Amiram Ariel

Saturated‐efferocytosis generates pro‐resolving CD11blow macrophages: Modulation by resolvins and glucocorticoids

During the resolution phase of inflammation, apoptotic leukocytes are efferocytosed by macrophages in a nonphlogistic fashion that results in diminished responses to bacterial moieties and production of anti‐inflammatory cytokines. Complement receptor 3 and pro‐resolving lipid mediators promote the engulfment of apoptotic leukocytes by macrophages. Here, we present evidence for the emergence of pro‐resolving, CD11blow macrophages in vivo during the resolution of murine peritonitis. These macrophages are distinct from the majority of peritoneal macrophages in terms of their functional protein expression profile, as well as pro‐resolving properties, such as apoptotic leukocyte engulfment, indifference to TLR ligands, and emigration to lymphoid organs. Notably, we also found macrophages convert from the CD11bhigh to the CD11blow phenotype upon interaction with apoptotic cells ex vivo. In addition, we found that the pro‐resolving lipid mediators resolvin E1 and D1, and the glucocorticoid dexamethasone regulated pro‐resolving macrophage functions in vivo. This regulation culminated in a novel pro‐resolving function, namely reducing the apoptotic leukocyte ingestion requirement for CD11blow macrophage generation. These new phenotype and molecular pathway markers define the new satiated macrophage. Thus, we suggest that satisfying efferocytosis generates CD11blow macrophages that are essential for complete nonphlogistic containment of inflammatory agents and the termination of acute inflammation.

Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

New Lives Given by Cell Death: Macrophage Differentiation Following Their Encounter with Apoptotic Leukocytes during the Resolution of Inflammation

Monocytes that migrate into tissues during inflammatory episodes and differentiate to macrophages were previously classified as classically (M1) or alternatively (M2) activated macrophages, based on their exposure to different fate-determining mediators. These macrophage subsets display distinct molecular markers and differential functions. At the same time, studies from recent years found that the encounter of apoptotic leukocytes with macrophages leads to the clearance of this cellular “debris” by the macrophages, while concomitantly reprogramming/immune-silencing the macrophages. While some of the features of M2 differentiation, such as arginase-1 (murine) and 15-lipoxygenases (human and murine) expression, were also displayed by macrophages following the engulfment of apoptotic cells, it was not clear whether apoptotic cells can be regarded as an M2-like differentiating signal. In this manuscript we review the recent information regarding the impact of apoptotic cells on macrophage phenotype changes in molecular terms. We will focus on recent evidence for the in vivo existence of distinct pro-resolving macrophages and the role of apoptotic cells, specialized lipid mediators, and glucocorticoids in their generation. Consequently, we will suggest that these pro-resolving CD11blow macrophages have metamorphed from M2-like macrophages, and modulated their protein profile to accommodate the changes in their function.

Macrophages, Meta-Inflammation, and Immuno-Metabolism

Current research depicts specific modes of immunity and energy metabolism as being interrelated at the molecular, cellular, organ and organism level. Hence, whereas M2 (alternatively-activated) macrophages dominate insulin-sensitive adipose tissue in the lean, M1-skewed (classically-activated) macrophages accumulate in parallel to adiposity in the obese, and promote inflammation and insulin resistance, that is, meta-inflammation. The latest frontier of immuno-metabolism explores the coregulation of energy metabolism and immune function within hematopoietic cells. M1-skewed macrophages are sustained in edematous, hypoxic tissues by anaerobic glycolysis, whereas mitochondrial biogenesis and respiration dominates in M2 cells. We review the underlying mechanisms and the consequences of the transition from M2 to M1 predominance in adipose tissue, as well as the extracellular signals and transcription factors that control macrophage phenotypes and impose distinct metabolic modes.

Hanging in the balance: endogenous anti-inflammatory mechanisms in tissue repair and fibrosis

Inflammation is the physiological response to tissue injury caused by pathogens or trauma. Nevertheless, inflammation should be resolved in a timely manner, resulting in elimination of the inflammatory cells and mediators from the injured tissue, to avoid its deleterious consequences. Uncontrolled inflammation can lead to inflammatory, autoimmune, and cancerous disorders that are the result of improper resolution. The healing of the injured tissue during the termination of inflammation must also be tightly controlled since excessive tissue repair can lead to fibrosis and scarring of the affected organ. In the last three decades, it has been revealed that the resolution of inflammation is tightly orchestrated by specific cells, protein, and lipid mediators that are produced at proper timing and distinct locations. The bioactivity of these anti‐inflammatory, pro‐resolving, and immunoregulatory agents results in clearance of the tissue from inflammatory leukocytes and their products, and the return of homeostatic tissue architecture and function. Here, we will survey the current endogenous mechanisms governing the resolution of inflammation and directing it towards injury healing and halting of acquired immune responses while preventing excessive tissue repair and fibrosis. We focus on the role played by apoptotic polymorphonuclear cells (PMNs), 15‐lipoxygenase (LO)‐derived lipid mediators, and TGFβ in this macrophage‐governed decision‐making process and suggest new modes of action for fibrosis prevention and return to homeostasis.

