Stat5B is required for IgE-Mediated mast cell function in vitro and in vivo.

Mast cells are found primarily at interfaces with the external environment, where they provide protection from pathogens but also elicit allergic inflammation. Mast cell activation by antigen-induced aggregation of IgE bound to the high affinity receptor, FcεRI, is a critical factor leading to inflammation and bronchoconstriction. We previously found that Stat5 is activated by FcεRI and that Stat5B suppression decreased IgE-induced cytokine production in vitro, but in vivo responses have not been assessed. We now show that Stat5B-deficient (KO) mice have reduced responses to IgE-mediated anaphylaxis, despite normal mast cell tissue distribution. Similarly, Stat5B KO mast cells have diminished IgE-induced degranulation and cytokine secretion in vitro. These mice have elevated IgE production that is not correlated with an intrinsic B cell defect. The current work demonstrates that the Stat5B isoform is required for normal mast cell function and suggests it limits IgE production in vivo.

Fluvastatin Induces Apoptosis in Primary and Transformed Mast Cells.

Statin drugs are widely employed in the clinic to reduce serum cholesterol. Because of their hydroxymethylglutaryl coenzyme A reductase antagonism, statins also reduce isoprenyl lipids necessary for the membrane anchorage and signaling of small G-proteins in the Ras superfamily. We previously found that statins suppress immunoglobulin E (IgE)-mediated mast cell activation, suggesting these drugs might be useful in treating allergic disease. Although IgE-induced function is critical to allergic inflammation, mast cell proliferation and survival also impact atopic disease and mast cell neoplasia. In this study, we describe fluvastatin-mediated apoptosis in primary and transformed mast cells. An IC50 was achieved between 0.8 and 3.5 µM in both cell types, concentrations similar to the reported fluvastatin serum Cmax value. Apoptosis was correlated with reduced stem cell factor (SCF)-mediated signal transduction, mitochondrial dysfunction, and caspase activation. Complementing these data, we found that p53 deficiency or Bcl-2 overexpression reduced fluvastatin-induced apoptosis. We also noted evidence of cytoprotective autophagy in primary mast cells treated with fluvastatin. Finally, we found that intraperitoneal fluvastatin treatment reduced peritoneal mast cell numbers in vivo These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell-associated allergic and neoplastic diseases. SIGNIFICANCE STATEMENT: Fluvastatin, a statin drug used to lower cholesterol, induces apoptosis in primary and transformed mast cells by antagonizing protein isoprenylation, effectively inhibiting stem cell factor (SCF)-induced survival signals. This drug may be an effective means of suppressing mast cell survival.

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.

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.

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.

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.

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.