Ileal Crohn's Disease Exhibits Reduced Activity of Phospholipase C-ß3-Dependent Wnt/ß-Catenin Signaling Pathway.

Crohn's disease is a chronic, debilitating, inflammatory bowel disease. Here, we report a critical role of phospholipase C-ß3 (PLC-ß3) in intestinal homeostasis. In PLC-ß3-deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-ß3 deficiency in multiple non-hematopoietic cell types. PLC-ß3 deficiency resulted in reduced Wnt/ß-catenin signaling, which is essential for homeostasis and the regeneration of the intestinal epithelium. PLC-ß3 regulated the Wnt/ß-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and, potentially, protein-protein interaction levels. PLC-ß3-deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/ß-catenin signaling. Reduced expression of PLC-ß3 and its signature genes was found in biopsies of patients with ileal Crohn's disease. PLC-ß regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that a reduction in PLC-ß3-mediated Wnt/ß-catenin signaling contributes to the pathogenesis of ileal Crohn's disease.

Immunomodulatory contribution of mast cells to the regenerative biomaterial microenvironment.

Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

Histamine-releasing factor in severe asthma and rhinovirus-associated asthma exacerbation.

BACKGROUND: Histamine-releasing factor (HRF) is implicated in allergic diseases. We previously showed its pathogenic role in murine models of asthma. OBJECTIVE: We aim to present data analysis from 3 separate human samples (sera samples from asthmatic patients, nasal washings from rhinovirus [RV]-infected individuals, and sera samples from patients with RV-induced asthma exacerbation) and 1 mouse sample to investigate correlates of HRF function in asthma and virus-induced asthma exacerbations. METHODS: Total IgE and HRF-reactive IgE/IgG as well as HRF in sera from patients with mild/moderate asthma or severe asthma (SA) and healthy controls (HCs) were quantified by ELISA. HRF secretion in culture media from RV-infected adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells and in nasal washings from experimentally RV-infected subjects was analyzed by Western blotting. HRF-reactive IgE/IgG levels in longitudinal serum samples from patients with asthma exacerbations were also quantified. RESULTS: HRF-reactive IgE and total IgE levels were higher in patients with SA than in HCs, whereas HRF-reactive IgG (and IgG1) level was lower in asthmatic patients versus HCs. In comparison with HRF-reactive IgElow asthmatic patients, HRF-reactive IgEhigh asthmatic patients had a tendency to release more tryptase and prostaglandin D2 on anti-IgE stimulation of bronchoalveolar lavage cells. RV infection induced HRF secretion from adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells, and intranasal RV infection of human subjects induced increased HRF secretion in nasal washes. Asthmatic patients had higher levels of HRF-reactive IgE at the time of asthma exacerbations associated with RV infection, compared with those after the resolution. This phenomenon was not seen in asthma exacerbations without viral infections. CONCLUSIONS: HRF-reactive IgE is higher in patients with SA. RV infection induces HRF secretion from respiratory epithelial cells both in vitro and in vivo. These results suggest the role of HRF in asthma severity and RV-induced asthma exacerbation.

Regulation of Syk activity by antiviral adaptor MAVS in FcεRI signaling pathway.

Background: Mast cells are the major effector cell type for IgE-mediated allergic reactions. Recent studies revealed a role for mast cells in orchestrating the host response to viral infections. Objective: We studied the relationship between FcεRI (high-affinity IgE receptor) and RIG-I-like receptor (RLR)-mediated antiviral signaling pathways. Methods: Mast cells (BMMCs) were cultured from bone marrow cells from mice deficient in MAVS or other RLR signaling molecules. MAVS expression was restored by retroviral transduction of MAVS-deficient BMMCs. These cells were stimulated with IgE and antigen and their activation (degranulation and cytokine production/secretion) was quantified. FcεRI-mediated signaling events such as protein phosphorylation and Ca2+ flux were analyzed by western blotting and enzyme assays. WT and mutant mice as well as mast cell-deficient KitW-sh/W-sh mice engrafted with BMMCs were subjected to passive cutaneous anaphylaxis. Results: Unexpectedly, we found that mast cells devoid of the adaptor molecule MAVS exhibit dramatically increased cytokine production upon FcεRI stimulation, despite near-normal degranulation. Consistent with these observations, MAVS inhibited tyrosine phosphorylation, thus catalytic activity of Syk kinase, the key signaling molecule for FcεRI-mediated mast cell activation. By contrast, mast cells deficient in RIG-I, MDA5 or IRF3, which are antiviral receptor and signaling molecules upstream or downstream of MAVS, exhibited reduced or normal mast cell activation. MAVS-deficient mice showed enhanced late-phase responses in passive cutaneous anaphylaxis. Conclusion: This study demonstrates that the adaptor MAVS in the RLR innate immune pathway uniquely intersects with the adaptive immune FcεRI signaling pathway.

Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation.

Allergens have been identified as potential triggers in patients with atopic dermatitis (AD). Patients with AD are highly sensitive to cockroach allergen. The underlying mechanism, however, remains undetermined. Here, we established a cockroach allergen-induced AD-like mouse model, and we demonstrate that repeated exposure to cockroach allergen led to aggravated mouse skin inflammation, characterized by increased type 2 immunity, type 2 innate lymphoid cells (ILC2s), and mast cells. Increased mast cells were also observed in patients with AD. Mast cell-deficient mice (KitW-sh/W-sh) showed diminished skin inflammation, suggesting that mast cells are required in allergen-induced skin inflammation. Furthermore, DC immunoreceptor (DCIR) is upregulated in skin mast cells of patients with AD and mediates allergen binding and uptake. DCIR-/- mice or reconstituted KitW-sh/W-sh mice with DCIR-/- mast cells showed a significant reduction in AD-like inflammation. Both in vitro and in vivo analyses demonstrate that DCIR-/- mast cells had reduced IgE-mediated mast cell activation and passive cutaneous anaphylaxis. Mechanistically, DCIR regulates allergen-induced IgE-mediated mast cell ROS generation and oxidation of calmodulin kinase II (ox-CaMKII). ROS-resistant CaMKII (MM-VVδ) prevents allergen-induced mast cell activation and inflammatory mediator release. Our study reveals a DCIR/ROS/CaMKII axis that controls allergen-induced mast cell activation and AD-like inflammation.

Short-term Change in Resting Energy Expenditure and Body Compositions in Therapeutic Process for Graves' Disease.

Objective In the medical treatment of Graves' disease, we sometimes encounter patients who gain weight after the onset of the disease. To estimate the energy required during the course of treatment when hyperthyroidism ameliorates, we measured the resting energy expenditure (REE) and body composition in patients with Graves' disease before and during treatment in the short-term. Methods Twenty patients with newly diagnosed Graves' disease were enrolled, and our REE data of 19 healthy volunteers were used. The REE was measured by a metabolic analyzer, and the basal energy expenditure (BEE) was estimated by the Harris-Benedict formula. The body composition, including body weight, fat mass (FM), muscle mass (MM) and lean body mass (LBM), were measured by a multi-frequency body composition analyzer. We tailored the nutritional guidance based on the measured REE. Results Serum thyrotropin levels were significantly increased at three and six months. Serum free thyroxine, free triiodothyronine and REE values were significantly decreased at one, three and six months. The REE/BEE ratio was 1.58±0.28 at the onset and significantly declined to 1.34±0.34, 1.06±0.19 and 1.01±0.16 at 1, 3 and 6 months, respectively. Body weight, MM and LBM significantly increased at three and six months. Conclusion The REE significantly decreased during treatment of Graves' disease. The decline was evident as early as one month after treatment. The REE after treatment was lower than in healthy volunteers, which may lead to weight gain. These data suggest that appropriate nutritional guidance is necessary with short-term treatment before the body weight normalizes in order to prevent an overweight condition and the emergence of metabolic disorders.

Cooperative Regulation of the Mucosal Mast Cell-Specific Protease Genes Mcpt1 and Mcpt2 by GATA and Smad Transcription Factors.