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Galectin-1 induces 12/15-lipoxygenase expression in murine macrophages and favors their conversion toward a pro-resolving phenotype

During the resolution of inflammation macrophages undergo functional changes upon exposure to pro-resolving agents in their microenvironment. Primarily, engulfment of apoptotic polymorphonuclear (PMN) cells promotes conversion of macrophages toward a pro-resolving phenotype characterized by reduced CD11b expression. These macrophages are not phagocytic, do not respond to TLR ligands, and express relatively high levels of the pro-resolving enzyme 12/15-lipoxygenase (LO). Here, we report that the immuno-regulatory lectin galectin-1 is selectively expressed by CD11b(high), but not CD11b(low) macrophages. Upon exposure in vivo and ex vivo, galectin-1 directly promoted macrophage conversion from a CD11b(high) to a CD11b(low) phenotype and up-regulated the expression and activity of 12/15-LO. Moreover, galectin-1 treatment in vivo promoted the loss of phagocytic capacity (efferocytic satiation) in peritoneal macrophages and down-regulated secretion of TNF-α, IL-1β, and IL-10 upon LPS exposure. Our results suggest that galectin-1 could be an essential mediator in the control of macrophage function during the resolution of inflammation.

Macrophages in inflammation and its resolution

Macrophages are highly plastic leukocytes that differentiate from monocytes following their entry into extravascular tissues. Macrophages can enter various tissues under inflammatory or non-inflammatory conditions and assume different functions and phenotypes according to the cues they receive from the environment. The notion that inflammation in general and macrophage responses in particular affect physiological phenomena that were previously considered to be not immune-related has enhanced and broadened our understanding of macrophage function during inflammation and its resolution. This volume brings together 14 manuscripts that cover various aspects of macrophage function during inflammation and its resolution, as well as in several pathologic states for which a significant, long-lasting, macrophage-mediated immune response plays a significant role. Two of the manuscripts present original research on macrophage phagocytosis and its implications. Five provide an overview of macrophage function during inflammation and its resolution, with an emphasis on the modulatory role of particular elements in this response, such as apoptotic leukocytes, specific pathogens, hypoxia, and hormone receptors. The remaining seven manuscripts outline the role of macrophages during inflammation and its resolution in different tissues, including the lung, cardiovascular and adipose tissues, injured skeletal muscle and neuronal tissues, and synovial and oral cavities. The two original research articles are devoted to the consequences of particle engulfment by macrophages. Labrousse et al. (2011) describe a novel experimental strategy in which they use micro-patterned immune complexes to trigger frustrated phagocytosis and thereby determine spatial parameters in lysosomal movement and fusion. Janko et al. (2011) report on the cumulative binding of CRP and anti-CRP antibodies to the surface of secondary necrotic cells. This binding leads to a pro-inflammatory cytokine response following engulfment by macrophages, implying a potential role for these elements in the etiology of systemic lupus erythematosus. Of the review articles that discuss the regulation of macrophage differentiation and function by discrete events, two cover the interaction between macrophages and apoptotic leukocytes during the resolution of inflammation. Korns et al. (2011) outline the regulation of apoptotic cell clearance by macrophages (efferocytosis) and the environmental cues that promote the efferocytic capabilities of macrophages. The second manuscript by Ariel and Serhan (2012) reviews the impact of apoptotic cell sensing and disposal by macrophages on the switches in functional phenotypes displayed by these cells. The effect of another environmental factor, hypoxia, on monocyte/macrophage activation, and differentiation through transcriptional and translational modulation is covered by Rahat et al. (2011). Lugo-Villarino et al. (2011) discuss the pathogenesis and co-mortality displayed by two macrophage-inhabiting microbes (HIV and Mycobacterium Tuberculosis) and their influence on macrophage polarization. Patel et al. (2011) review the role of melanocortin receptor expression by macrophages in anti-inflammation and the resolution of inflammation, with attention given to melanocortin receptor agonists as therapeutic agents. Several review articles discuss the function of macrophages during inflammation and/or its resolution within distinct anatomical sites, taking into account the unique features of these tissue-specific macrophages, in particular the distinct environments in which they reside and their interactions with neighboring cells. Claria et al. (2011) review current knowledge on the contribution of macrophages to the inflammatory state characterizing adipose tissue and the phenotypic changes observed in macrophages during obesity. Kennedy et al. (2011) discuss macrophage polarization occurring within the synovial space of arthritic joints and its modulation by cytokines, transcription factors, and pro-resolving lipid mediators. The article from Bosurgi et al. (2011) describes the multiple actions of macrophages in injured skeletal muscle, where the effects of these cells are a double-edged sword and can either promote healing and repair or lead to fibrosis and fat replacement. Herold et al. (2011) survey the indispensable role of macrophages in the resolution and termination of inflammation in lung infection and injury as well as the molecular pathways involved in these processes. Proper termination of inflammatory events and clearance of apoptotic cells are also critical to the cardiovascular system, as reviewed by Thorp (2012), and defects in macrophage efferocytosis can lead to atherosclerosis and myocardial infarction. While monocyte-derived macrophages and resident microglia were previously considered to be detrimental in brain inflammation and injury, recent advances reviewed by Jung and Schwartz (2012) suggest an opposite role for these macrophage-like cells, with a positive impact on brain maintenance and repair. Finally, Hasturk et al. (2012) outline the reciprocal interaction between periodontal disease and chronic inflammatory illnesses and the role that macrophages play in mediating these chronic inflammatory diseases. Altogether, the articles in this volume portray the complexity of the multiple roles played by macrophages and members of their lineage during inflammation and its resolution, and their manipulation by the injured milieu. These topics are currently heavily studied, and advances in the field, facilitated by state-of-the-art genetics and optical technologies, will undoubtedly continue to contribute to our understanding of the immune systems response to foreign insults, trauma, and inflammatory disorders.

