Alena P. Chumanevich

Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor

Inflammatory bowel diseases (IBDs), mainly Crohns disease and ulcerative colitis, are dynamic, chronic inflammatory conditions that are associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism for regulating IBD. Peptidylarginine deiminases (PADs) catalyze the posttranslational conversion of peptidylarginine to peptidylcitrulline in a calcium-dependent, irreversible reaction and mediate the effects of proinflammatory cytokines. Because PAD levels are elevated in mouse and human colitis, we hypothesized that a novel small-molecule inhibitor of the PADs, i.e., chloramidine (Cl-amidine), could suppress colitis in a dextran sulfate sodium mouse model. Results are consistent with this hypothesis, as demonstrated by the finding that Cl-amidine treatment, both prophylactic and after the onset of disease, reduced the clinical signs and symptoms of colitis, without any indication of toxic side effects. Interestingly, Cl-amidine drives apoptosis of inflammatory cells in vitro and in vivo, providing a mechanism by which Cl-amidine suppresses colitis. In total, these data help validate the PADs as therapeutic targets for the treatment of IBD and further suggest Cl-amidine as a candidate therapy for this disease.

Chronic epithelial NF-κB activation accelerates APC loss and intestinal tumor initiation through iNOS up-regulation

The role of NF-κB activation in tumor initiation has not been thoroughly investigated. We generated Ikkβ(EE)IEC transgenic mice expressing constitutively active IκB kinase β (IKKβ) in intestinal epithelial cells (IECs). Despite absence of destructive colonic inflammation, Ikkβ(EE)IEC mice developed intestinal tumors after a long latency. However, when crossed to mice with IEC-specific allelic deletion of the adenomatous polyposis coli (Apc) tumor suppressor locus, Ikkβ(EE)IEC mice exhibited more β-catenin+ early lesions and visible small intestinal and colonic tumors relative to Apc+/ΔIEC mice, and their survival was severely compromised. IEC of Ikkβ(EE)IEC mice expressed high amounts of inducible nitric oxide synthase (iNOS) and elevated DNA damage markers and contained more oxidative DNA lesions. Treatment of Ikkβ(EE)IEC/Apc+/ΔIEC mice with an iNOS inhibitor decreased DNA damage markers and reduced early β-catenin+ lesions and tumor load. The results suggest that persistent NF-κB activation in IEC may accelerate loss of heterozygocity by enhancing nitrosative DNA damage.

A Hexane Fraction of American Ginseng Suppresses Mouse Colitis and Associated Colon Cancer: Anti-inflammatory and Proapoptotic Mechanisms

Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4+/CD25− primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis. Cancer Prev Res; 5(4); 685–96. ©2012 AACR.

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.

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.