Molecular features of macrophage activation

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Molecular features of macrophage activation Joachim L. Schultze ∗ , Susanne V. Schmidt Genomics & Immunoregulation, LIMES-Institute, University of Bonn, Carl-Troll-Str. 31, D-53115 Bonn, Germany

a r t i c l e

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Article history: Received 7 August 2015 Accepted 16 March 2016 Available online xxx Keywords: Macrophage Activation Transcriptional regulation Epigenetic regulation

a b s t r a c t Cellular activation is mainly defined as the response of a cell to exogenous signals eventually leading to changes in protein expression and cellular function. Originally, macrophage activation was mainly associated with phagocytic function. Later other effector functions such as cytokine secretion, upregulation of cell surface receptors came into focus. For a while macrophage activation was classified as being either pro-inflammatory or anti-inflammatory and certain signal transduction pathways were associated with these two conditions. Most recent findings on transcriptional and epigenetic level, however, suggest that the molecular features of macrophage activation are significantly more complex. Here, we will introduce a novel and integrative model of macrophage activation. Albeit recognizing that macrophage activation cannot be reduced to nuclear processes, we will focus in this review on the most recent findings concerning transcriptional and epigenetic regulation of macrophage activation. Understanding the complexity of the central regulatory mechanisms in the nucleus will form a foundation for deciphering all the different effector functions that are associated with macrophage activation. © 2016 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 How do we define macrophage activation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Transcriptional regulation in tissue macrophages under homeostatic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Basic principles of transcription during macrophage activation induced by stress signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Transcriptional regulation in tissue macrophages under inflammatory conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Epigenetic regulation in tissue macrophages under homeostatic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Epigenetic regulation during stress-signal induced activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Two special forms of macrophage activation: trained immunity and tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Macrophages are key cellular elements of tissue homeostasis and play central roles in defense against pathogens, but also during many chronic inflammatory responses associated with diseases such as atherosclerosis, obesity, diabetes, cancer, skin diseases, but also neurodegenerative diseases [1–5]. Activation of macrophages has always been seen as a major pre-requisite of macrophage function [6–9]. However, for a long time macrophage activa-

∗ Corresponding author. E-mail address: [email protected] (J.L. Schultze).

tion was mainly seen in context of immune responses whether they were beneficial like in successful resolution of many acute infections [5] or destructive as in many chronic inflammatory response [5,10,11]. During the last two decades a rather simplistic model of macrophage activation evolved suggesting that these cells react either in an anti-inflammatory or a pro-inflammatory fashion [9,12,13]. This bipolar model, often also described as classical macrophage activation (or M1 macrophages) and alternative macrophage activation (or M2 macrophages) has been widely applied although many discrepant findings such as simultaneous expression of effector molecules being described as being either pro- or anti-inflammatory could not be explained by the bipolar model. Since there has been enormous progress in our understanding of macrophage activation both in tissue homeostasis

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[14,15] but also under physiological and non-physiological stress [16–19] we here propose a new integrated and multi-dimensional model of macrophage activation. We will focus on transcriptional and epigenetic aspects of this new model, since most progress in this field indeed derived from the application of high throughput technologies such as microarrays and next generation sequencing generating transcriptome and epigenome data from tissue macrophages as well as stress-signal induced activation programs.