Mouse mast cell proteases (mMCP)-1 and -2 are specifically expressed in mucosal mast cells (MCs). However, the transcriptional regulation mechanism of the Mcpt1 and Mcpt2 genes induced in mucosal MCs is largely unknown. In the current study, we found that TGF-ß stimulation drastically induced upregulation of Mcpt1 and Mcpt2 mRNA in mouse bone marrow-derived MCs (BMMCs). TGF-ß-induced expression of Mcpt1 and Mcpt2 was markedly suppressed by transfection with small interfering RNA targeting Smad2 or Smad4 and moderately reduced by Smad3 small interfering RNA. We next examined the roles of the hematopoietic cell-specific transcription factors GATA1 and GATA2 in the expression of Mcpt1 and Mcpt2 and demonstrated that knockdown of GATA1 and GATA2 reduced the mRNA levels of Mcpt1 and Mcpt2 in BMMCs. The recruitment of GATA2 and acetylation of histone H4 of the highly conserved GATA-Smad motifs, which were localized in the distal regions of the Mcpt1 and Mcpt2 genes, were markedly increased by TGF-ß stimulation, whereas the level of GATA2 binding to the proximal GATA motif was not affected by TGF-ß. A reporter assay showed that TGF-ß stimulation upregulated GATA2-mediated transactivation activity in a GATA-Smad motif-dependent manner. We also observed that GATA2 and Smad4 interacted in TGF-ß-stimulated BMMCs via immunoprecipitation and Western blotting analysis. Taken together, these results demonstrate that TGF-ß induced mMCP-1 and -2 expression by accelerating the recruitment of GATA2 to the proximal regions of the Mcpt1 and Mcpt2 genes in mucosal MCs.

Effect of Simulated Patient Practice on the Self-Efficacy of Japanese Undergraduate Dietitians in Nutrition Care Process Skills.

OBJECTIVE: To examine the effect of an adapted simulated patient (SP) intervention on self-efficacy in nutrition care process skills. DESIGN: A repeated-measures design using a 25-item survey divided into 7 nutrition professional practice competencies (PPCs) employing a 5-point self-efficacy scale (1 = lowest to 5 = highest) administered immediately before and after the intervention. SETTING: A private Japanese university. PARTICIPANTS: Ninety Japanese third-year dietetics undergraduates aged 20-38 years. INTERVENTION: An adapted SP activity practicing nutrition care process skills for the infirm elderly population. MAIN OUTCOME MEASURES: Pre- to postintervention self-efficacy response scores and feedback. ANALYSIS: Mean preintervention survey scores were used to divide participants into statistical quartiles (Q1 indicated lowest mean scores and Q3, highest mean scores). Wilcoxon signed-rank tests compared each PPC's pre- and postintervention means. Kruskal-Wallis tests examined changes in quartiles' scores within each PPC. RESULTS: Self-efficacy improved significantly in PPCs relating to application of appropriate medical ethics and interpersonal skills (P = .02), appropriate nutrition assessment (P = .04), and creation of a nutrition management plan and nutrition intervention (P = .03). Self-efficacy of Q1 and Q2 rose significantly in most PPCs, although not for acting as a dietitian within a medical care team, whereas that of Q3 decreased for all PPCs. CONCLUSIONS AND IMPLICATIONS: Among initially low self-efficacy dietetics undergraduates, the SP intervention enhanced self-efficacy in 3 of the 6 PPCs practiced directly and may facilitate more realistic self-views among initially high self-efficacy students. However, further research in the design, implementation, and efficacy of this type of training is recommended to gauge its effects on the quality of related professional practice.

LIGHT-HVEM signaling in keratinocytes controls development of dermatitis.

