Carole A. Oskeritzian

Mast cell plasticity and sphingosine-1-phosphate in immunity, inflammation and cancer

Mast cells (MC) are found in all vascularized tissues at homeostasis and, until recently, were viewed only as effector cells of allergic reactions via degranulation, the canonical process through which MC release mediators, including histamine and pre-formed proteases and cytokines such as TNF. Cross-linking of IgE bound to surface high affinity receptors for IgE (FcɛRI) by a specific antigen (Ag) triggers signaling events leading to degranulation. We and others have reported the concomitant production and export of an influential multifaceted sphingolipid mediator, sphingosine-1-phosphate (S1P) transported outside of MC by ATP-binding cassettes (ABC) transporters, i.e., independently of degranulation. Indeed, the MC horizon expanded by the discovery of their unique ability to selectively release mediators depending upon the stimulus and receptors involved. Aside from degranulation and transporter usage, MC are also endowed with piecemeal degranulation, a slower process during which mediator release occurs with minor morphological changes. The broad spectrum of pro- and anti-inflammatory bioactive substances MC produce and release, their amounts and delivery pace render these cells bona fide fine-tuners of the immune response. In this viewpoint article, MC developmental, phenotypic and functional plasticity, its modulation by microRNAs and its relevance to immunity, inflammation and cancer will be discussed.

IL-10–Induced miR-155 Targets SOCS1 To Enhance IgE-Mediated Mast Cell Function

IL-10 is an important regulatory cytokine that modulates a wide range of immune cells. Whereas it is best known for its ability to suppress immune responses, IL-10 has been found to be pathogenic in several human and animal studies of immune-mediated diseases. There is a considerable gap in our understanding of the molecular mechanisms behind the stimulatory effects of IL-10 during allergic inflammation. IL-10 treatment has been shown to suppress mast cell TNF production. In this study, we report that whereas TNF secretion was reduced, IL-10 surprisingly enhanced IgE-mediated protease and cytokine production both in vitro and in vivo. This stimulatory effect was consistent in mouse and human skin mast cells. IL-10 enhanced activation of the key FcεRI signaling proteins Stat5, JNK, and ERK. We demonstrate that IL-10 effects are dependent on Stat3 activation, eliciting miR-155 expression, with a resulting loss of suppressor of cytokine signaling-1. The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155–deficient mice. Taken together, our results reveal an IL-10–induced, Stat3–miR-155 signaling pathway that can promote mast cell responses.

Sphingosine-1 Phosphate: A New Modulator of Immune Plasticity in the Tumor Microenvironment.

In the last 15 years, increasing evidences demonstrate a strong link between sphingosine-1-phosphate (S1P) and both normal physiology and progression of different diseases, including cancer and inflammation. Indeed, numerous studies show that tissue levels of this sphingolipid metabolite are augmented in many cancers, affecting survival, proliferation, angiogenesis, and metastatic spread. Recent insights into the possible role of S1P as a therapeutic target has attracted enormous attention and opened new opportunities in this evolving field. In this review, we will focus on the role of S1P in cancer, with particular emphasis in new developments that highlight the many functions of this sphingolipid in the tumor microenvironment. We will discuss how S1P modulates phenotypic plasticity of macrophages and mast cells, tumor-induced immune evasion, differentiation and survival of immune cells in the tumor milieu, interaction between cancer and stromal cells, and hypoxic response.

TGF-β1 Suppresses IL-33–Induced Mast Cell Function

TGF-β1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-β1 can suppress IgE-mediated mast cell activation of human and mouse mast cells. IL-33 is a member of the IL-1 family capable of inducing mast cell responses and enhancing IgE-mediated activation. In this study, we investigated the effects of TGF-β on IL-33–mediated mast cell activation. Bone marrow–derived mast cells cultured in TGF-β1, β2, or β3 showed reduced IL-33–mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-β1 inhibited IL-33–mediated Akt and ERK phosphorylation as well as NF-κB– and AP-1–mediated transcription. These effects were functionally important, as TGF-β1 injection suppressed IL-33–induced systemic cytokines in vivo and inhibited IL-33–mediated cytokine release from human mast cells. TGF-β1 also suppressed the combined effects of IL-33 and IgE-mediated activation on mouse and human mast cells. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGF-β1 on IgE-mediated activation, demonstrate that TGF-β1 can provide broad inhibitory signals to activated mast cells.

Fluvastatin Suppresses Mast Cell and Basophil IgE Responses: Genotype-Dependent Effects

Mast cell (MC)– and basophil-associated inflammatory diseases are a considerable burden to society. A significant portion of patients have symptoms despite standard-of-care therapy. Statins, used to lower serum cholesterol, have immune-modulating activities. We tested the in vitro and in vivo effects of statins on IgE-mediated MC and basophil activation. Fluvastatin showed the most significant inhibitory effects of the six statins tested, suppressing IgE-induced cytokine secretion among mouse MCs and basophils. The effects of fluvastatin were reversed by mevalonic acid or geranylgeranyl pyrophosphatase, and mimicked by geranylgeranyl transferase inhibition. Fluvastatin selectively suppressed key FcεRI signaling pathways, including Akt and ERK. Although MCs and basophils from the C57BL/6J mouse strain were responsive to fluvastatin, those from 129/SvImJ mice were completely resistant. Resistance correlated with fluvastatin-induced upregulation of the statin target HMG-CoA reductase. Human MC cultures from eight donors showed a wide range of fluvastatin responsiveness. These data demonstrate that fluvastatin is a potent suppressor of IgE-mediated MC activation, acting at least partly via blockade of geranyl lipid production downstream of HMG-CoA reductase. Importantly, consideration of statin use for treating MC–associated disease needs to incorporate genetic background effects, which can yield drug resistance.

Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2

Mast cells (MC) are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P) functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2-) mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF-) A and matrix metalloproteinase- (MMP-) 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC) or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation.

Dexamethasone rapidly suppresses IL‐33‐stimulated mast cell function by blocking transcription factor activity

Mast cells are critical effectors of allergic disease and can be activated by IL‐33, a proinflammatory member of the IL‐1 cytokine family. IL‐33 worsens the pathology of mast cell–mediated diseases, but therapies to antagonize IL‐33 are still forthcoming. Because steroids are the mainstay of allergic disease treatment and are well known to suppress mast cell activation by other stimuli, we examined the effects of the steroid dexamethasone on IL‐33‐mediated mast cell function. We found that dexamethasone potently and rapidly suppressed cytokine production elicited by IL‐33 from murine bone marrow–derived and peritoneal mast cells. IL‐33 enhances IgE‐mediated mast cell cytokine production, an activity that was also antagonized by dexamethasone. These effects were consistent in human mast cells. We additionally observed that IL‐33 augmented migration of IgE‐sensitized mast cells toward antigen. This enhancing effect was similarly reversed by dexamethasone. Simultaneous addition of dexamethasone with IL‐33 had no effect on the phosphorylation of MAP kinases or NFκB p65 subunit; however, dexamethasone antagonized AP‐1‐ and NFκB‐mediated transcriptional activity. Intraperitoneal administration of dexamethasone completely abrogated IL‐33‐mediated peritoneal neutrophil recruitment and prevented plasma IL‐6 elevation. These data demonstrate that steroid therapy may be an effective means of antagonizing the effects of IL‐33 on mast cells in vitro and in vivo, acting partly by suppressing IL‐33‐induced NFκB and AP‐1 activity.

Lactic Acid Suppresses IL-33–Mediated Mast Cell Inflammatory Responses via Hypoxia-Inducible Factor-1α–Dependent miR-155 Suppression

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments with elevated IL-33 and mast cell infiltration. Although IL-33 is a potent mast cell activator, how LA affects IL-33–mediated mast cell function is unknown. To investigate this, mouse bone marrow–derived mast cells were cultured with or without LA and activated with IL-33. LA reduced IL-33–mediated cytokine and chemokine production. Using inhibitors for monocarboxylate transporters (MCT) or replacing LA with sodium lactate revealed that LA effects are MCT-1– and pH-dependent. LA selectively altered IL-33 signaling, suppressing TGF-β–activated kinase-1, JNK, ERK, and NF-κB phosphorylation, but not p38 phosphorylation. LA effects in other contexts have been linked to hypoxia-inducible factor (HIF)-1α, which was enhanced in bone marrow–derived mast cells treated with LA. Because HIF-1α has been shown to regulate the microRNA miR-155 in other systems, LA effects on miR-155-5p and miR-155-3p species were measured. In fact, LA selectively suppressed miR-155-5p in an HIF-1α–dependent manner. Moreover, overexpressing miR-155-5p, but not miR-155-3p, abolished LA effects on IL-33–induced cytokine production. These in vitro effects of reducing cytokines were consistent in vivo, because LA injected i.p. into C57BL/6 mice suppressed IL-33–induced plasma cytokine levels. Lastly, IL-33 effects on primary human mast cells were suppressed by LA in an MCT-dependent manner. Our data demonstrate that LA, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation.

A New Image Analysis Method Based on Morphometric and Fractal Parameters for Rapid Evaluation of In Situ Mammalian Mast Cell Status

Apart from their effector functions in allergic disorders, tissue-resident mast cells (MC) are gaining recognition as initiators of inflammatory events through their distinctive ability to secrete many bioactive molecules harbored in cytoplasmic granules. Activation triggers mediator release through a regulated exocytosis named degranulation. MC activation is still substantiated by measuring systemic levels of MC-restricted mediators. However, identifying the anatomical location of MC activation is valuable for disease diagnosis. We designed a computer-assisted morphometric method based on image analysis of methylene blue (MB)-stained normal mouse skin tissue sections that quantitates actual in situ MC activation status. We reasoned MC cytoplasm could be viewed as an object featuring unique relative mass values based on activation status. Integrated optical density and area (A) ratios were significantly different between intact and degranulated MC (p<0.001). The examination of fractal characteristics is of translational diagnostic/prognostic value in cancer and readily applied to quantify cytoskeleton morphology and vasculature. Fractal dimension (D), a measure of their comparative space filling capacity and structural density, also differed significantly between intact and degranulated MC (p<0.001). Morphometric analysis provides a reliable and reproducible method for in situ quantification of MC activation status.

Mast cells and sphingosine-1-phosphate underlie prelesional remodeling in a mouse model of eczema

Atopic dermatitis (AD) is a chronic skin inflammation that affects children and adults worldwide, but its pathogenesis remains ill‐understood.