support this concept, deletion of the anti-apoptotic protein c-Flip in circulating monocytes (the precursors of monocyte-derived AMs) in mice showed that the number of monocyte-derived AMs decreased, which was accompanied by a reduction in lung fibrosis with bleomycin injury [112] Gene expression studies of resident AMs and monocyte-derived AMs also indicate that only monocyte-derived AMs have a profibrotic gene profile in bleomycin induced model of lung fibrosis [112] Single cell transcriptomic studies support these findings [113]. Transgenic reporter mice that marked the Cx3cr1-expressing transitional macrophages showed that these macrophages localize to fibrotic niches suggesting that these transitional macrophages arise from monocytes and interact with fibroblasts to drive fibrosis, in concordance to studies of Misharin et al. and McCubbrey et al. [111, 112].

In human interstitial lung diseases, AMs show greater heterogeneity compared with healthy lungs. In subjects with idiopathic pulmonary fibrosis, lung tissues have a higher proportions of AMs lacking CD71, a transferrin receptor. These CD71 negative macrophages were also more immature and showed impaired phagocytosis and enhanced expression of profibrotic genes [114]. Single cell RNA sequencing analysis from eight normal compared to eight lungs from advanced lung fibrosis subjects (from patients undergoing lung transplantation) supports the idea of increased heterogeneity of macrophages in fibrotic lungs and subsets of AMs that were enriched with pro-fibrotic genes [115]. Together these studies suggest lung macrophages contribute significantly to the pathogenesis of interstitial fibrotic lung disease. However, it is still unclear whether these macrophages are from the resident pool or newly recruited into the lung, what signals attract these macrophages or whether they are just secondary to the change in microenvironment, and lastly, what their contribution is to progression of the disease [116].

Interstitial macrophages (IMs) are ideally positioned to participate in the lung fibrotic processes. In a radiation-induced lung fibrosis (RIF) mouse models, IMs have acquired a pro-fibrotic phenotype, and express high levels of CD206, a marker of alternatively activated M2 macrophages [117]. Interstitial macrophages isolated from RIF lungs promote fibrosis by inducing the differentiation myocytes to myofibroblasts. Myofibroblasts have been shown to be key players in the initiation and progression of lung fibrosis [118]. Depletion of IMs in the RIF mouse model with CSF1-R specific mAb exerts an anti-fibrotic effect, while the depletion of AMs by intranasal administration of clodronate liposomes has no effects on RIF [117]. Recent studies showed that lung fibrosis was exacerbated after depletion of Lyve1_hiMHCII_lo IM1s during the induction of fibrosis, which suggested that these IM1 might have an early antifibrotic role. This is in keeping with expression of high levels of genes associated with wound healing, repair, and fibrosis in this subset of IMs [20].

8. Conclusion

It is clear that lung macrophages have an essential role in both lung homeostasis and in disease states. Here we have highlighted the specific origins, unique phenotypes and functions of the two main populations of lung macrophages, AMs and IMs, and emphasized the distinct roles in common lung diseases. It is still not clear if lung macrophages derived from circulating monocytes eventually become indistinguishable from embryonically derived resident AMs in chronic lung diseases or if they are long lived in the lungs with a slightly different genomic and functional profile thereby changing the lung macrophage landscape. There is still a lack of knowledge of IMs in terms of how they are maintained and their importance in lung conditions. With the increasing power of phenotyping and genomic techniques, there will be opportunities to better characterize the origin, subtypes and functions of AMs and IMs in both health and disease. There is a pressing need to focus strongly on macrophage function, especially in regards to mechanisms of their role in inflammatory and anti-inflammatory pathways, their turnover and survival during the immune response, and their interactions with recruited macrophages in promoting wound healing. Refining our understanding of macrophage plasticity and the role of distinct populations of macrophages in various pulmonary diseases will lead to the identification of novel macrophage-targeted therapies.

Acknowledgments

This work was grant supported by Canadian Institute of Health Research, the British Columbia Lung Association, and the Providence Airway Centre. Stephan F. van Eeden is the Canadian Institute for Health Research/Glaxo Smith Kline (CIHR/GSK) Professor in Chronic Obstructive Pulmonary Disease and Don D. Sin holds a Tier 1 Canada Research Chair in COPD and the HLI De Lazzari Family Chair.