Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6Clow Monocytes while Preserving Macrophage Gene Function.

Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry.

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.

Human Blood Monocyte Subsets: A New Gating Strategy Defined Using Cell Surface Markers Identified by Mass Cytometry.

OBJECTIVE: Human monocyte subsets are defined as classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16+). Alterations in monocyte subset frequencies are associated with clinical outcomes, including cardiovascular disease, in which circulating intermediate monocytes independently predict cardiovascular events. However, delineating mechanisms of monocyte function is hampered by inconsistent results among studies. APPROACH AND RESULTS: We use cytometry by time-of-flight mass cytometry to profile human monocytes using a panel of 36 cell surface markers. Using the dimensionality reduction approach visual interactive stochastic neighbor embedding (viSNE), we define monocytes by incorporating all cell surface markers simultaneously. Using viSNE, we find that although classical monocytes are defined with high purity using CD14 and CD16, intermediate and nonclassical monocytes defined using CD14 and CD16 alone are frequently contaminated, with average intermediate and nonclassical monocyte purity of ≈86.0% and 87.2%, respectively. To improve the monocyte purity, we devised a new gating scheme that takes advantage of the shared coexpression of cell surface markers on each subset. In addition to CD14 and CD16, CCR2, CD36, HLA-DR, and CD11c are the most informative markers that discriminate among the 3 monocyte populations. Using these additional markers as filters, our revised gating scheme increases the purity of both intermediate and nonclassical monocyte subsets to 98.8% and 99.1%, respectively. We demonstrate the use of this new gating scheme using conventional flow cytometry of peripheral blood mononuclear cells from subjects with cardiovascular disease. CONCLUSIONS: Using cytometry by time-of-flight mass cytometry, we have identified a small panel of surface markers that can significantly improve monocyte subset identification and purity in flow cytometry. Such a revised gating scheme will be useful for clinical studies of monocyte function in human cardiovascular disease.

Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis.

OBJECTIVE: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature. APPROACH AND RESULTS: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein. CONCLUSIONS: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.

2014 Jeffrey M. Hoeg Award Lecture: Transcriptional Control of Monocyte Development.

Monocytes and macrophages are key immune cells involved in the early progression of atherosclerosis. Transcription factors that control their development in the bone marrow are important therapeutic targets to control the numbers and functions of these cells in disease. This review highlights what is currently known about the transcription factors that are critical for monocyte development.

Cutting Edge: The Orphan Nuclear Receptor Nr4a1 Regulates CD8+ T Cell Expansion and Effector Function through Direct Repression of Irf4.

The transcription factor IFN regulatory factor (IRF)4 was shown to play a crucial role in the protective CD8(+) T cell response; however, regulation of IRF4 expression in CD8(+) T cells remains unclear. In this article, we report a critical role for Nr4a1 in regulating the expansion, differentiation, and function of CD8(+) T cells through direct transcriptional repression of Irf4. Without Nr4a1, the regulation of IRF4 is lost, driving an increase in Irf4 expression and, in turn, resulting in a faster rate of CD8 T cell proliferation and expansion. Nr4a1-deficient mice show increases in CD8 T cell effector responses with improved clearance of Listeria monocytogenes. Our data support a novel and critical role for Nr4a1 in the regulation of CD8(+) T cell expansion and effector function through transcriptional repression of Irf4.

Modulation of dendritic cell alternative activation and function by the vitamin A metabolite retinoic acid.

The archetypal Th2 cytokine IL-4 has previously been shown to alternatively activate murine macrophages and, more recently, dendritic cells (DCs) both in vitro and in vivo. IL-4 has also been shown to induce Aldh1a2 (aldehyde dehydrogenase 1a2) expression in murine macrophages recruited to the peritoneal cavity. However, the influence of IL-4 on DC Aldh1a2 induction in vivo has not yet been addressed. In this work, we found that DCs show enhanced aldehyde dehydrogenase enzyme activity in vivo, which led us to investigate the impact of the vitamin A metabolite all-trans retinoic acid (RA) on DC alternative activation and function. Antagonism of RA receptors reduced production of resistin-like molecule alpha by DCs responding to IL-4, while addition of exogenous RA enhanced production of this marker of alternative activation. Functionally, RA increased DC induction of CD4(+) T-cell IL-10, while reducing CD4(+) T-cell IL-4 and IL-13, revealing a previously unidentified role for RA in regulating the ability of alternatively activated DCs to influence Th2 polarization.

The biology of nematode- and IL4Rα-dependent murine macrophage polarization in vivo as defined by RNA-Seq and targeted lipidomics.

