DOI: 10.1111/exd.12727
Commentary
www.wileyonlinelibrary.com/journal/EXD
Deciphering the alternatively activated (M2) phenotype of macrophages in scleroderma Mirko Manetti Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy Correspondence: Mirko Manetti, PhD, Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, I-50134 Florence, Italy, Tel.: +39-055-2758077, Fax: +39-055-4379500, e-mails: [email protected] or mirko. [email protected]
Key words: alternative macrophage activation – dermal fibrosis – localized scleroderma – M2 macrophages – systemic sclerosis
Accepted for publication 9 April 2015
Scleroderma refers to a heterogeneous group of autoimmune fibrosing disorders which includes systemic sclerosis (SSc) and localized scleroderma, also known as morphea (1). SSc is characterized by widespread vascular injury, autoimmunity and progressive fibrosis of the skin and multiple internal organs, resulting in multifaceted and severe clinical manifestations (2). Hence, SSc has the highest disease-specific mortality of all autoimmune connective tissue diseases. In localized scleroderma, the lesions are usually limited to the skin and subcutaneous tissue, as fatty tissue, muscle and sometimes bone beneath the cutaneous lesions, causing significant morbidity but without affecting mortality (1). In the past decade, substantial advances have been made in deciphering the molecular bases of these disorders; however, their aetiologies and pathogenesis remain incompletely understood (1,2). As a consequence, to date there is no effective standard treatment to reverse or slow down the progression of fibrosis. Although important differences in signalling pathways and gene signatures indicate that SSc and localized scleroderma are likely biologically distinct processes, there is also evidence of activation of similar inflammatory and profibrotic pathways (1). In particular, in both disorders an early inflammatory process clustered around small vessels is thought to evolve into an overt and irreversible fibrotic process. Indeed, histopathological hallmarks of early disease stages are perivascular inflammatory infiltrates dominated by macrophages and T cells. An altered balance between Th1 and Th2 cytokines towards a Th2-polarized immune response and the release of several mediators, including transforming growth factor (TGF)-b, connective tissue growth factor/CCN2, platelet-derived growth factor, monocyte chemoattractant protein1/CCL2, interleukin (IL)-4 and IL-13, ultimately leads to a constitutive activation of fibroblasts, which transdifferentiate into apoptosis-resistant profibrotic myofibroblasts and produce an exaggerated amount of collagen and other extracellular matrix components (2,3). Myofibroblasts further secrete growth factors and fibrogenic cytokines that perpetuate the fibrotic process in an autocrine or paracrine manner (3). A growing body of evidence suggests that macrophage plasticity may have a pivotal role in this complex scenario (4,5). In fact, macrophages have an ability to efficiently respond to environmental signals with remarkable plasticity and undergo different forms
of polarized activation that can be roughly categorized as classically activated (M1) and alternatively activated (M2) macrophages (4–6). Classically activated macrophages are effector phagocytes activated by interferon-c, lipopolysaccharide and microbial products. M1 macrophages have the IL-12high, IL-23high and IL-10low phenotype, produce inducible nitric oxide synthase and inflammatory cytokines such as IL-1b, tumour necrosis factor-a and IL-6 to promote active inflammation, and exhibit enhanced microbicidal or tumouricidal capacity (4). M2-polarized macrophages are activated mostly by IL-4, IL-13 and IL-10, as well as by CCL2 and IL-6 (4,6,7). These M2 macrophages have the IL-12low, IL-23low and IL-10high phenotype and express elevated levels of several receptors such as haemoglobin scavenger receptor (CD163), class A scavenger receptor (CD204) and c-type mannose receptor-1 (CD206) (3,4,6). Moreover, in vitro and in vivo studies in mice have shown that the M2 phenotype is characterized by the elevated expression of chitinase-3-like protein-3 (also known as