2. How do we define macrophage activation? Early on, macrophage activation has been defined by the induction of effector function such as phagocytosis, cytokine production or induction of cell surface molecules as a consequence of the recognition of exogenous signals. More precisely, macrophages were described to be activated once they received stress-related signals via pattern recognition receptors (PRR) or via cytokine, chemokine or growth factor receptors. This is then followed by the activation of specific signal transduction pathways such as the NF-␬B pathway or the STAT signaling pathways that subsequently induce transcription and translation of effector molecules associated with macrophage function [20]. Macrophages that did not receive such stress-related signals were termed baseline macrophages in vitro and homeostatic tissue macrophages in vivo. Following a very simplistic view macrophage activation was then further subdivided into pro-inflammatory (often termed classical or M1 macrophages) and anti-inflammatory (termed alternatively activated or M2 macrophages) polarization as a consequence of macrophage activation [9,12,13]. Albeit not entirely wrong, most recent findings from transcriptomics and epigenomics studies suggests that this stress-signal centered model of macrophage activation might not reflect the true biology of macrophages [18]. Indeed, considering newer findings of transcriptional regulation of tissue macrophages, it is rather obvious that a ‘baseline’ macrophage does not exist. Moreover, stress-signal induced macrophage activation cannot be simply divided into pro- or anti-inflammatory activation. As a consequence, we propose to re-define macrophage activation (Fig. 1). First of all, macrophages are always in an activated state since every environmental signal sensed by receptors on the surface of macrophages is processed leading to transcriptional and subsequent translational changes. As has been recently demonstrated, macrophages derived from different tissues show a rather distinct transcriptional and epigenetic landscape demonstrating that these cells sense, integrate and process signals from their respective microenvironments thereby inducing specialized transcriptional programs (see below) [14,15]. Even an in vitro ‘baseline’ macrophage derived from monocytes has reacted towards signals provided by either M-CSF or GM-CSF with significant changes in transcription and translation. If macrophages are always activated, we need to classify the different aspects of macrophage activation. First, depending on their origin and ontogeny, macrophages might differ in their reactivity towards tissue or stress-induced signals (Fig. 1). At least two routes of development have been determined, the yolk-sac derived hematopoiesis leading to primitive macrophages and the fetal liver hematopoiesis leading to fetal liver derived monocytes that further develop towards tissue macrophages. In different organs, different distributions of both routes have been identified (for excellent reviews see Refs. [21–24]). Second, there is a tissue homeostasis-related activation status integrating signals from the microenvironment allowing the macrophage to fulfill its tissue-specific functions. E.g. red pulp macrophages are required for splenic iron homeostasis and to fulfill this function, macrophages have to switch on the transcription factor Spi-C that regulates important genes involved in iron homeo-

stasis [25]. Moreover, heme itself as an environmental signal is able to drive the induction of Spi-C in monocytes thereby driving the differentiation towards iron-recycling macrophages [25,26]. Third, there is a state of acute stress-signal related macrophage activation. During any kind of insult that leads to the induction of inflammatory signals within the local environment, macrophages sense these changes and integrate these new stress-related signals to change their overall transcriptional program allowing them to change their biological effector repertoire [18]. For example, signaling via TLR4 by elevated levels of LPS leads to a significant upregulation of NF-␬B signaling and the very fast induction of effector cytokines such as TNF␣ or IL-6 [27]. Such acute stress signals can be rather diverse. Even non-physiological concentrations of nutrients including free fatty acids [28,29] and even elevated salt concentrations [30,31] can lead to acute changes of the macrophage activation state. Moreover, one also needs to consider significant influx of monocyte-derived macrophages that might be actually the main force reacting towards the inflammatory stimuli [21]. There might be even division of labor between tissueresident macrophages and monocyte-derived macrophages during such a stressor-mediated tissue insult. A recent example provided strong evidence for division of labor. Daniel Engels and colleagues demonstrated that tissue-resident Ly6c− macrophages attracted Ly6C+ monocyte-derived macrophages, which subsequently were instructed to produce TNF causing Ly6C− macrophages to secrete CXCL2, thereby activating matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria [32]. To which extend tissue-resident macrophages can fulfill their homeostatic functions also under inflammatory conditions is currently not well understood and requires further exploration. Another form of deviation from homeostatic activation is chronic activation by stress-related signals. Chronic activation, e.g. during chronic inflammatory processes, is characterized by a prolonged presence of non-physiological signals in the microenvironment [33]. Moreover, very often, these signals are much weaker than during an acute stress-response. Nevertheless, very often the same sensors (such as the PRRs) are triggered during acute and chronic exposure of microenvironmental stress-signals [34]. A particularly interesting form of chronic activation is induced by ‘metaflammation’, which is related to non-physiological uptake of nutrients, particularly carbohydrates, salt, cholesterol and free fatty acids [35]. Yet other activation states are associated with tissue repair mechanisms [36]. Macrophages are involved in the restoration of tissue integrity, function, and structure and depending on the organ their role can vary quite dramatically [37]. Moreover, there are different outcomes of tissue damage and depending on the overall result (complete restoration of tissue function versus fibrosis) macrophage activation states can differ quite significantly in a qualitative manner but also over time. Macrophage activation states related to tissue repair should not be confused with activation during the resolution phase of an acute inflammatory insult. Depending on the damage on the tissue, there might be quite some overlap, however, acute immune response without major tissue damage also require reprogramming of macrophages from a stress-signal induced activation back to a tissue homeostasis program. Activation of AMP-activated protein kinase-␣1 (AMPK ␣1), a sensor for cellular energy levels, was found to be necessary for a metabolic switch of murine inflammation associated macrophages into resolution-phase macrophages which express anti-inflammatory molecules like TGF␤, CD163 and CD206 [38] but can also possess pro-inflammatory features enabled by COX2 and iNOS [39]. Particularly during the resolution phase there are also alternative options. One could be that the inflammatory response itself is mainly executed by infiltrating monocyte-derived