Alternatively activated macrophages (AAMϕ) are a major component of the response to helminth infection; however, their functions remain poorly defined. To better understand the helminth-induced AAMϕ phenotype, we performed a systems-level analysis of in vivo derived AAMϕ using an established mouse model. With next-generation RNA sequencing, we characterized the transcriptomes of peritoneal macrophages from BALB/c and IL4Rα(-/-) mice elicited by the nematode Brugia malayi, or via intraperitoneal thioglycollate injection. We defined expression profiles of AAMϕ-associated cytokines, chemokines, and their receptors, providing evidence that AAMϕ contribute toward recruitment and maintenance of eosinophilia. Pathway analysis highlighted complement as a potential AAMϕ-effector function. Up-regulated mitochondrial genes support in vitro evidence associating mitochondrial metabolism with alternative activation. We mapped macrophage transcription start sites, defining over-represented cis-regulatory motifs within AAMϕ-associated promoters. These included the binding site for PPAR transcription factors, which maintain mitochondrial metabolism. Surprisingly PPARγ, implicated in the maintenance of AAMϕ, was down-regulated on infection. PPARδ expression, however, was maintained. To explain how PPAR-mediated transcriptional activation could be maintained, we used lipidomics to quantify AAMϕ-derived eicosanoids, potential PPAR ligands. We identified the PPARδ ligand PGI(2) as the most abundant AAMϕ-derived eicosanoid and propose a PGI(2)-PPARδ axis maintains AAMϕ during B malayi implantation.

Nonclassical monocytes potentiate anti-tumoral CD8+ T cell responses in the lungs.

CD8+ T cells drive anti-cancer immunity in response to antigen-presenting cells such as dendritic cells and subpopulations of monocytes and macrophages. While CD14+ classical monocytes modulate CD8+ T cell responses, the contributions of CD16+ nonclassical monocytes to this process remain unclear. Herein we explored the role of nonclassical monocytes in CD8+ T cell activation by utilizing E2-deficient (E2-/-) mice that lack nonclassical monocytes. During early metastatic seeding, modeled by B16F10-OVA cancer cells injected into E2-/- mice, we noted lower CD8+ effector memory and effector T cell frequencies within the lungs as well as in lung-draining mediastinal lymph nodes in the E2-/- mice. Analysis of the myeloid compartment revealed that these changes were associated with depletion of MHC-IIloLy6Clo nonclassical monocytes within these tissues, with little change in other monocyte or macrophage populations. Additionally, nonclassical monocytes preferentially trafficked to primary tumor sites in the lungs, rather than to the lung-draining lymph nodes, and did not cross-present antigen to CD8+ T cells. Examination of the lung microenvironment in E2-/- mice revealed reduced CCL21 expression in endothelial cells, which is chemokine involved in T cell trafficking. Our results highlight the previously unappreciated importance of nonclassical monocytes in shaping the tumor microenvironment via CCL21 production and CD8+ T cell recruitment.

Reversing T-cell Exhaustion in Cancer: Lessons Learned from PD-1/PD-L1 Immune Checkpoint Blockade.

Anti-PD-1/PD-L1 immune checkpoint blockade (ICB) therapy has revolutionized the treatment of many types of cancer over the past decade. The initial therapeutic hypothesis underlying the mechanism of anti-PD-1/PD-L1 ICB was built around the premise that it acts locally in the tumor, reversing the exhaustion of PD-1hiCD8+ T cells by "releasing the brakes." However, recent studies have provided unprecedented insight into the complexity within the CD8+ T-cell pool in the tumor microenvironment (TME). Single-cell RNA sequencing and epigenetic profiling studies have identified novel cell surface markers, revealing heterogeneity within CD8+ T-cell states classified as unique. Moreover, these studies highlighted that following ICB, CD8+ T-cell states within and outside the TME possess a differential capacity to respond, mobilize to the TME, and seed an effective antitumor immune response. In aggregate, these recent developments have led to a reevaluation of our understanding of both the underlying mechanisms and the sites of action of ICB therapy. Here, we discuss the evidence for the reversibility of CD8+ T-cell exhaustion after ICB treatment and its implication for the further development of cancer immunotherapy.

CD8+ T Cell Exhaustion in Cancer.

A paradigm shift in the understanding of the exhausted CD8+ T cell (Tex) lineage is underway. Originally thought to be a uniform population that progressively loses effector function in response to persistent antigen, single-cell analysis has now revealed that CD8+ Tex is composed of multiple interconnected subpopulations. The heterogeneity within the CD8+ Tex lineage is comprised of immune checkpoint blockade (ICB) permissive and refractory subsets termed stem-like and terminally differentiated cells, respectively. These populations occupy distinct peripheral and intratumoral niches and are characterized by transcriptional processes that govern transitions between cell states. This review presents key findings in the field to construct an updated view of the spatial, transcriptional, and functional heterogeneity of anti-tumoral CD8+ Tex. These emerging insights broadly call for (re-)focusing cancer immunotherapies to center on the driver mechanism(s) underlying the CD8+ Tex developmental continuum aimed at stabilizing functional subsets.