The atypical chemokine receptor D6 controls macrophage efferocytosis and cytokine secretion during the resolution of inflammation

The resolution of acute inflammation is hallmarked by the apoptotic death of inflammatory polymorphonuclear (PMN) cells, followed by their clearance by macrophages. In turn, resolution‐phase macrophages exert reduced proinflammatory cytokine production, termed immune silencing. In this study, we found that the atypical chemokine receptor D6 plays an important and chemokine scavenging‐independent role in promoting macrophage‐mediated resolution. D6–/– mice displayed increased numbers of macrophages (2.2‐fold increase), but not neutrophils, in their peritonea during the resolution of murine zymosan A‐initiated peritonitis, in comparison to D6+/+ animals. Moreover, D6‐deficient macrophages engulfed higher numbers of apoptotic PMN cells in vivo (1.6‐fold increase), and secreted higher amounts of TNF‐α, CCL3, and CCL5 ex vivo than their wild‐type (WT) counterparts. In addition, D6 was found to be expressed on apoptotic neutrophils from healthy humans and rodents. Moreover, the immune silencing of LPS‐stimulated macrophages following their incubation with senescent PMN cells ex vivo (in terms of TNF‐α, IL‐1β, and CCL5 secretion) was diminished (50–65% decrease) when D6–/– PMN cells were applied. Accordingly, the adhesive responses induced by macrophage interactions with senescent PMN cells were reduced with D6‐deficient PMN cells. Thus, our results indicate a novel mode of action for D6 during the resolution of inflammation that is instrumental to the shaping of resolving macrophage phenotypes and the completion of resolution.—Pashover‐Schallinger, E., Aswad, M., Schif‐Zuck, S., Shapiro, H., Singer, P., Ariel, A. The atypical chemokine receptor D6 controls macrophage efferocytosis and cytokine secretion during the resolution of inflammation. FASEB J. 26, 3891–3900 (2012). www.fasebj.org

15-Lipoxygenases in cancer: A double-edged sword?

Among the lipoxygenases, a diverse family of fatty acid dioxygenases with varying tissue-specific expression, 15-lipoxygenase (15-LOX) was found to be involved in many aspects of human cancer, such as angiogenesis, chronic inflammation, metastasis formation, and direct and indirect tumor suppression. Herein, evidence for the expression and action of 15-LOX and its orthologs in various neoplasms, including solid tumors and hematologic malignancies, is reviewed. The debate surrounding the impact of 15-LOX as either a tumor-promoting or a tumor-suppressing enzyme is highlighted and discussed in the context of its role in other biological systems.