Macrophage Identity Research Articles

Prolonged fasting drives a program of metabolic inflammation in human adipose tissue

ObjectiveThe human adaptive fasting response enables survival during periods of caloric deprivation. A crucial component of the fasting response is the shift from glucose metabolism to utilization of lipids, underscoring the importance of adipose tissue as the central lipid-storing organ. The objective of this study was to investigate the response of adipose tissue to a prolonged fast in humans. MethodsWe performed RNA sequencing of subcutaneous adipose tissue samples longitudinally collected during a 10-day, 0-calorie fast in humans. We further investigated observed transcriptional signatures utilizing cultured human monocytes and Thp1 cells. We examined the cellularity of adipose tissue biopsies with transmission electron microscopy and tested for associated changes in relevant inflammatory mediators in the systemic circulation by ELISA assays of longitudinally collected blood samples. ResultsCoincident with the expected shift away from glucose utilization and lipid storage, we demonstrated downregulation of pathways related to glycolysis, oxidative phosphorylation, and lipogenesis. The canonical lipolysis pathway was also downregulated, whereas fasting drove alternative lysosomal paths to lipid digestion. Unexpectedly, the dominant induced pathways were associated with immunity and inflammation, although this only became evident at the 10-day time point. Among the most augmented transcripts were those associated with macrophage identity and function, such as members of the erythroblast transformation-specific (ETS) transcription factor family. Key components of the macrophage transcriptional signal in fasting adipose tissue were recapitulated with induced expression of two of the ETS transcription factors via cultured macrophages, SPIC and SPI1. The inflammatory signal was further reflected by an increase in systemic inflammatory mediators. ConclusionsCollectively, this study demonstrates an unexpected role of metabolic inflammation in the human adaptive fasting response.

The angiocrine Rspondin3 instructs interstitial macrophage transition via metabolic–epigenetic reprogramming and resolves inflammatory injury

Macrophages demonstrate remarkable plasticity that is essential for host defense and tissue repair. The tissue niche imprints macrophage identity, phenotype and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages remains unknown. The lung is a highly vascularized organ and replete with a large population of resident macrophages. We found that, in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3, which activates β-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation. Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti-inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury.

Peripheral nerve resident macrophages share tissue-specific programming and features of activated microglia

Whereas microglia are recognized as fundamental players in central nervous system (CNS) development and function, much less is known about macrophages of the peripheral nervous system (PNS). Here, by comparing gene expression across neural and conventional tissue-resident macrophages, we identified transcripts that were shared among neural resident macrophages as well as selectively enriched in PNS macrophages. Remarkably, PNS macrophages constitutively expressed genes previously identified to be upregulated by activated microglia during aging, neurodegeneration, or loss of Sall1. Several microglial activation-associated and PNS macrophage-enriched genes were also expressed in spinal cord microglia at steady state. We further show that PNS macrophages rely on IL-34 for maintenance and arise from both embryonic and hematopoietic precursors, while their expression of activation-associated genes did not differ by ontogeny. Collectively, these data uncover shared and unique features between neural resident macrophages and emphasize the role of nerve environment for shaping PNS macrophage identity.

Speckled Protein (SP)140, an immune-specific chromatin reader, contains a CARD and chromatin-interacting domains that dictate subnuclear localization and macrophage identity

Abstract Chromatin regulators that “read” histone post-translational modifications are desirable therapeutic targets in inflammation and cancer. The Speckled Protein (SP) family of chromatin “readers” consists of SP100, SP110, SP140 and SP140L and share high homology with autoimmune regulator (AIRE). SPs possess a caspase activation and recruitment domain (CARD) and multiple chromatin “reader” domains (Plant Homeodomain (PHD), Bromodomain, and SAND). SP140 is preferentially expressed in immune cells and loss-of-function single nucleotide polymorphisms (SNPs) within SP140 significantly associate with immune disorders, such as Crohn’s disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). We have previously shown that SP140 is a critical orchestrator of macrophage identity and function by occupying and repressing silenced lineage-inappropriate genes. We also demonstrated that PBMCs from patients carrying SP140 SNPs lose SP140 expression and are hypo-responsive to TLR stimulation. How SP140 functions to interact with or maintain heterochromatin for cell identity and function, what SP140 protein domains drive activity, and how disease variants alter SP140 function remain to be determined. Here we show that human SP140 localizes to promyelocytic leukemia (PML) nuclear bodies, sites of transcriptional repression, and we delineate the SP140 protein domains required for this. Furthermore, we determined the impact of SP140 disease-associated SNPs on PML localization and macrophage identity and functional responses. Collectively, our work provides functional characterization of this important chromatin protein and how SP140 disease variants contribute to immunological disease.