Dermatitis is often associated with an allergic reaction characterized by excessive type 2 responses leading to epidermal acanthosis, hyperkeratosis, and dermal inflammation. Although factors like IL-4, IL-13, and thymic stromal lymphopoietin (TSLP) are thought to be instrumental for the development of this type of skin disorder, other cytokines may be critical. Here, we show that the tumor necrosis factor (TNF) superfamily protein LIGHT (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes) is required for experimental atopic dermatitis, and LIGHT directly controls keratinocyte hyperplasia, and production of periostin, a matricellular protein that contributes to the clinical features of atopic dermatitis as well as other skin diseases such as scleroderma. Mice with a conditional deletion of the LIGHT receptor HVEM (herpesvirus entry mediator) in keratinocytes phenocopied LIGHT-deficient mice in exhibiting reduced epidermal thickening and dermal collagen deposition in a model of atopic dermatitis driven by house dust mite allergen. LIGHT signaling through HVEM in human epidermal keratinocytes directly induced proliferation and periostin expression, and both keratinocyte-specific deletion of HVEM or antibody blocking of LIGHT-HVEM interactions after disease onset prevented expression of periostin and limited atopic dermatitis symptoms. Developing reagents that neutralize LIGHT-HVEM signaling might be useful for therapeutic intervention in skin diseases where periostin is a central feature.

Histamine-releasing factor enhances food allergy.

Food allergy occurs due to IgE- and mast cell-dependent intestinal inflammation. Previously, we showed that histamine-releasing factor (HRF), a multifunctional protein secreted during allergy, interacts with a subset of IgE molecules and that the HRF dimer activates mast cells in an HRF-reactive IgE-dependent manner. In this study, we investigated whether HRF plays any role in food allergy. Specifically, we determined that prophylactic and therapeutic administration of HRF inhibitors that block HRF-IgE interactions reduces the incidence of diarrhea and mastocytosis in a murine model of food allergy. Food allergy-associated intestinal inflammation was accompanied by increased secretion of the HRF dimer into the intestine in response to proinflammatory, Th2, and epithelial-derived cytokines and HRF-reactive IgE levels at the elicitation phase. Consistent with these data, patients with egg allergy had higher blood levels of HRF-reactive IgE compared with individuals that were not hypersensitive. Successful oral immunotherapy in egg-allergy patients and food-allergic mice reduced HRF-reactive IgE levels, thereby suggesting a pathological role for HRF in food allergy. Together, these results suggest that antigen and HRF dimer amplify intestinal inflammation by synergistically activating mast cells and indicate that HRF has potential as a therapeutic target in food allergy.

TWEAK mediates inflammation in experimental atopic dermatitis and psoriasis.

Atopic dermatitis (AD) and psoriasis are driven by alternate type 2 and type 17 immune responses, but some proteins might be critical to both diseases. Here we show that a deficiency of the TNF superfamily molecule TWEAK (TNFSF12) in mice results in defective maintenance of AD-specific T helper type 2 (Th2) and psoriasis-specific Th17 cells in the skin, and impaired expression of disease-characteristic chemokines and cytokines, such as CCL17 and TSLP in AD, and CCL20 and IL-19 in psoriasis. The TWEAK receptor, Fn14, is upregulated in keratinocytes and dermal fibroblasts, and TWEAK induces these cytokines and chemokines alone and in synergy with the signature T helper cytokines of either disease, IL-13 and IL-17. Furthermore, subcutaneous injection of recombinant TWEAK into naive mice induces cutaneous inflammation with histological and molecular signs of both diseases. TWEAK is therefore a critical contributor to skin inflammation and a possible therapeutic target in AD and psoriasis.

Defective natural killer cell activity in a mouse model of eczema herpeticum.

BACKGROUND: Patients with atopic dermatitis (AD) are susceptible to several viruses, including herpes simplex virus (HSV). Some patients experience 1 or more episodes of a severe skin infection caused by HSV termed eczema herpeticum (EH). There are numerous mouse models of AD, but no established model exists for EH. OBJECTIVE: We sought to establish and characterize a mouse model of EH. METHODS: We infected AD-like skin lesions with HSV1 to induce severe skin lesions in a dermatitis-prone mouse strain of NC/Nga. Gene expression was investigated by using a microarray and quantitative PCR; antibody titers were measured by means of ELISA; and natural killer (NK) cell, cytotoxic T-cell, regulatory T-cell, and follicular helper T-cell populations were evaluated by using flow cytometry. The role of NK cells in HSV1-induced development of severe skin lesions was examined by means of depletion and adoptive transfer. RESULTS: Inoculation of HSV1 induced severe erosive skin lesions in eczematous mice, which had an impaired skin barrier, but milder lesions in small numbers of normal mice. Eczematous mice exhibited lower NK cell activity but similar cytotoxic T-cell activity and humoral immune responses compared with normal mice. The role of NK cells in controlling HSV1-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. CONCLUSION: A murine model of EH with an impaired skin barrier was established in this study. We demonstrated a critical role of defective NK activities in the development of HSV1-induced severe skin lesions in eczematous mice.