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Fig. 1. Overview of different states of macrophage activation. During adult live, macrophage activation under homeostatic conditions is determined mainly by environmental signals derived from the tissue origin, and to a certain extent to their ontogeny (here exemplified for four different organs). During tissue insults, stress signals are integrated into the activation program leading to new or enhanced functions while dispensing others at least temporarily. Each tissue will have a different pattern of stress-signal induced changes of activation. Monocytes entering tissues during inflammation or other tissue insults differentiate into monocyte-derived macrophages fulfilling diverse functions during such insults. It can by speculated that monocyte-derived macrophages play a bigger role later in live.

macrophages and these cells die as a consequence of resolution which leaves the primary tissue macrophages fulfilling their normal homeostatic function. Last, macrophage activation can also be suppressed. Here we can distinguish between suppression of stress-signal induced activation and repression of homeostatic function. While both forms of suppression might often be tidily linked, molecularly it is important to distinguish between both forms particularly when interrogating targets that would allow interference with inflammatory macrophage responses but not their homeostatic functions. Suppression of macrophage activation can be part of a physiological process during the restoration phase of an immune response. However, often suppression is associated with paralysis of macrophage function. For example, HIF1␣ and Arginase-1 expressing tumor associated macrophages have been shown to be deteriorated from their normal tissue function nurturing rather than fighting tumor cells by secretion of VEGF supporting neovascularization [40].

Another example is the temporary inability to induce stress-signal related responses following high-dose LPS exposure (so-called LPS tolerance) via MyD88-dependent and −independent signaling pathways [41], with subsequent abrogation of pro-inflammatory cytokine expression, like TNF␣, IL-6 or IL-12 to avoid overshooting immune responses [42,43]. Furthermore, corticosteroids like dexamethasone and other immunoregulatory molecules have been shown mostly in the murine system to reduce stress-signal induced macrophage activation by the inhibition of NF-␬B signaling and apoptosis [44,45]. In this context, it will be of great interest to learn more about the role of these immunoregulatory or suppressive molecules on homeostatic functions of tissue macrophages. It will be particularly interesting to learn, whether there are differences in suppression of macrophage function in different tissues. Finally, accepting this more integrated view of macrophage activation makes previous polar activation models dispensable.

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3. Transcriptional regulation in tissue macrophages under homeostatic conditions During the last years an increasing number of reports indicated that microenvironmental signals activate sets of transcription factors (TFs) in macrophages that are then inducing tissue-specific functions. Red pulp macrophages were shown to upregulate the TF Spi-C in response to heme and Spi-C was important for heme metabolism by these macrophages [25,26]. Another example is the elevated expression of TGF-␤ in the brain, which has been shown to be responsible for the regulation of Smad TFs in microglia cells [46,47]. In peritoneal macrophages, Gata6 expression was shown to be induced by local retinoic acid thereby contributing to the regulatory program of these cells [48]. Induction of the free fatty acid sensor peroxisome proliferator-activated receptor ␦ (PPAR␦) by IL4 results in Kupffer cells that control the expression of Arginase-1 [49]. These studies were recently complemented by two hallmark studies published by the groups of C. Glass and F. Geissmann [14] and I. Amit, S. Jung, and M. Merad [15]. While Amit and colleagues analyzed RNA-seq profiles of macrophages from 7 different tissues, Glass and colleagues studied microglia, large MHCIIlow and small MHChigh peritoneal macrophages as well as in vitro cultured bone-marrow derived macrophages and thioglycollate-elicited peritoneal macrophages. Together, these studies revealed significant differences of overall gene expression in the macrophage compartment which was mainly due to different signals derived from the local microenvironment. Moreover, a set of TFs clearly showed tissue-specific expression patterns. For example, Mef2c was only expressed in microglia, Gata6 only in peritoneal macrophages, Runx3 in intestinal macrophages and Lxra only in liver and spleen macrophages. Furthermore, small and large peritoneal macrophages differed in the expression of Ciita, the critical TF upstream of MHCII expression further strengthening that differential TF expression patterns guide differential biological functions of macrophages at different tissue locations. We have further extended these findings applying co-regulation network analysis on TFs expressed in tissue macrophages and could demonstrate that the differences are not restricted to single TFs but that there are clusters of TFs that show a tissue-specific distribution in macrophages derived from different locations [50] strongly arguing that many biological functions of macrophages are shaped by their local environment. These novel data strongly support that numerous different tissue homeostasis-related activation states exist in our new model of macrophage activation.