Ym1), resistin-like molecule-a (also known as Fizz1), major histocompatibility complex class II antigens and the enzyme arginase-1 (3). The expression of arginase-1 by M2 macrophages is of particular interest because this enzyme controls L-proline production, which is required for collagen synthesis by activated fibroblasts/myofibroblasts. In addition, M2 macrophages have been implicated in the development of Th2 effector responses, suppression of M1 responses, production of fibrogenic cytokines (e.g. TGF-b), recruitment of circulating fibroblast precursors (fibrocytes) and promotion of angiogenesis (3–6). Because they are commonly observed during the peak of the profibrotic immune response, M2 macrophages have been proposed to be important inducers of wound healing and tissue fibrosis and remodelling (3). In recent years, M2 macrophage polarization has increasingly been implicated in the pathogenesis of SSc and localized scleroderma (4,5,8–11). The possible mechanisms and functions of alternative activation of macrophages in dermal fibrosis are depicted in Fig. 1. Infiltrating macrophages in skin lesions of both SSc and localized scleroderma express either CD163 or CD204, suggesting that M2 macrophages may play a pivotal role in the development of dermal fibrosis in these conditions (4,5). Moreover, it has been reported that in SSc patients, a significant portion of circulating
576
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 576–578
Commentary
Figure 1. Possible mechanisms and functions of alternative activation of macrophages in dermal fibrosis. In the early inflammatory phase of the fibrotic process, in situ M2 polarization of macrophages may be induced by infiltrating Th2 cells via the release of several mediators, mainly interleukin (IL)-4 and IL-13. Moreover, M2 macrophages may also arise from a distinct subset of activated monocytes (CD14brightCD163+CD204+) recruited into the dermis from the bloodstream. M2 macrophages highly express several cell-surface receptors, such as CD163, CD204 and CD206, and may take centre stage in the induction and progression of fibrosis. In particular, M2 macrophages may further promote Th2 effector responses, neovascularization, and contribute to the transition of fibroblasts into apoptosis-resistant profibrotic myofibroblasts via the production and release of fibrogenic cytokines and growth factors, such as transforming growth factor (TGF)-b.
monocytes abnormally differentiates into a distinct CD14brightCD163+CD204+ cell subset which may represent the source of CD163+ and CD204+ infiltrating macrophages observed in the affected skin (4). Therefore, it is conceivable that lesional M2 macrophages arise either from the recruitment of activated monocytic precursors or from in situ polarization. Serum levels of soluble CD163 are increased in patients with SSc and may reflect the ongoing alternative macrophage activation (8). Interestingly, several investigators have shown increased serum levels of the M2 inducers IL-4, IL-13 and IL-10 in SSc patients (4). Lung microarray data from patients with SSc-related interstitial lung disease revealed a strong upregulation of several alternatively activated macrophage markers, such as CD163 and the chemokine CCL18, which correlated with pulmonary disease progression (9). Another study suggested an important role for M2-polarized alveolar macrophages in the pathogenesis of different forms of pulmonary fibrosis, including SSc-related interstitial lung disease, and indicated that both IL-4 and IL-10 may account for human alveolar macrophage phenotype shift to M2 (10). In addition, the expression of the alternative monocyte/macrophage activation marker CD206 was found to be highly increased in monocytes from limited cutaneous SSc patients with pulmonary arterial hypertension (PAH) and correlated with pulmonary artery pressure and PAH-related mortality (11). In the light of the aforementioned observations, the study by Kato et al. (12) in this issue of the journal provides further insight into the mechanisms that may drive alternative macrophage
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 576–578
