Fernando O. Martinez

Alternative Activation of Macrophages: Mechanism and Functions

The concept of an alternative pathway of macrophage activation has stimulated interest in its definition, mechanism, and functional significance in homeostasis and disease. We assess recent research in this field, argue for a restricted definition, and explore pathways by which the T helper 2 (Th2) cell cytokines interleukin-4 (IL-4) and IL-13 mediate their effects on macrophage cell biology, their biosynthesis, and responses to a normal and pathological microenvironment. The stage is now set to gain deeper insights into the role of alternatively activated macrophages in immunobiology.

Macrophage activation and polarization.

Macrophages are widely distributed immune system cells that play an indispensable role in homeostasis and defense. They can be phenotypically polarized by the microenvironment to mount specific functional programs. Polarized macrophages can be broadly classified in two main groups: classically activated macrophages (or M1), whose prototypical activating stimuli are IFNgamma and LPS, and alternatively activated macrophages (or M2), further subdivided in M2a (after exposure to IL-4 or IL-13), M2b (immune complexes in combination with IL-1beta or LPS) and M2c (IL-10, TGFbeta or glucocorticoids). M1 exhibit potent microbicidal properties and promote strong IL-12-mediated Th1 responses, whilst M2 support Th2-associated effector functions. Beyond infection M2 polarized macrophages play a role in resolution of inflammation through high endocytic clearance capacities and trophic factor synthesis, accompanied by reduced pro-inflammatory cytokine secretion. Similar functions are also exerted by tumor-associated macrophages (TAM), which also display an alternative-like activation phenotype and play a detrimental pro-tumoral role. Here we review the main functions of polarized macrophages and discuss the perspectives of this field.

Alternative Activation of Macrophages: An Immunologic Functional Perspective

Macrophages are innate immune cells with well-established roles in the primary response to pathogens, but also in tissue homeostasis, coordination of the adaptive immune response, inflammation, resolution, and repair. These cells recognize danger signals through receptors capable of inducing specialized activation programs. The classically known macrophage activation is induced by IFN-gamma, which triggers a harsh proinflammatory response that is required to kill intracellular pathogens. Macrophages also undergo alternative activation by IL-4 and IL-13, which trigger a different phenotype that is important for the immune response to parasites. Here we review the cellular sources of these cytokines, receptor signaling pathways, and induced markers and gene signatures. We draw attention to discrepancies found between mouse and human models of alternative activation. The evidence for in vivo alternative activation of macrophages is also analyzed, with nematode infection as prototypic disease. Finally, we revisit the concept of macrophage activation in the context of the immune response.

Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression

Comprehensive analysis of the gene expression profiles associated with human monocyte-to-macrophage differentiation and polarization toward M1 or M2 phenotypes led to the following main results: 1) M-CSF-driven monocyte-to-macrophage differentiation is associated with activation of cell cycle genes, substantiating the underestimated proliferation potential of monocytes. 2) M-CSF leads to expression of a substantial part of the M2 transcriptome, suggesting that under homeostatic conditions a default shift toward M2 occurs. 3) Modulation of genes involved in metabolic activities is a prominent feature of macrophage differentiation and polarization. 4) Lipid metabolism is a main category of modulated transcripts, with expected up-regulation of cyclo-oxygenase 2 in M1 cells and unexpected cyclo-oxygenase 1 up-regulation in M2 cells. 5) Each step is characterized by a different repertoire of G protein-coupled receptors, with five nucleotide receptors as novel M2-associated genes. 6) The chemokinome of polarized macrophages is profoundly diverse and new differentially expressed chemokines are reported. Thus, transcriptome profiling reveals novel molecules and signatures associated with human monocyte-to-macrophage differentiation and polarized activation which may represent candidate targets in pathophysiology.

The M1 and M2 paradigm of macrophage activation: time for reassessment

Macrophages are endowed with a variety of receptors for lineage-determining growth factors, T helper (Th) cell cytokines, and B cell, host, and microbial products. In tissues, macrophages mature and are activated in a dynamic response to combinations of these stimuli to acquire specialized functional phenotypes. As for the lymphocyte system, a dichotomy has been proposed for macrophage activation: classic vs. alternative, also M1 and M2, respectively. In view of recent research about macrophage functions and the increasing number of immune-relevant ligands, a revision of the model is needed. Here, we assess how cytokines and pathogen signals influence their functional phenotypes and the evidence for M1 and M2 functions and revisit a paradigm initially based on the role of a restricted set of selected ligands in the immune response.

Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells

Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.