RXRs control serous macrophage neonatal expansion and identity and contribute to ovarian cancer progression

Tissue-resident macrophages (TRMs) populate all tissues and play key roles in homeostasis, immunity and repair. TRMs express a molecular program that is mostly shaped by tissue cues. However, TRM identity and the mechanisms that maintain TRMs in tissues remain poorly understood. We recently found that serous-cavity TRMs (LPMs) are highly enriched in RXR transcripts and RXR-response elements. Here, we show that RXRs control mouse serous-macrophage identity by regulating chromatin accessibility and the transcriptional regulation of canonical macrophage genes. RXR deficiency impairs neonatal expansion of the LPM pool and reduces the survival of adult LPMs through excess lipid accumulation. We also find that peritoneal LPMs infiltrate early ovarian tumours and that RXR deletion diminishes LPM accumulation in tumours and strongly reduces ovarian tumour progression in mice. Our study reveals that RXR signalling controls the maintenance of the serous macrophage pool and that targeting peritoneal LPMs may improve ovarian cancer outcomes.

Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche

SummaryMacrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.

Transcriptional repressors as guardians of tissue macrophage identity.

The association between specific transcription factors (TFs) and defined tissue-specific macrophage phenotypes is far from being univocal. Many TFs that have been associated with tissue-specific macrophages have relatively broad expression profiles suggesting the critical involvement of combinatorial regulation by multiple TFs in bringing about specific phenotypes. In the current issue of The EMBO Journal, Rauschmeier etal (2019) report the identification of the transcriptional repressors BHLHE40 and BHLHE41 as novel regulators of tissue-specific macrophage properties that work both to promote alveolar macrophage (AM) identity and to suppress alternative tissue-specific phenotypes.

Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages

Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure.

Does niche competition determine the origin of tissue-resident macrophages?

Most tissue-resident macrophages are derived from embryonic precursors but, under certain circumstances, circulating monocytes can differentiate into self-maintaining tissue-resident macrophages that resemble their embryonic counterparts. In this Opinion article, we propose that distinct macrophage precursors have an almost identical potential to develop into resident macrophages but they compete for a restricted number of niches. The tight regulation of the niche ensures that monocytes do not differentiate into macrophages when the niche is full but that these cells can differentiate efficiently into macrophages when the niche is available. Imprinting by the niche would be the dominant factor conferring macrophage identity and self-maintenance capacity, rather than origin as was previously proposed.

The transcription factor basic helix loop helix, member e40 is required for establishment of proper peritoneal macrophage identity

Abstract Tissue-resident macrophages occupy key roles in immunity and physiology, both as sensors of and first responders to homeostatic perturbations. The majority of resident macrophages derive from embryonic progenitors and self-maintain locally as unique tissue-specific populations. The transcriptional basis for tissue-resident macrophage identity is poorly understood. We have observed that the transcription factor basic helix loop helix, member e40 (Bhlhe40) is highly expressed in a subset of hematopoietic cell types, including peritoneal macrophages. Based on these data, we hypothesized that Bhlhe40 is a key part of the peritoneal macrophage transcriptional network and adapts nascent macrophages to the functional demands of the peritoneal niche. We have found that peritoneal macrophages are selectively reduced in Bhlhe40−/− mice, in contrast to other resident macrophages. Furthermore, in the absence of Bhlhe40 the remaining large peritoneal macrophages (LPMs) are replaced by monocyte-derived cells to a greater extent than in wildtype mice. Bhlhe40-deficient peritoneal macrophages exhibit altered polarization and self-renewal. Mixed bone marrow chimeras and other approaches demonstrated a specific, intrinsic defect in mature Bhlhe40−/− LPMs. Using models of peritoneal immunity, we observed impaired responses of Bhlhe40-deficient peritoneal macrophages. Our preliminary findings demonstrate a role for Bhlhe40 in peritoneal macrophage biology and lead us to propose that Bhlhe40 transcriptionally controls a tissue-specific program in macrophages tailored to the peritoneal environment.