activation in scleroderma. The authors show for the first time that glycosylation of cell-surface proteins by N-acetylglucosaminyltransferase-V (GnT-V) may substantially contribute to a macrophage shift towards the M2 phenotype during dermal fibrosis. In fact, strong expression of GnT-V was observed in CD163+ M2 macrophages and T cells in the lesional skin of SSc and localized scleroderma patients (12). Further, to determine the functional relevance of GnT-V in the induction of dermal fibrosis the authors employed the bleomycin-induced scleroderma mouse model. Strikingly, mice deficient for the gene encoding GnT-V (MGAT5 / ) were resistant to bleomycin-induced scleroderma, as shown by significant lower dermal thickness and reduced myofibroblast counts and collagen content compared with wild-type mice (12). Of note, although total macrophages in early inflammatory skin lesions were not different between the two groups, the number of M2 macrophages was greatly reduced in MGAT5 / mice. Further in vitro investigation revealed that bone marrowderived macrophages from MGAT5 / mice displayed a significantly reduced expression of Fizz1 and Ym1 M2 markers upon challenge with IL-4. Thus, the authors conclude that activation towards the M2 phenotype might be disturbed in MGAT5 / mice (12). The absence of GnT-V might crucially interfere with M2 macrophage polarization, which in turn may counteract the development of dermal fibrosis. Interestingly, the GnT-V enzyme catalyses the formation of cell-surface oligosaccharide structures that can be associated with galectin-3, a b-galactoside-binding lectin modulating intracellular signal transduction which has also been implicated in the regulation of alternative macrophage activation and fibrosis (13). Further in-depth studies are now required to ascertain whether targeting GnT-V and/or galectin-3 might represent a reliable strategy to block the M1/M2 macrophage phenotypical shift in the early inflammatory phase of the fibrotic remodelling process. It is apparent that although there has been striking progress in understanding the mechanisms and functions of alternatively activated macrophages, the majority of data available to date arise from in vitro studies. Much remains to be discovered using in vivo translational approaches, without, however, forgetting that human being is not a mouse. In fact, it appears that important M2 macrophage markers in the mouse, such as Fizz1 and Ym1, lack homologues in humans, and others such as arginase-1 are not upregulated in human macrophages by IL-4 and IL-13, despite clear gene homology (6). To date, the mannose receptor CD206, which is highly induced by IL-4/IL-13 in human monocytes, is considered the most reliable marker of alternative activation of monocytes/macrophages in humans (11). Additional studies will likely contribute to identify additional selective M2 markers worth targeting. Considering the evidence that M2 macrophage polarization may participate in different pathogenetic mechanisms and clinical manifestations of SSc, such as skin and lung fibrosis and PAH (4,8–11), there is realistic hope that targeting this process will provide a substantial advance in the treatment of this heterogeneous disorder.
Conflict of interest The author has declared no conflicting interests.
577
Commentary
References 1 Fett N. Clin Dermatol 2013: 31: 432–437. 2 Hunzelmann N, Krieg T. Exp Dermatol 2010: 19: 393–400. 3 Wynn T A, Ramalingam T R. Nat Med 2012: 18: 1028–1040. 4 Higashi-Kuwata N, Jinnin M, Makino T et al. Arthritis Res Ther 2010: 12: R128. 5 Higashi-Kuwata N, Makino T, Inoue Y et al. Exp Dermatol 2009: 18: 727–729.
578
6 Gordon S, Martinez F O. Immunity 2010: 32: 593–604. 7 Roca H, Varsos Z S, Sud S et al. J Biol Chem 2009: 284: 34342–34354. 8 Nakayama W, Jinnin M, Makino K et al. Rheumatol Int 2012: 32: 403–407. 9 Christmann R B, Sampaio-Barros P, Stifano G et al. Arthritis Rheumatol 2014: 66: 714– 725.
10 Pechkovsky D V, Prasse A, Kollert F et al. Clin Immunol 2010: 137: 89–101. 11 Christmann R B, Hayes E, Pendergrass S et al. Arthritis Rheum 2011: 63: 1718–1728. 12 Kato A, Yutani M, Terao M et al. Exp Dermatol 2015: doi: 10.1111/exd.12730. 13 MacKinnon A C, Farnworth S L, Hodkinson P S et al. J Immunol 2008: 180: 2650–2658.
