Macrophage polarization: Reaching across the aisle?

Macrophage polarization can influence the pathogenesis of many human diseases; however, the transcriptional program regulating this process remains poorly characterized. Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar highlight the high diversity of M1-to-M2 repolarization exerted by distinct M2 stimuli through transcriptome-based pathway analysis and provide a new approach to phenotype human macrophages in clinically relevant disease states, such as cystic fibrosis (CF) and asthma. An extensive literature documents the high versatility of macrophages, which are able to display a wide range of functional states in a tissue- and time-dependent manner. Macrophage phenotype is dictated by a network of environmental signals.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar, 4Becker M. De Bastiani M.A. Parisi M.M. Guma F.T. Markoski M.M. Castro M.A. et al.Integrated transcriptomics establish macrophage polarization signatures and have potential applications for clinical health and disease.Sci Rep. 2015; 5: 13351Crossref PubMed Scopus (36) Google Scholar Two main subsets of polarized macrophages have been defined based on the type of stimulation, surface molecule and secreted cytokine patterns, and functional properties: the classically activated M1 and the alternatively activated M2 macrophages.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 5Xue J. Schmidt S.V. Sander J. Draffehn A. Krebs W. Quester I. et al.Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.Immunity. 2014; 40: 274-288Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 6Martinez F.O. Helming L. Milde R. Varin A. Melgert B.N. Draijer C. et al.Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.Blood. 2013; 121: e57-e69Crossref PubMed Scopus (349) Google Scholar M1 macrophages are induced by proinflammatory TH1 cytokines, microbial factors, or both; display effector, proinflammatory, and TH1-oriented immunostimulatory properties; and mediate antimicrobial defense, tissue destruction, and antitumor resistance.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar The M2 subset comprises a collection of many functionally different macrophage populations, some of which exhibit overlapping features with the M1 subset: M2a induced by TH2 cytokines, M2b induced by immune complexes, M2c induced by anti-inflammatory cytokines or glucocorticoids, and M2d induced by IL-6–like cytokines. M2 macrophages are oriented toward TH2-type immunoregulation and resolution of inflammation and promote wound repair, angiogenesis, resistance to parasites, and tumor growth.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar Macrophage plasticity is exemplified by their ability to rapidly and reversibly shift among different functional phenotypes in response to changes in the activating stimulus, overriding the initial M1/M2 polarization.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 4Becker M. De Bastiani M.A. Parisi M.M. Guma F.T. Markoski M.M. Castro M.A. et al.Integrated transcriptomics establish macrophage polarization signatures and have potential applications for clinical health and disease.Sci Rep. 2015; 5: 13351Crossref PubMed Scopus (36) Google Scholar, 7Tarique A.A. Logan J. Thomas E. Holt P.G. Sly P.D. Fantino E. Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.Am J Respir Cell Mol Biol. 2015; 53: 676-688Crossref PubMed Scopus (302) Google Scholar In vivo macrophages can be polarized into specialized functional subsets under both physiologic (eg, ontogenesis and pregnancy) and pathologic (eg, allergic and chronic inflammation, infection, tissue repair, and cancer) conditions, exerting a beneficial or detrimental role on these processes.3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar The simplistic in vitro–based M1/M2 dichotomous classification provides a conceptual framework for the description of in vivo macrophage polarization and identification of polarizing stimuli; however, it does not fully recapitulate the complex spectrum and overlapping features of tissue macrophages dictated by the interplay among a plethora of stimuli concomitantly present in the environment.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar, 5Xue J. Schmidt S.V. Sander J. Draffehn A. Krebs W. Quester I. et al.Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.Immunity. 2014; 40: 274-288Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar Over the past several years, evidence has been provided on the coexistence of macrophages exhibiting mixed polarization states in many pathologic situations and on the ability of macrophages to switch their phenotypes over time throughout the course of a disease.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar, 4Becker M. De Bastiani M.A. Parisi M.M. Guma F.T. Markoski M.M. Castro M.A. et al.Integrated transcriptomics establish macrophage polarization signatures and have potential applications for clinical health and disease.Sci Rep. 2015; 5: 13351Crossref PubMed Scopus (36) Google Scholar, 8Raggi F. Pelassa S. Pierobon D. Penco F. Gattorno M. Novelli F. et al.Regulation of human macrophage M1-M2 polarization balance by hypoxia and the triggering receptor expressed on myeloid cells-1.Front Immunol. 2017; 8: 1097Crossref PubMed Scopus (148) Google Scholar The generation of a reliable approach to categorize major macrophage phenotypes in the context of complex clinical conditions would be extremely relevant to unravel the different roles played by these cells in their development and outcome and represent a basis for macrophage-centered diagnostic and therapeutic strategies. Despite many advances in the understanding of macrophage polarization,2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 8Raggi F. Pelassa S. Pierobon D. Penco F. Gattorno M. Novelli F. et al.Regulation of human macrophage M1-M2 polarization balance by hypoxia and the triggering receptor expressed on myeloid cells-1.Front Immunol. 