4. Basic principles of transcription during macrophage activation induced by stress signals In the proposed new model of macrophage activation (Fig. 1), we suggest to distinguish between tissue homeostasis-related activation and stress-signal related activation. In essence, any signal that would not be part of the normal tissue-related repertoire of input-signals can be seen as a stress signal. This includes inflammatory cytokines, chemokines and growth factors, pathogen(PAMP) and danger- (DAMP) associated molecular patterns, but also non-physiological concentrations of otherwise homeostatic signals. We recently generated a large transcriptome dataset of human monocyte-derived macrophages stimulated with 28 different combinations of stress signals [18]. Although every condition induced an input-signal specific transcriptional program we were also able to bio-mathematically determine a common core signature of macrophage activation in response to stress signals. We identified a set of transcriptional regulators (NFKB1, JUNB, CREB1, HIVEP1, HBP1) being potentially involved in activation processes

irrespective of stimulus. Numerous previous studies would support a general involvement of the NF-␬B pathway in macrophage activation [27,51–53]. However, much less is known about the other candidates identified. Further studies are necessary to functionally proof that stress-signal-mediated activation always induces these regulators. Our large transcriptome study also allowed to determine specific sets of TRs associated with every stimulation condition studied. Importantly, TFs identified to mediate particular functions in response to well-studied stimuli such as IFNy (e.g. STAT1, IRF1) [54,55], LPS (e.g. JunD, STAT1) [56,57] or IL-4 (STAT6, IRF4) [58,59] were all identified by our analysis. However, for each input signal, we identified many more TFs to be regulated indicating a significantly more complex transcriptional regulation of gene transcription and expression in response to stress-signals as previously suggested. To determine hierarchies of these TF candidates and to link certain TFs to certain functions perturbation experiments combined with global gene expression analysis followed by assessment of predicted functions are now required. These studies will build on previous models of the integration of different classes of transcription factors working at different time points of macrophage activation [20,60]. Lineage-determining TFs, particularly PU.1 and C/EBP ensure that promoters and enhancers of macrophage-associated genes are becoming accessible. Stresssignal associated TFs of the NF-kB family have been shown to bind to enhancer sites of responsive target genes [53]. Signal-specificity is mainly provided by the activation of TFs directly downstream of major signal input receptors. Good examples are activation of STAT1 following IFN-mediated signaling or STAT4 as a consequence of IL4-induced signaling [54,55,61]. These signal-specific TFs have also been shown to be involved in the establishment of so-called latent enhancer sites, not present in macrophages in the absence of stress signals [17]. Although all these studies have tremendously enriched our knowledge about macrophage activation, we are far from understanding the combinatorics of TFs in response to other stress-signals and their integration into homeostatic activation in the different tissues. Furthermore, kinetic analysis of changes in gene transcription in response to stress signals suggests that at least a secondary wave of transcription factors is induced, which can further contribute in combination with the earlier mentioned TFs to shape the overall biological program of such activated macrophages (Schultze, unpublished results). Moreover, we also do not fully understand, which of the TFs changed in expression and activation status are involved in stabilizing a stress signal-induced transcriptional program over time. The FANTOM research consortium profiled transcriptional changes during differentiation of IFNy and IL-4/IL-13 activated bone-marrow derived murine macrophages and correlated these to changes in activities of promoter and enhancer regions using the CAGE technology [62]. TF binding predictions were performed to evaluate the regulatory capacity of TFs on promoter activities via binding to sequences in enhancer regions (MARA, Motif Activity Response Analysis) and combined with data on differentially expressed TFs. The consortium identified active binding motifs for the TFs Nf␬b1, Rel, Rela, Irf1, Irf2, Irf7 and Tbp in both activation states of macrophages, yet the activity dynamics and expression profiles of these TFs were different. A transient expression of these common TFs were observed in IFNy activated macrophages, whereas IL-4/IL-13 activated macrophages displayed a rather moderate expression of these TF which prolonged over 24 h during differentiation. Furthermore, proteins such as TFs are not the only regulators of transcription during macrophage activation. A class of small RNAs, so called enhancer RNAs (eRNAs) has been recently identified and shown to be transcribed very early during activation at enhancer sites of specific gene loci [63] (for further review see Ref. [64]).