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 576–578
Commentary
www.wileyonlinelibrary.com/journal/EXD
Deciphering the alternatively activated (M2) phenotype of macrophages in scleroderma Mirko Manetti Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy Correspondence: Mirko Manetti, PhD, Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, I-50134 Florence, Italy, Tel.: +39-055-2758077, Fax: +39-055-4379500, e-mails: [email protected] or mirko. [email protected]
Key words: alternative macrophage activation – dermal fibrosis – localized scleroderma – M2 macrophages – systemic sclerosis
Accepted for publication 9 April 2015
Scleroderma refers to a heterogeneous group of autoimmune fibrosing disorders which includes systemic sclerosis (SSc) and localized scleroderma, also known as morphea (1). SSc is characterized by widespread vascular injury, autoimmunity and progressive fibrosis of the skin and multiple internal organs, resulting in multifaceted and severe clinical manifestations (2). Hence, SSc has the highest disease-specific mortality of all autoimmune connective tissue diseases. In localized scleroderma, the lesions are usually limited to the skin and subcutaneous tissue, as fatty tissue, muscle and sometimes bone beneath the cutaneous lesions, causing significant morbidity but without affecting mortality (1). In the past decade, substantial advances have been made in deciphering the molecular bases of these disorders; however, their aetiologies and pathogenesis remain incompletely understood (1,2). As a consequence, to date there is no effective standard treatment to reverse or slow down the progression of fibrosis. Although important differences in signalling pathways and gene signatures indicate that SSc and localized scleroderma are likely biologically distinct processes, there is also evidence of activation of similar inflammatory and profibrotic pathways (1). In particular, in both disorders an early inflammatory process clustered around small vessels is thought to evolve into an overt and irreversible fibrotic process. Indeed, histopathological hallmarks of early disease stages are perivascular inflammatory infiltrates dominated by macrophages and T cells. An altered balance between Th1 and Th2 cytokines towards a Th2-polarized immune response and the release of several mediators, including transforming growth factor (TGF)-b, connective tissue growth factor/CCN2, platelet-derived growth factor, monocyte chemoattractant protein1/CCL2, interleukin (IL)-4 and IL-13, ultimately leads to a constitutive activation of fibroblasts, which transdifferentiate into apoptosis-resistant profibrotic myofibroblasts and produce an exaggerated amount of collagen and other extracellular matrix components (2,3). Myofibroblasts further secrete growth factors and fibrogenic cytokines that perpetuate the fibrotic process in an autocrine or paracrine manner (3). A growing body of evidence suggests that macrophage plasticity may have a pivotal role in this complex scenario (4,5). In fact, macrophages have an ability to efficiently respond to environmental signals with remarkable plasticity and undergo different forms
of polarized activation that can be roughly categorized as classically activated (M1) and alternatively activated (M2) macrophages (4–6). Classically activated macrophages are effector phagocytes activated by interferon-c, lipopolysaccharide and microbial products. M1 macrophages have the IL-12high, IL-23high and IL-10low phenotype, produce inducible nitric oxide synthase and inflammatory cytokines such as IL-1b, tumour necrosis factor-a and IL-6 to promote active inflammation, and exhibit enhanced microbicidal or tumouricidal capacity (4). M2-polarized macrophages are activated mostly by IL-4, IL-13 and IL-10, as well as by CCL2 and IL-6 (4,6,7). These M2 macrophages have the IL-12low, IL-23low and IL-10high phenotype and express elevated levels of several receptors such as haemoglobin scavenger receptor (CD163), class A scavenger receptor (CD204) and c-type mannose receptor-1 (CD206) (3,4,6). Moreover, in vitro and in vivo studies in mice have shown that the M2 phenotype is characterized by the elevated expression of chitinase-3-like protein-3 (also known as