2017; 8: 1097Crossref PubMed Scopus (148) Google Scholar the underlying transcriptional programs remain poorly characterized. In particular, current knowledge on the transcriptional regulation of human macrophage repolarization from a classical toward an alternative activation state is far from complete. The study by Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar published in this issue of the Journal addresses this topic in detail by analyzing the effects of both well-known (IL-4, IL-10, and glucocorticoids, such as methylprednisolone) and not previously characterized (apoptotic neutrophils and the drug azithromycin) M2 stimuli on the transcriptional response of human monocyte-derived macrophages (hMDMs) previously induced to express an M1 (LPS-stimulated) state. The authors derive stimulus-specific gene expression signatures, highlighting the high transcriptional diversity of hMDMs repolarized from an M1- to an M2-skewed phenotype in response to distinct M2 stimuli. Transcriptome-based network analysis revealed a spectrum of common and divergent pathway profiles among which only suppression of interferon-associated pathways was shared by all M2-polarizing stimuli. In parallel, the authors provide functional validation of the transcriptional programs in terms of restoration of LPS responsiveness and clearance of apoptotic neutrophils (efferocytosis), suggesting that diverse M2 stimuli can alter key immune functions in the repolarizing macrophage in different ways (Fig 1). These data expand the current M1-to-M2 repolarization model, demonstrating how similar types of stimuli can impart a highly distinct activation state to macrophages. IL-10 has long been considered a hallmark of M2 polarization, mainly based on studies in mice2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar; however, recent findings demonstrated greater IL-10 expression in M1- than M2-polarized hMDMs, highlighting potential interspecies variability with regard to M1/M2-produced cytokines.7Tarique A.A. Logan J. Thomas E. Holt P.G. Sly P.D. Fantino E. Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.Am J Respir Cell Mol Biol. 2015; 53: 676-688Crossref PubMed Scopus (302) Google Scholar, 8Raggi F. Pelassa S. Pierobon D. Penco F. Gattorno M. Novelli F. et al.Regulation of human macrophage M1-M2 polarization balance by hypoxia and the triggering receptor expressed on myeloid cells-1.Front Immunol. 2017; 8: 1097Crossref PubMed Scopus (148) Google Scholar Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar underline the high complexity of the hMDM transcriptional signature elicited by IL-10, which was associated on the one hand with downregulation of several gene members of the interferon signaling pathways, similar to what was observed for all the other M2-polarizing stimuli analyzed, and on the other hand with unexpected upregulation of multiple gene sets belonging to the immune and inflammatory pathways (in particular Toll-like receptor signaling), which were suppressed by the other M2 signals. Accordingly, IL-10 exerted opposite effects with respect to IL-4 on the hMDM functional response to LPS rechallenge, increasing macrophage production of proinflammatory cytokines and thus allowing restoration of LPS responsiveness, whereas IL-4 reduced proinflammatory responses, thereby prolonging LPS tolerance. Improvement in efferocytosis, paralleled by enrichment of phagocytosis-associated processes, was also displayed by hMDMs after stimulation with IL-10 compared with the other signals (Fig 1). The evidence that IL-10 can “prime” hMDMs toward a proinflammatory phenotype is consistent with an M1-polarizing attribution for IL-10 in human macrophages and further strengthens the evolving concept that LPS can affect macrophage functions in different ways depending on the type of costimulation.8Raggi F. Pelassa S. Pierobon D. Penco F. Gattorno M. Novelli F. et al.Regulation of human macrophage M1-M2 polarization balance by hypoxia and the triggering receptor expressed on myeloid cells-1.Front Immunol. 2017; 8: 1097Crossref PubMed Scopus (148) Google Scholar A number of comparative genome transcriptional profile and proteomic studies reported in the literature have identified several differences between mice and human subjects in the gene and protein repertoire defining the M1-M2 states, cautioning against direct mouse-to-human data translation.5Xue J. Schmidt S.V. Sander J. Draffehn A. Krebs W. Quester I. et al.Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.Immunity. 2014; 40: 274-288Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 6Martinez F.O. Helming L. Milde R. Varin A. Melgert B.N. Draijer C. et al.Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.Blood. 2013; 121: e57-e69Crossref PubMed Scopus (349) Google Scholar A merit of the study by Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar concerns integration of human and murine transcriptome data sets. In murine bone marrow–derived macrophages, they validate IL-4 and IL-10 transcriptional differences identified in hMDMs, establishing molecular signatures shared by human and mouse macrophages. In fact, murine macrophages exhibited impaired and stimulated proinflammatory response pathways when repolarized by IL-4 and IL-10, respectively, suggesting that the opposite dependency of LPS tolerance to critical signaling components of IL-4 and IL-10 stimuli were conserved across species. A major challenge of macrophage studies is to understand how plasticity in gene expression regulates macrophage function in vivo.9Murray P.J. Allen J.E. Biswas S.K. Fisher E.A. Gilroy D.W. Goerdt S. et al.Macrophage activation and polarization: nomenclature and experimental guidelines.Immunity. 