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Negative regulation of eRNAs occurs in a cell-type-specific manner in macrophages by the nuclear receptors NR1D1, (REV-ERB␣) and NR1D2 (REV-ERB␤), which provide a mechanism for repression or negative regulation [65]. 5. Transcriptional regulation in tissue macrophages under inflammatory conditions There have been extensive studies in the past addressing the role of tissue macrophages during inflammation. Most of these studies have not assessed genome-wide changes during the different phases of inflammatory processes, but rather focused on the assessment of few markers associated with the extremes of the previous polarization model of macrophage activation. Furthermore, in many studies the inflammatory phenotype of macrophages in a particular tissue was compared to thioglycollate-elicited peritoneal macrophages, to splenic macrophages or even in vitro generated bone-marrow derived macrophages, but rarely to macrophages derived from the same tissue under homeostatic conditions. In light of the recent findings in tissue macrophage homeostasis [14,15] clearly demonstrating significant differences between all tissues, such comparisons are actually not legitimate anymore. We have recently compiled the few currently available public RNA-seq datasets of murine tissue macrophages derived from different pathophysiological conditions, including pneumonia as well as lung and breast cancer models (J. Schultze, unpublished results). Our preliminary analysis revealed several important aspects. First, in general RNA-seq data from different studies can be compiled into larger datasets for comparative studies. Second the global transcriptomes of splenic F4/80+ macrophages derived from different mouse strains seem to be more similar to each other than to macrophages derived from other tissues further supporting the significant differences between different tissues. Third, for comparative control studies, only tissue macrophages from the same organ can be reliably used. Fourth, including several time points during inflammatory conditions will be most informative to understand the overall changes occurring in tissue macrophages during such processes (J. Schultze, unpublished results). Taking all these recent observations into account, we conclude that it will be necessary to re-evaluate macrophage activation in tissue pathophysiology including inflammatory conditions applying state-of-the-art technologies assessing transcriptional and epigenetic regulation on a genome-wide level in a spatio-temporal fashion. Once the technology is widely applicable we probably want to study tissue macrophage activation under inflammatory conditions even at the fundamental unit of investigation − the single cell [66–68]. 6. Epigenetic regulation in tissue macrophages under homeostatic conditions Transcriptional changes are complemented by chromatin remodeling and it is therefore not too surprising that differential transcriptomes of tissue macrophages are accompanied by significant differences in the epigenetic landscapes [14,15]. Interestingly, at the promoter level, macrophages, monocytes and neutrophils show an enormous overlap sharing more than 82% of the ∼10.800 myeloid cell-associated active promoters which were defined by tri-methylation at lysine 4 of histone 3 (H3K4me3). In contrast, at the level of active cis-enhancers (as defined by mono-methylation at lysine 4 of H3 (H3K4me1) and acetylation at lysine 27 of H3 (H3K27ac), there was a significant difference between the different tissue macrophages strongly suggesting that differential gene expression is related to different cis-enhancer usage. Thus, in addition to cell origin, signals from the local microenvironment strongly influence the enhancer landscape of tissue macrophages. It had