Ym1), resistin-like molecule-a (also known as Fizz1), major histocompatibility complex class II antigens and the enzyme arginase-1 (3). The expression of arginase-1 by M2 macrophages is of particular interest because this enzyme controls L-proline production, which is required for collagen synthesis by activated fibroblasts/myofibroblasts. In addition, M2 macrophages have been implicated in the development of Th2 effector responses, suppression of M1 responses, production of fibrogenic cytokines (e.g. TGF-b), recruitment of circulating fibroblast precursors (fibrocytes) and promotion of angiogenesis (3–6). Because they are commonly observed during the peak of the profibrotic immune response, M2 macrophages have been proposed to be important inducers of wound healing and tissue fibrosis and remodelling (3). In recent years, M2 macrophage polarization has increasingly been implicated in the pathogenesis of SSc and localized scleroderma (4,5,8–11). The possible mechanisms and functions of alternative activation of macrophages in dermal fibrosis are depicted in Fig. 1. Infiltrating macrophages in skin lesions of both SSc and localized scleroderma express either CD163 or CD204, suggesting that M2 macrophages may play a pivotal role in the development of dermal fibrosis in these conditions (4,5). Moreover, it has been reported that in SSc patients, a significant portion of circulating
576
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 576–578
Commentary
Figure 1. Possible mechanisms and functions of alternative activation of macrophages in dermal fibrosis. In the early inflammatory phase of the fibrotic process, in situ M2 polarization of macrophages may be induced by infiltrating Th2 cells via the release of several mediators, mainly interleukin (IL)-4 and IL-13. Moreover, M2 macrophages may also arise from a distinct subset of activated monocytes (CD14brightCD163+CD204+) recruited into the dermis from the bloodstream. M2 macrophages highly express several cell-surface receptors, such as CD163, CD204 and CD206, and may take centre stage in the induction and progression of fibrosis. In particular, M2 macrophages may further promote Th2 effector responses, neovascularization, and contribute to the transition of fibroblasts into apoptosis-resistant profibrotic myofibroblasts via the production and release of fibrogenic cytokines and growth factors, such as transforming growth factor (TGF)-b.
monocytes abnormally differentiates into a distinct CD14brightCD163+CD204+ cell subset which may represent the source of CD163+ and CD204+ infiltrating macrophages observed in the affected skin (4). Therefore, it is conceivable that lesional M2 macrophages arise either from the recruitment of activated monocytic precursors or from in situ polarization. Serum levels of soluble CD163 are increased in patients with SSc and may reflect the ongoing alternative macrophage activation (8). Interestingly, several investigators have shown increased serum levels of the M2 inducers IL-4, IL-13 and IL-10 in SSc patients (4). Lung microarray data from patients with SSc-related interstitial lung disease revealed a strong upregulation of several alternatively activated macrophage markers, such as CD163 and the chemokine CCL18, which correlated with pulmonary disease progression (9). Another study suggested an important role for M2-polarized alveolar macrophages in the pathogenesis of different forms of pulmonary fibrosis, including SSc-related interstitial lung disease, and indicated that both IL-4 and IL-10 may account for human alveolar macrophage phenotype shift to M2 (10). In addition, the expression of the alternative monocyte/macrophage activation marker CD206 was found to be highly increased in monocytes from limited cutaneous SSc patients with pulmonary arterial hypertension (PAH) and correlated with pulmonary artery pressure and PAH-related mortality (11). In the light of the aforementioned observations, the study by Kato et al. (12) in this issue of the journal provides further insight into the mechanisms that may drive alternative macrophage
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 576–578