Analysis of the Gene Expression Profile Activated by the CC Chemokine Ligand 5/RANTES and by Lipopolysaccharide in Human Monocytes

The gene expression profile induced by the CC chemokine ligand (CCL) 5/RANTES in human monocytes was examined using the oligonucleotide array technology. Of 5600 transcripts examined, 42 were consistently induced by CCL5, and none were suppressed. Chemokine-inducible transcripts could be clustered in functional groups, including selected cytokines and receptors (e.g., IL-1β, CCL2/monocyte chemotactic protein-1, and the CCL5 receptor CCR1) and molecules involved in extracellular matrix recognition and digestion (e.g., CD44 splice transcripts, urokinase-type plasminogen activator receptor, matrix metalloprotease (MMP)-9 , and MMP-19). Transcript expression, confirmed by quantitative real-time PCR analysis for selected genes, was associated with protein induction for some (e.g., CCL2), but not all (e.g., IL-1β), transcripts examined. The chemokine-induced gene profile was distinct from that activated by LPS, a prototypic phagocyte activator. Although certain transcripts were stimulated by both agonists (e.g., IL-1β and CCL2), others were induced only by either LPS (e.g., TNF-α and IL-6) or CCL5 (e.g., MMP-19) or were divergently regulated (e.g., CCR1). Thus, CCL5, a prototypic CC inflammatory chemokine, activates a restricted transcriptional program in monocytes distinct from that induced by the prototypic pathogen-derived proinflammatory stimulant LPS. Chemokine-induced chemokines production could represent a novel amplification loop of leukocyte recruitment, while a subset of chemokine-inducible transcripts could be involved in monocyte extravasation and tissue invasion.

Transcriptional profiling reveals complex regulation of the monocyte IL-1 beta system by IL-13.

IL-4 and IL-13 are prototypic Th2 cytokines that generate an “alternatively activated” phenotype in macrophages. We used high-density oligonucleotide microarrays to investigate the transcriptional profile induced in human monocytes by IL-13. After 8-h stimulation with IL-13, 142 genes were regulated (85 increased and 57 decreased). The majority of these genes were related to the inflammatory response and innate immunity; a group of genes related to lipid metabolism was also identified, with clear implications for atherosclerosis. In addition to characteristic markers of alternatively activated macrophages, a number of novel IL-13-regulated genes were seen. These included various pattern recognition receptors, such as CD1b/c/e, TLR1, and C-type lectin superfamily member 6. Several components of the IL-1 system were regulated. IL-1RI, IL-1RII, and IL-1Ra were all up-regulated, whereas the IL-1β-converting enzyme, caspase 1, and IRAK-M were down-regulated. LPS-inducible caspase 1 enzyme activity was also reduced in IL-13-stimulated monocytes, with a consequent decrease in pro-IL-1β processing. These data reveal that IL-13 has a potent effect on the transcriptional profile in monocytes. The IL-13-induced modulation of genes related to IL-1 clearly highlights the tightly controlled and complex levels of regulation of the production and response to this potent proinflammatory cytokine.

Distinct Transcriptional Programs Activated by Interleukin-10 with or without Lipopolysaccharide in Dendritic Cells: Induction of the B Cell-Activating Chemokine, CXC Chemokine Ligand 13

To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Affymetrix technology (Santa Clara, CA) in human monocyte-derived DC treated with IL-10, alone or in combination with LPS. The modulation of selected genes was validated by real-time PCR, Northern blot, and protein production. IL-10 regulated in DC the expression of a limited number of genes, including IL-7, the receptors for transferrin and vitamin D3, structural matrix proteins, and signal transduction elements. The combined treatment with LPS plus IL-10 modulated a number of genes comparable to LPS alone, but the expression profiles were distinct. As expected, IL-10 suppressed the expression of several LPS-inducible proinflammatory molecules. Among genes uniquely modulated by the concomitant treatment with LPS plus IL-10, phosphatidylinositol 3-kinase γ was down-regulated while the suppressor of cytokine signaling 3, signaling lymphocytic activation molecule, regulator of G protein signaling 16, and the chemokine, CXC chemokine ligand (CXCL) 13, were up-regulated. Overall, four distinct transcriptional programs were identified, related to: 1) control of immunity and inflammation; 2) tuning of cytokine receptor and G protein-coupled receptor signaling; 3) remodeling of extracellular matrix; and 4) B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrate that IL-10 synergizes with TLR ligands for the production of functionally active B cell-attracting chemokine, CXCL13, in both myeloid and plasmacytoid DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production in APCs of the chemokine, CXCL13, which amplifies B cell recruitment and promotes lymphoid tissue neogenesis.