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been previously demonstrated that the lineage-determining transcription factor (LDTF) PU.1 together with other LDTFs defines the open chromatin landscape of macrophages [16]. In the more recent studies [14,15], these findings were extended to tissue macrophage-specific enhancers that can be explained at least in part by a collaborative interaction between PU.1 and alternative sets of TFs in each tissue subset. An additional finding was the detection of so-called super enhancers (SEs) in a tissue-macrophage specific fashion. SEs are defined as regions with disproportionately high activation-associated histone marks, for example H3K27Ac peaks, and TF binding sites [69,70]. Genes in close proximity are often characterized by an elevated expression level as a consequence of the cooperative accessibility of promoters and enhancers driving gene expression in a cell type specific fashion. While Glass and colleagues identified common SEs at loci of genes important to macrophage function and ontology (Spi1, Cebpa, Csf1r and others) in all tissue macrophages studied, they also detected tissue macrophage specific SEs, for example for Gata6 in large peritoneal macrophages [14]. These findings are strongly suggestive for environmental signals influencing the development of tissue-specific SEs in macrophages. In fact, when isolating large peritoneal macrophages and culturing these cells in absence of retinoic acid (RA), a known environmental signal for these cells [48], the macrophages dramatically changed gene expression and lost many subtype-specific enhancer marks, particularly SEs. However, when adding RA to the cultures, gene expression and chromatin landscape at least in part remained similar to the in vivo situation. The authors concluded that both RA-dependent but also RA-independent signals shape the epigenome and the transcriptome in peritoneal macrophages. These findings have two further implications. First, there seem to be rather dominant signals within the environment explaining a large part of the observed transcriptional program. Second, if we know the major dominant signals, we can successfully mimic the in vivo situation in vitro, which would be beneficial for example for large-scale perturbation assays, target identification, or drug screening assays.

7. Epigenetic regulation during stress-signal induced activation Since differential epigenetic regulation due to different environmental signals exists, it was very likely that stress-signal induced signals could further change the epigenetic landscape of macrophages as a consequence of sensing such stress-signals [20,71,72]. In 2010, two studies independently addressed global epigenetic changes in response to LPS [16,19]. They could elegantly demonstrate that a combinatorial assembly of tissue- and signal-specific transcription factors was required to activate a distinct group of enhancers during macrophage activation. Particularly the LDTF PU.1 in concert with stress signal-inducible transcription factors including NF-kB, IRF, and AP-1 was identified at these enhancer sites. Accumulating evidence supports the hypothesis that chromatin-remodeling actively contributes to stimulus-specificity of inducible transcription. As exemplified for LPS-induced genes, their gene loci contain variable chromatin properties correlating with the variable requirement for SWI/SNF-dependent nucleosome remodeling [73–75]. Most SWI/SNF-dependent genes are characterized by promoters with a low content of CpG dinucleotides while SWI/SNF-independent genes contain CpG island promoters [73–76]. As determined by RNA-sequencing, CpG island genes can be found at many genes throughout the transcriptional cascade [77]. In a more recent study Natoli and colleagues extended the initial findings concerning the combinatorial effect of LDTFs and stimulus-specific TFs by defining a new class of enhancers, so-called latent enhancers that are

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not detectable in the absence of stress signals, but induced and stabilized in response to stress signals [17]. Moreover, the induction of latent enhancers was not restricted to an LPS response, but also apparent in response to other stimuli such as TNF, IL1␤, IFNy, IL-4, TGF␤, CpG (TLR9 ligand) or MALP2 (TLR2 ligand). Of note, once these latent enhancers were induced, many of them persisted even when stimulation ceased. Moreover, re-stimulation of genes in close proximity of these enhancers were characterized by a faster and stronger response suggesting that these latent enhancers might be part of an innate memory response (see below). While all these findings were derived from murine model systems, experiments using human monocyte subsets [78] and monocyte-derived macrophages stimulated via M-CSF [60] also suggested that human macrophages might follow similar rules when stimulated with stress signals. In fact, we recently established evidence that human monocyte-derived inflammatory macrophages exposed to stress signals such as IFNy or TNF are also characterized by the induction of a stimulus-specific patterns of novel enhancers including SEs [50]. Applying computational approaches linking transcriptome and epigenome data, we observed another peculiarity during stress-signal mediated macrophage activation. We identified a network of transcriptional regulators (TRs) that showed strong dynamics in gene expression during stress-signal mediated activation, yet, this was not accompanied with changes in epigenetic marks since this TR network was already characterized by an open epigenetic landscape prior stimulation with stress signals [50]. We postulate that this is yet another mechanism contributing to functional macrophage plasticity. Taken together, the multidimensional model of macrophage activation further suggests that different combinations of stress-signal induced transcription factors together with LTDFs such as PU.1 act in concert to drive specific macrophage functions during inflammatory conditions. Future research will be targeted at deciphering this combinatorial system by performing perturbation experiments to establish the role of individual TRs.