activation in scleroderma. The authors show for the first time that glycosylation of cell-surface proteins by N-acetylglucosaminyltransferase-V (GnT-V) may substantially contribute to a macrophage shift towards the M2 phenotype during dermal fibrosis. In fact, strong expression of GnT-V was observed in CD163+ M2 macrophages and T cells in the lesional skin of SSc and localized scleroderma patients (12). Further, to determine the functional relevance of GnT-V in the induction of dermal fibrosis the authors employed the bleomycin-induced scleroderma mouse model. Strikingly, mice deficient for the gene encoding GnT-V (MGAT5 / ) were resistant to bleomycin-induced scleroderma, as shown by significant lower dermal thickness and reduced myofibroblast counts and collagen content compared with wild-type mice (12). Of note, although total macrophages in early inflammatory skin lesions were not different between the two groups, the number of M2 macrophages was greatly reduced in MGAT5 / mice. Further in vitro investigation revealed that bone marrowderived macrophages from MGAT5 / mice displayed a significantly reduced expression of Fizz1 and Ym1 M2 markers upon challenge with IL-4. Thus, the authors conclude that activation towards the M2 phenotype might be disturbed in MGAT5 / mice (12). The absence of GnT-V might crucially interfere with M2 macrophage polarization, which in turn may counteract the development of dermal fibrosis. Interestingly, the GnT-V enzyme catalyses the formation of cell-surface oligosaccharide structures that can be associated with galectin-3, a b-galactoside-binding lectin modulating intracellular signal transduction which has also been implicated in the regulation of alternative macrophage activation and fibrosis (13). Further in-depth studies are now required to ascertain whether targeting GnT-V and/or galectin-3 might represent a reliable strategy to block the M1/M2 macrophage phenotypical shift in the early inflammatory phase of the fibrotic remodelling process. It is apparent that although there has been striking progress in understanding the mechanisms and functions of alternatively activated macrophages, the majority of data available to date arise from in vitro studies. Much remains to be discovered using in vivo translational approaches, without, however, forgetting that human being is not a mouse. In fact, it appears that important M2 macrophage markers in the mouse, such as Fizz1 and Ym1, lack homologues in humans, and others such as arginase-1 are not upregulated in human macrophages by IL-4 and IL-13, despite clear gene homology (6). To date, the mannose receptor CD206, which is highly induced by IL-4/IL-13 in human monocytes, is considered the most reliable marker of alternative activation of monocytes/macrophages in humans (11). Additional studies will likely contribute to identify additional selective M2 markers worth targeting. Considering the evidence that M2 macrophage polarization may participate in different pathogenetic mechanisms and clinical manifestations of SSc, such as skin and lung fibrosis and PAH (4,8–11), there is realistic hope that targeting this process will provide a substantial advance in the treatment of this heterogeneous disorder.
Conflict of interest The author has declared no conflicting interests.
577
Commentary
References 1 Fett N. Clin Dermatol 2013: 31: 432–437. 2 Hunzelmann N, Krieg T. Exp Dermatol 2010: 19: 393–400. 3 Wynn T A, Ramalingam T R. Nat Med 2012: 18: 1028–1040. 4 Higashi-Kuwata N, Jinnin M, Makino T et al. Arthritis Res Ther 2010: 12: R128. 5 Higashi-Kuwata N, Makino T, Inoue Y et al. Exp Dermatol 2009: 18: 727–729.
578
6 Gordon S, Martinez F O. Immunity 2010: 32: 593–604. 7 Roca H, Varsos Z S, Sud S et al. J Biol Chem 2009: 284: 34342–34354. 8 Nakayama W, Jinnin M, Makino K et al. Rheumatol Int 2012: 32: 403–407. 9 Christmann R B, Sampaio-Barros P, Stifano G et al. Arthritis Rheumatol 2014: 66: 714– 725.
10 Pechkovsky D V, Prasse A, Kollert F et al. Clin Immunol 2010: 137: 89–101. 11 Christmann R B, Hayes E, Pendergrass S et al. Arthritis Rheum 2011: 63: 1718–1728. 12 Kato A, Yutani M, Terao M et al. Exp Dermatol 2015: doi: 10.1111/exd.12730. 13 MacKinnon A C, Farnworth S L, Hodkinson P S et al. J Immunol 2008: 180: 2650–2658.
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 576–578