Corrigendum: The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis.

Nat. Immunol. 17, 18–25 (2016); published online 17 December 2015; corrected after print 24 October 2016 In the version of this article initially published, the citation of Figure 3 in the second paragraph of the third subsection (Chromatin: the nexus of phenotype and the environment) is incorrect. That should cite Figure 2, as follows: “From an evolutionary perspective, two extreme models of how such complexity might be generated and regulated can be envisaged95 (Fig.

Genome-Wide Approaches to Defining Macrophage Identity and Function.

Macrophages play essential roles in the response to injury and infection and contribute to the development and/or homeostasis of the various tissues they reside in. Conversely, macrophages also influence the pathogenesis of metabolic, neurodegenerative, and neoplastic diseases. Mechanisms that contribute to the phenotypic diversity of macrophages in health and disease remain poorly understood. Here we review the recent application of genome-wide approaches to characterize the transcriptomes and epigenetic landscapes of tissue-resident macrophages. These studies are beginning to provide insights into how distinct tissue environments are interpreted by transcriptional regulatory elements to drive specialized programs of gene expression.

Abstract 47: Quaking Post-Transcriptionally Promotes Differentiation of Monocytes Into Pro-Atherogenic Macrophages by Controling Pre-mRNA Splicing and Gene Expression

Aim: Atherosclerosis is accelerated by excessive monocyte recruitment, influx and differentiation into pro-inflammatory macrophages. While the genome-wide mRNA expression profiles of human monocytes and pro-inflammatory macrophages are well-established, their transcriptomes are ultimately defined by factors, such as RNA-binding proteins, that modulate pre-mRNA splicing patterns and mRNA transcript abundance. This prompted us to investigate the role of the RNA-binding protein Quaking in regulating global changes in pre-mRNA splicing and mature mRNA expression as human monocytes acquire the pro-inflammatory macrophage identity. Methods: We employed RNA-sequencing and splicing-sensitive microarrays to determine genome-wide changes in pre-mRNA splicing and mRNA expression upon conversion of human monocytes into pro-inflammatory macrophages, including those derived from a unique Quaking haploinsufficient patient. Results: Using laser-capture micro-dissection and immunohistochemistry, we discovered that expression levels of Quaking mRNA and protein are low in monocytes of early human atherosclerotic lesions, but abundant in macrophages of advanced plaques. Depletion of Quaking protein using both siRNA and GapmeR technology significantly impaired monocyte adhesion and migration; delayed differentiation into pro-inflammatory macrophages while maintaining the capacity to adopt the anti-inflammatory phenotype; and diminished foam cell formation in vitro and in vivo. RNA-sequencing and microarray analysis of human monocyte and macrophage transcriptomes revealed striking changes in Quaking-dependent pre-mRNA splicing and mRNA transcript levels, with gene ontology analyses identifying an enrichment in transcripts involved in cellular migration and lipid metabolism. Furthermore, these studies uncovered common as well as novel alternatively spliced transcripts with unknown biological functions in monocytes and macrophages. Conclusions: Our studies illustrate a central role for Quaking in post-transcriptionally guiding pro-inflammatory macrophage identity and function.

Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.

IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation.

