Heather L. Caslin

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.

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.

The effect of obesity on inflammatory cytokine and leptin production following acute mental stress.

Obesity may contribute to cardiovascular disease (CVD) risk by eliciting chronic systemic inflammation and impairing the immune response to additional stressors. There has been little assessment of the effect of obesity on psychological stress, an independent risk factor for CVD. Therefore, it was of interest to examine interleukin-6, tumor necrosis factor-α, interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1Ra), and leptin following an acute mental stress task in nonobese and obese males. Twenty college-aged males (21.3 ± 0.56 years) volunteered to participate in a 20-min Stroop color-word and mirror-tracing task. Subjects were recruited for obese (body mass index: BMI > 30) and nonobese (BMI < 25) groups, and blood samples were collected for enzyme-linked immunosorbent assay analysis. The acute mental stress task elicited an increase in heart rate, catecholamines, and IL-1β in all subjects. Additionally, acute mental stress increased cortisol concentrations in the nonobese group. There was a significant reduction in leptin in obese subjects 30 min posttask compared with a decrease in nonobese subjects 120 min posttask. Interestingly, the relationship between the percent change in leptin and IL-1Ra at 120 min posttask in response to an acute mental stress task was only observed in nonobese individuals. This is the first study to suggest that adiposity in males may impact leptin and inflammatory signaling mechanisms following acute mental stress.

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.

The effect of acute physical and mental stress on soluble cellular adhesion molecule concentration.

AIMS This study investigated the impact of acute physical and mental stress on serum concentrations of vascular cell adhesion molecule (VCAM)-1 and CX3CL1/fractalkine. MATERIALS AND METHODS Male volunteers (n=20; 21.3±0.55years of age) completed a graded treadmill test to exhaustion and a 20-minute mental stress task (Stroop Color-Word Test, mental arithmetic) on separate, non-consecutive days. Heart rate (HR) was measured at baseline and throughout exercise and mental stress. Blood was collected at baseline (PRE), immediately following (POST) and 30min after (POST30) exercise and mental stress. Soluble VCAM-1 and fractalkine were quantified in participant serum via enzyme-linked immunosorbent assays. KEY FINDINGS Both treadmill exercise and the mental stress task significantly increased participant HR; although, exercise resulted in a substantially greater increase in participant HR compared to mental stress (197.82±11.99 vs. 38.67±3.10% [p<0.001]). VCAM-1 (815.74±139.55 vs. 738.67±131.59ng/mL [p=0.002]) and fractalkine (1.032±0.33 vs. 0.59±0.20ng/mL [p<0.001]) were significantly elevated in participant serum POST maximal exercise before returning to values similar to baseline at POST30. The acute mental stress task did not significantly alter serum VCAM-1 or fractalkine at any time point. SIGNIFICANCE In conclusion, maximal aerobic exercise results in a significant elevation of the soluble adhesion molecules VCAM-1 and fractalkine in the serum of adult males that does not occur following laboratory-induced mental stress. The findings of the current investigation may suggest a novel protective role for acute aerobic exercise in vascular health via exercise-induced CAM proteolysis.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells

While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.

Controlling Mast Cell Activation and Homeostasis: Work Influenced by Bill Paul That Continues Today

Mast cells are tissue resident, innate immune cells with heterogenous phenotypes tuned by cytokines and other microenvironmental stimuli. Playing a protective role in parasitic, bacterial, and viral infections, mast cells are also known for their role in the pathogenesis of allergy, asthma, and autoimmune diseases. Here, we review factors controlling mast cell activation, with a focus on receptor signaling and potential therapies for allergic disease. Specifically, we will discuss our work with FcεRI and FγR signaling, IL-4, IL-10, and TGF-β1 treatment, and Stat5. We conclude with potential therapeutics for allergic disease. Much of these efforts have been influenced by the work of Bill Paul. With many mechanistic targets for mast cell activation and different classes of therapeutics being studied, there is reason to be hopeful for continued clinical progress in this area.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription

Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.

G protein-coupled receptor kinase-2 in peripheral blood mononuclear cells following acute mental stress.

AIMS This study investigated G-protein-coupled receptor kinase-2 (GRK2) density in peripheral blood mononuclear cells (PBMC) and its relationship to plasma pro-inflammatory cytokine concentrations following acute mental stress. MAIN METHODS Apparently healthy males (n=20; 21.3±0.55years) participated in an acute mental stress task. Heart rate was measured at baseline and throughout mental stress. Plasma epinephrine (EPI), norepinephrine (NE), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were assessed via enzyme-linked immunosorbent assays before, immediately following, and 30, 60 and 120min after the mental stress task. GRK2 density was measured by western blot technique at the same time points. KEY FINDINGS Acute mental stress elicited significant elevations in HR, and plasma EPI and NE. Additionally, GRK2 density increased significantly across time following the stress task. Post hoc analyses revealed that GRK2 density was significantly elevated at 30 and 60min following acute stress. A significant positive correlation was observed between GRK2 density and plasma EPI, while a significant negative correlation was revealed between GRK2 density and TNF-α across all time points. SIGNIFICANCE Acute mental stress significantly increased GRK2 density in PBMCs of young adult males. Furthermore, although plasma IL-6 and TNF-α did not change following mental stress, it remains unknown whether a longer time period was needed to observe a pro-inflammatory state associated with the desensitization of β-adrenergic receptor activity. Our findings that GRK2 expression is promptly increased in PBMC following an acute stress task, may suggest a link between stress and intracellular inflammatory signaling.

The Use of Human and Mouse Mast Cell and Basophil Cultures to Assess Type 2 Inflammation

Mast cells and basophils are important innate immune cells involved in resistance to parasitic infection and are critical orchestrators of allergic disease. The relative ease with which they are cultured from mouse or human tissues allows one to work with primary cells that maintain a differentiated and functional phenotype. In this chapter, we describe the methods by which mouse mast cells and basophils can be cultured from bone marrow. We also provide methods for isolating and expanding mouse peritoneal mast cells and human skin mast cells.