8. Two special forms of macrophage activation: trained immunity and tolerance For a long time, immune memory has been assigned solely to the adaptive immune system. However, recent findings concerning epigenetic changes in macrophages following stress-signal induced activation suggested that innate immune cells can also exert a form of immune memory [75,79–81]. Probably the two extremes are the loss of reactivity towards a secondary stimulus as exemplified by LPS tolerance [81] and what has been termed trained immunity characterized by elevated or more robust responses towards secondary stimuli [79,80,82,83]. The gene loci of a subgroup of LPS-induced genes were demonstrated to assemble into repressive chromatin structures following an initial activation period [84,85]. In fact, repressive chromatin landscapes might reflect a molecular mechanism of memory that prevents induction of transcription to secondary stimuli. So far, it has been difficult to dissect epigenetic from other molecular mechanisms that might be involved in LPS induced tolerance. A first hint that trained immunity is associated with epigenetic regulatory mechanisms came from two studies demonstrating in human in vitro and in vivo studies that BCG vaccination and infection with Candida albicans induces long-term activation of monocytes via epigenetic reprogramming, particularly histone trimethylation [86,87]. Further evidence that trained immunity is at least in part regulated epigenetically came from the observation that the above described latent enhancers maintain their H3K4me1 marks, albeit loosing histone acetylation and release of the TFs contributing to their activation [17]. Two recent reports

have extended on this finding [82,83]. It was shown that ␤-glucan (BG) priming of human monocyte-derived macrophages induced significant changes of the histone marks H3K4me1, H3K4me3, and H3K27ac, of chromatin accessibility as assessed by DNase I accessibility assays on a global scale which all together was related to significant changes in global gene transcription [82]. Interestingly, a group of genes involved in glucose metabolism showed significant epigenetic changes in trained macrophages compared to their un-trained counterpart [83]. These changes were accompanied with high glucose consumption, high lactate production, and a high ratio of nicotinamide adenine dinucleotide (NAD+ ) to its reduced form NADH. Furthermore, this shift in metabolism could be linked to elevated glycolysis due to activation of a signaling cascade involving CLEC7a (Dectin-1), AKT, mTOR (mechanistic target of rapamycin), and HIF-1␣ [83]. These data strongly support that the metabolic basis of trained immunity is aerobic glycolysis induced through an AKT − mTOR − HIF1␣ pathway. So far, trained immunity has been mainly discussed in context of BCG vaccination [80] and atherosclerosis [79,88]. However, many question concerning trained immunity and tolerance remain to be answered. For example, is it possible to define classes of molecules that induce trained immunity? How long is trained immunity lasting in vitro and in vivo? Are epigenetic mechanisms the major molecular mechanisms driving trained immunity or are other regulatory molecules such as small and long non-coding RNAs also involved? Is trained immunity also relevant in other disease and vaccine settings? These are only some of the open questions that urgently need further studies.

9. Conclusion and outlook We have made considerable progress in the understanding of macrophage activation during the last years. Application of high-throughput technologies, particularly sequencing technologies have been the major drivers and it is therefore not surprising that novel findings in transcriptional and epigenetic control are leading areas in our progress. The most important change is the development of a new and integrative model of macrophage activation (Fig. 1) accepting different activation states even during tissue homeostasis, dismissing any kind of resting macrophages completely. Furthermore, stress signal-mediated activation can also be integrated into this new model. However, in contrast to previous models this new model suggests that any stress-signal mediated reprogramming of macrophages has to be seen in context of existing tissue-specific signals. We therefore dismiss previous oversimplified models of macrophage polarization since such models cannot reflect the true biology sufficiently. Although it will probably never be possible in vitro to mimic the myriad of signals macrophages are exposed to in their natural environments, we are convinced that reductionist in vitro experiments are still valid to study major regulatory mechanisms of macrophage activation. Moreover, we propose a hierarchy of signals that are dominant over others that are mainly guiding the overall activation programs in a given tissue. Identifying these major or dominant input signals for each tissue macrophage will allow us to mimic the in vivo situation sufficiently to study basic principles of macrophage activation in vitro or to develop larger cellular screening assays for the identification of bioactive compounds enhancing or inhibiting macrophage function. The identification of clusters of transcription factors that are expressed in macrophages at a particular tissue location or a specific combination of stress-related input signals opens the opportunity to study their role for different macrophage functions. As shown for example for Spi-C in red pulp macrophages [25,26], gain- and loss-of-function experiments are now required