2014; 41: 14-20Abstract Full Text Full Text PDF PubMed Scopus (3561) Google Scholar Published reports demonstrated the value of analyzing transcriptome-based pathways during macrophage polarization to define possible gene expression alterations associated with human diseases.5Xue J. Schmidt S.V. Sander J. Draffehn A. Krebs W. Quester I. et al.Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.Immunity. 2014; 40: 274-288Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 6Martinez F.O. Helming L. Milde R. Varin A. Melgert B.N. Draijer C. et al.Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.Blood. 2013; 121: e57-e69Crossref PubMed Scopus (349) Google Scholar A critical aspect addressed in the study by Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar regards the applicability of in vitro–derived M2 repolarization transcriptional signatures to discriminate the macrophage activation state in the context of CF and asthma. Loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) in macrophages is associated with increased lung inflammation, suggesting the potential role of CFTR in macrophage activation.10Bruscia E.M. Zhang P.X. Satoh A. Caputo C. Medzhitov R. Shenoy A. et al.Abnormal trafficking and degradation of TLR4 underlie the elevated inflammatory response in cystic fibrosis.J Immunol. 2011; 186: 6990-6998Crossref PubMed Scopus (101) Google Scholar However, the association of CFTR mutation with a specific macrophage phenotype has not been previously assessed. Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar demonstrate that hMDMs from patients with CF with the most common CFTR mutation responded to all M2 stimuli similarly to those from healthy subjects at the gene expression, pathway enrichment, and functional levels (Fig 1). On the basis of these data, the authors conclude that macrophage response patterns in patients with CF-related lung disease is shaped by the local environmental milieu and not by the patient's genotype status. M2-polarized macrophages have been implicated in the immunopathogenesis of asthma,4Becker M. De Bastiani M.A. Parisi M.M. Guma F.T. Markoski M.M. Castro M.A. et al.Integrated transcriptomics establish macrophage polarization signatures and have potential applications for clinical health and disease.Sci Rep. 2015; 5: 13351Crossref PubMed Scopus (36) Google Scholar but the transcriptional profile of alveolar macrophages after acute allergen exposure has not been explored previously. In the study by Gharib et al,1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar in vitro–derived M2 transcriptional signatures were used to fingerprint the alveolar macrophage polarization state in an established human model of acute allergen-induced asthma using gene set enrichment analysis. Results of this analysis provide the first evidence that the transcriptional program of alveolar macrophages in patients with allergic asthma is concordant with IL-4– and IL-10–induced and discordant with azithromycin- and methylprednisolone-induced hMDM expression profiles (Fig 2). These observations emphasize the complex activation state of alveolar macrophages in asthmatic patients, which is likely due to the presence of different subpopulations and represents a reflection of the dynamic interplay among signals present in their local environment.2Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2900) Google Scholar, 3Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3910) Google Scholar, 8Raggi F. Pelassa S. Pierobon D. Penco F. Gattorno M. Novelli F. et al.Regulation of human macrophage M1-M2 polarization balance by hypoxia and the triggering receptor expressed on myeloid cells-1.Front Immunol. 2017; 8: 1097Crossref PubMed Scopus (148) Google Scholar As acknowledged by Gharib et al,1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar a limitation of the study is the use of gene set enrichment analysis because this analysis relies on predefined gene sets that might not accurately capture the complexity of human disease states and are prone to biases in selection of gene members. However, this approach has been effectively used in other similar studies,4Becker M. De Bastiani M.A. Parisi M.M. Guma F.T. Markoski M.M. Castro M.A. et al.Integrated transcriptomics establish macrophage polarization signatures and have potential applications for clinical health and disease.Sci Rep. 2015; 5: 13351Crossref PubMed Scopus (36) Google Scholar, 5Xue J. Schmidt S.V. Sander J. Draffehn A. Krebs W. Quester I. et al.Transcriptome-based network analysis reveals a spectrum model of human macrophage activation.Immunity. 2014; 40: 274-288Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar and the exploratory analysis that integrates the in vitro signatures of hMDMs with alveolar macrophage responses in vivo demonstrates the feasibility and promise of the procedure. Another weakness of the study is the small sample size analyzed; given the high interindividual variability of macrophage polarization responses, further validation of these findings in larger studies is required. Overall, the results by Gharib et al1Gharib S.A. McMahan R.S. Eddy W.E. Long M.E. Parks W.C. Aitken M.L. et al.Transcriptional and functional diversity of human macrophage repolarization.J Allergy Clin Immunol. 2019; 143: 1536-1548Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar represent an important step forward in understanding the transcriptional and functional diversity of human macrophages repolarized to an M2 phenotype in response to distinct stimuli and provide a new approach to guide the interrogation of human macrophage phenotypes in the context of clinically relevant inflammatory diseases, with critical implications for the development of novel and more effective therapeutic strategies. Transcriptional and functional diversity of human macrophage repolarizationJournal of Allergy and Clinical ImmunologyVol. 143Issue 4PreviewMacrophage plasticity allows cells to adopt different phenotypes, a property with important implications in disorders such as cystic fibrosis (CF) and asthma. Full-Text PDF