Guanine nucleotide exchange factor RABGEF1 regulates keratinocyte-intrinsic signaling to maintain skin homeostasis.

Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target.

Hypothetical Atopic Dermatitis-Myeloproliferative Neoplasm Syndrome.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Myeloproliferative neoplasms (MPNs) are hematopoietic malignancies caused by uncontrolled proliferation of hematopoietic stem/progenitor cells. Recent studies have described several mutant mice exhibiting both AD-like skin inflammation and MPN. Common pathways for skin inflammation encompass overexpression of thymic stromal lymphopoietin and reduced signaling of epidermal growth factor receptor in the epidermis, while overproduction of granulocyte-colony-stimulating factor by keratinocytes and constitutive activation of Stat5 in hematopoietic stem cells are important for the development of MPN. The murine studies suggest the existence of a similar human disease tentatively termed as the atopic dermatitis-myeloproliferative neoplasm syndrome.

Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation.

Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell-cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell-cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC-DC synapse suggest a new role for intercellular crosstalk in defining the immune response.

The proportion of genes in a functional category is linked to mass-specific metabolic rate and lifespan.

Metabolic rate and lifespan are important biological parameters that are studied in a wide range of research fields. They are known to correlate with body mass, but their association with gene (protein) functions is poorly understood. In this study, we collected data on the metabolic rate and lifespan of various organisms and investigated the relationship of these parameters with their genomes. We showed that the proportion of genes in a functional category, but not genome size, was correlated with mass-specific metabolic rate and maximal lifespan. In particular, the proportion of genes in oxic reactions (which occur in the presence of oxygen) was significantly associated with these two biological parameters. Additionally, we found that temperature, taxonomy, and mode-of-life traits had little effect on the observed associations. Our findings emphasize the importance of considering the biological functions of genes when investigating the relationships between genome, metabolic rate, and lifespan. Moreover, this provides further insights into these relationships, and may be useful for estimating metabolic rate and lifespan in individuals and the ecosystem using a combination of body mass measurements and genomic data.

Basic techniques to study FcεRI signaling in mast cells.

Mast cells are the crucial effector cells for allergic reactions. They are activated through the aggregation of the high-affinity IgE receptor (FcεRI) with allergen and allergen-specific IgE. Tyrosine phosphorylation of FcεRI subunits and various signaling proteins is an initial triggering event, leading to the activation of several signaling pathways in mast cells. Much has been learned from analysis of mast cells derived from gene-targeted mice. Therefore, in this chapter we will first describe how to generate mast cells from mouse bone marrow cells and how to correct the genetic defect by retroviral transduction. Then we will describe how to assess early activation events by measuring several protein-tyrosine kinases (PTKs) and serine/threonine kinases (PS/TKs) such as Akt (protein kinase B), protein kinase C (PKC), and JNK. As signal transduction is highly dependent on protein-protein interactions, we will describe experimental details of co-immunoprecipitation methods that are used to confirm such interactions.

A mouse model of atopic dermatitis.

Atopic dermatitis (AD) is a chronic or chronically relapsing, pruritic inflammatory skin disease. The incidence of AD has dramatically increased for the past three decades in industrialized countries. We established a highly efficient method to induce AD-like skin lesions using repeated epicutaneous treatments with house dust mite allergen and staphylococcal enterotoxin B (SEB). The dermatitis-induced mice showed increased serum IgE levels that were similar to human AD patients and also treatable with dexamethasone. This mouse AD model has been used in a vaccinia virus infection study. It will also be useful to study pathogenic processes of AD and to evaluate the efficacy of a drug candidate. In this chapter, we describe the detailed method that can induce AD-like skin inflammation in multiple mouse strains.