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to address the role of the many other TFs that have been discovered to be expressed in macrophages under specific conditions or at distinct locations. We also have to learn more about the epigenetic regulation during macrophage activation. Our preliminary findings that the gene loci of many expressed transcription factors in macrophages are characterized by an open chromatin landscape [50] suggests additional levels of regulation of gene transcription. For example, we need to understand, whether the fast response of macrophages in response to stress-signals is − at least in part − due to this open chromatin state. Furthermore, is macrophage plasticity explained by open chromatin states of important regulatory genes such as transcription factors? Moreover, what is the regulation of these gene loci during tolerance induction, chronification or the induction of innate memory? Are there differences in macrophage activation to be discovered in the elderly? Are there clear differences in macrophage activation between monocytederived macrophages and yolk-sac derived macrophages? Last but not least, accepting this novel integrative model of macrophage activation will also require to revisit many of our findings in context of human diseases. So far, we have mainly focused on describing such responses as pro- or anti-inflammatory. We strongly argue to globally assess specific regulations of macrophage activation in any given pathology in a tissue-associated manner. Moreover, a novel aspect of such studies could also be to assess whether homeostatic functions of tissue macrophages are altered when stress-signals and tissue insults occur. Taken together, we are at the beginning of an exciting era of research in macrophage biology, since we are finally ready to address physiological and pathophysiological conditions both in a very specific fashion including tissue-related aspects but at the same time generating large, genome-wide datasets allowing a global, integrative and more holistic view on processes such as macrophage activation. Acknowledgements JLS is a member of the Excellence Cluster ImmunoSensation. This work was supported by Sonderforschungsbereiche SFB704 and SFB645 to JLS. Research leading to this work has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2077-2013 under REA grant agreement no. 317445. References [1] M.T. Heneka, M.P. Kummer, E. Latz, Innate immune activation in neurodegenerative disease, Nat. Rev. Immunol. 14 (2014) 463–477. [2] J. Viola, O. Soehnlein, Atherosclerosis—a matter of unresolved inflammation, Semin. Immunol. (2015). [3] A.R. Tall, L. Yvan-Charvet, Cholesterol, inflammation and innate immunity, Nat. Rev. Immunol. 15 (2015) 104–116. [4] F.G. De Felice, S.T. Ferreira, Inflammation, defective insulin signaling, and mitochondrial dysfunction as common molecular denominators connecting type 2 diabetes to Alzheimer disease, Diabetes 63 (2014) 2262–2272. [5] T.A. Wynn, A. Chawla, J.W. Pollard, Macrophage biology in development, homeostasis and disease, Nature 496 (2013) 445–455. [6] D.M. Mosser, J.P. Edwards, Exploring the full spectrum of macrophage activation, Nat. Rev. Immunol. 8 (2008) 958–969. [7] D.M. Mosser, The many faces of macrophage activation, J. Leukoc. Biol. 73 (2003) 209–212. [8] P.J. Murray, T.A. Wynn, Protective and pathogenic functions of macrophage subsets, Nat. Rev. Immunol. 11 (2011) 723–737. [9] A. Sica, A. Mantovani, Macrophage plasticity and polarization: in vivo veritas, J. Clin. Invest. 122 (2012) 787–795. [10] J.W. Pollard, Trophic macrophages in development and disease, Nat. Rev. Immunol. 9 (2009) 259–270. [11] B.Z. Qian, J.W. Pollard, Macrophage diversity enhances tumor progression and metastasis, Cell 141 (2010) 39–51. [12] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes, Trends Immunol. 23 (2002) 549–555. [13] A. Mantovani, A. Vecchi, P. Allavena, Pharmacological modulation of monocytes and macrophages, Curr. Opin. Pharmacol. 17c (2014) 38–44.

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