BackgroundThe role of cytokines in establishing specific transcriptional programmes in innate immune cells has long been recognized. However, little is known about how these extracellular factors instruct innate immune cell epigenomes to engage specific differentiation states. Human monocytes differentiate under inflammatory conditions into effector cells with non-redundant functions, such as dendritic cells and macrophages. In this context, interleukin 4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF) drive dendritic cell differentiation, whereas GM-CSF alone leads to macrophage differentiation.ResultsHere, we investigate the role of IL-4 in directing functionally relevant dendritic-cell-specific DNA methylation changes. A comparison of DNA methylome dynamics during differentiation from human monocytes to dendritic cells and macrophages identified gene sets undergoing dendritic-cell-specific or macrophage-specific demethylation. Demethylation is TET2-dependent and is essential for acquiring proper dendritic cell and macrophage identity. Most importantly, activation of the JAK3-STAT6 pathway, downstream of IL-4, is required for the acquisition of the dendritic-cell-specific demethylation and expression signature, following STAT6 binding. A constitutively activated form of STAT6 is able to bypass IL-4 upstream signalling and instruct dendritic-cell-specific functional DNA methylation changes.ConclusionsOur study is the first description of a cytokine-mediated sequence of events leading to direct gene-specific demethylation in innate immune cell differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0863-2) contains supplementary material, which is available to authorized users.

The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis.

Macrophages provide a critical systemic network cells of the innate immune system. Emerging data suggest that in addition, they have important tissue-specific functions that range from clearance of surfactant from the lungs to neuronal pruning and establishment of gut homeostasis. The differentiation and tissue-specific activation of macrophages require precise regulation of gene expression, a process governed by epigenetic mechanisms such as DNA methylation, histone modification and chromatin structure. We argue that epigenetic regulation of macrophages is determined by lineage- and tissue-specific transcription factors controlled by the built-in programming of myeloid development in combination with signaling from the tissue environment. Perturbation of epigenetic mechanisms of tissue macrophage identity can affect normal macrophage tissue function and contribute to pathologies ranging from obesity and autoimmunity to neurodegenerative diseases.

Tissue-resident versus monocyte-derived macrophages in the tumor microenvironment

The tumor-promoting role of macrophages has been firmly established in most cancer types. However, macrophage identity has been a matter of debate, since several levels of complexity result in considerable macrophage heterogeneity. Ontogenically, tissue-resident macrophages derive from yolk sac progenitors which either directly or via a fetal liver monocyte intermediate differentiate into distinct macrophage types during embryogenesis and are maintained throughout life, while a disruption of the steady state mobilizes monocytes and instructs the formation of monocyte-derived macrophages. Histologically, the macrophage phenotype is heavily influenced by the tissue microenvironment resulting in molecularly and functionally distinct macrophages in distinct organs. Finally, a change in the tissue microenvironment as a result of infectious or sterile inflammation instructs different modes of macrophage activation. These considerations are relevant in the context of tumors, which can be considered as sites of chronic sterile inflammation encompassing subregions with distinct environmental conditions (for example, hypoxic versus normoxic). Here, we discuss existing evidence on the role of macrophage subpopulations in steady state tissue and primary tumors of the breast, lung, pancreas, brain and liver.

Abstract 217: Quaking Post-Transcriptionally Guides Monocyte Adhesion and Differentiation into the Pro-Inflammatory Macrophage

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce new transcription necessary for macrophage identity, but post-transcriptional control of human macrophage differentiation is less well understood. Here, we detail our discovery that levels of the RNA-binding protein Quaking (QKI) are low in monocytes of early atherosclerotic lesions, but abundant in macrophages of advanced plaques. Specific depletion of QKI protein impaired monocyte adhesion, migration and differentiation into macrophages, and lesion formation. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal developmental changes in RNA levels and alternative splicing of RNA transcripts enriched in QKI-bound sequence elements. The importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in post-transcriptionally guiding macrophage identity and function. These studies implicate QKI as a novel target for therapeutic intervention in inflammatory diseases.

Genetic and genomic approaches to understanding macrophage identity and function.

A major goal of our laboratory is to understand the molecular mechanisms that underlie the development and functions of diverse macrophage phenotypes in health and disease. Recent studies using genetic and genomic approaches suggest a relatively simple model of collaborative and hierarchical interactions between lineage-determining and signal-dependent transcription factors that enable selection and activation of transcriptional enhancers that specify macrophage identity and function. In addition, we have found that it is possible to use natural genetic variation as a powerful tool for advancing our understanding of how the macrophage deciphers the information encoded by the genome to attain specific phenotypes in a context-dependent manner. Here, I will describe our recent efforts to extend genetic and genomic approaches to investigate the roles of distinct tissue environments in determining the phenotypes of different resident populations of macrophages.

Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local Microenvironment

Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogrammed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.