EZH2 Inhibition Compromises α4-1BB-Mediated Antitumor Efficacy by Reducing the Survival and Effector Programming of CD8+ T Cells.

Enhancer of Zeste Homolog 2 (EZH2) inhibitors (EZH2i) are approved to treat certain cancer types. Previous studies have suggested the potential to combine EZH2i with immune checkpoint blockade targeting coinhibitory receptors like PD-(L)1 and CTLA-4, but whether it can also enhance the activity of agents targeting costimulatory receptors is not known. Here, we explore the combination between EZH2i and an agonist antibody targeting the T cell costimulatory receptor 4-1BB (α4-1BB). Our data show that EZH2i compromise the efficacy of α4-1BB in both CT26 colon carcinoma and in an in vivo protein immunization model. We link this to reduced effector survival and increased BIM expression in CD8+ T cells upon EZH2i treatment. These data support the requirement of EZH2 function in 4-1BB-mediated CD8+ T cell expansion and effector programming and emphasize the consideration that must be given when combining such antitumoral therapies.

EZH2 as a Regulator of CD8+ T Cell Fate and Function.

Enhancer of zeste 2 (EZH2) is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) that mediates di- and trimethylation of histone 3 lysine 27 effectively precluding successful gene transcription at these loci. This class of epigenetic modifications facilitates the maintenance of tissue-specific cellular transcriptional programs as cells undergoing successive rounds of proliferation. CD8+ T cells are effective mediators of adaptive immunity and function to eliminate virus- and bacteria-infected cells as well as tumor cells. Upon recognition of cognate antigen, T cells undergo activation/proliferation to clear the target cells. The heterogeneous population of responding T cells formed during these proliferative events thus rely on epigenetic modifications to ensure identity and confer functional capabilities. In this review, we will focus on the role of the dynamic expression EZH2 in shaping the epigenetic landscape of CD8+ T cell fate and function, with a particular emphasis on infection and cancer. We also explore competing hypotheses pertaining to EZH2 function and the prospects of clinical EZH2 inhibitors in fine-tuning T cell responses.

The nuclear receptor nr4a1 controls CD8 T cell development through transcriptional suppression of runx3.

The NR4A nuclear receptor family member Nr4a1 is strongly induced in thymocytes undergoing selection, and has been shown to control the development of Treg cells; however the role of Nr4a1 in CD8(+) T cells remains undefined. Here we report a novel role for Nr4a1 in regulating the development and frequency of CD8(+) T cells through direct transcriptional control of Runx3. We discovered that Nr4a1 recruits the corepressor, CoREST to suppress Runx3 expression in CD8(+) T cells. Loss of Nr4a1 results in increased Runx3 expression in thymocytes which consequently causes a 2-fold increase in the frequency and total number of intrathymic and peripheral CD8(+) T cells. Our findings establish Nr4a1 as a novel and critical player in the regulation of CD8 T cell development through the direct suppression of Runx3.

Patrolling monocytes control tumor metastasis to the lung.

The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical "patrolling" monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells.

Dendritic cells (DCs) direct CD4(+) T-cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a markedly impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to the activation of CD4(+) T-cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.

IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1.

Macrophages (MΦs) colonize tissues during inflammation in two distinct ways: recruitment of monocyte precursors and proliferation of resident cells. We recently revealed a major role for IL-4 in the proliferative expansion of resident MΦs during a Th2-biased tissue nematode infection. We now show that proliferation of MΦs during intestinal as well as tissue nematode infection is restricted to sites of IL-4 production and requires MΦ-intrinsic IL-4R signaling. However, both IL-4Rα-dependent and -independent mechanisms contributed to MΦ proliferation during nematode infections. IL-4R-independent proliferation was controlled by a rise in local CSF-1 levels, but IL-4Rα expression conferred a competitive advantage with higher and more sustained proliferation and increased accumulation of IL-4Rα(+) compared with IL-4Rα(-) cells. Mechanistically, this occurred by conversion of IL-4Rα(+) MΦs from a CSF-1-dependent to -independent program of proliferation. Thus, IL-4 increases the relative density of tissue MΦs by overcoming the constraints mediated by the availability of CSF-1. Finally, although both elevated CSF1R and IL-4Rα signaling triggered proliferation above homeostatic levels, only CSF-1 led to the recruitment of monocytes and neutrophils. Thus, the IL-4 pathway of proliferation may have developed as an alternative to CSF-1 to increase resident MΦ numbers without coincident monocyte recruitment.