Kim A. T. Verheijden

Deoxynivalenol: a trigger for intestinal integrity breakdown

Disintegration of the colonic epithelial barrier is considered a key event in the initiation and progression of inflammatory bowel and celiac disease. As the primary etiology of these diseases remains unknown, we hypothesized that the trichothecene deoxynivalenol (DON), a fungal metabolite found in grain‐based human diets, might be one of the triggers resulting in an impairment of the intestinal tight junction network preceding an inflammatory response. Using horizontal impedance measurements, we demonstrate that DON disintegrates a human Caco‐2 cell monolayer within <1 h after exposure to concentrations as low as 1.39 μM. This initial trigger is followed by a decrease in transepithelial resistance and an increased permeability of marker molecules, such as lucifer yellow and FITC‐labeled dextran. In parallel, the increase in paracellular transport of FITC‐dextran is demonstrated in vivo in B6C3F1 mice, challenged orally with DON. In vitro claudin protein levels are decreased and correlated with a displacement within the cells in vitro and in vivo, accompanied by a compensatory up‐regulation of mRNA levels of claudins and their binding partner ZO‐1. In treated mice, alterations in villus architecture in the entire intestinal tract resemble the disintegration of the epithelial barrier, a characteristic of chronic inflammatory bowel disease.—Akbari, P., Braber, S., Gremmels, H., Koelink, P. J., Verheijden, K. A. T., Garssen, J., Fink‐Gremmels, J. Deoxynivalenol: a trigger for intestinal integrity breakdown. FASEB J. 28, 2414–2429 (2014). www.fasebj.org

Galacto-oligosaccharides Protect the Intestinal Barrier by Maintaining the Tight Junction Network and Modulating the Inflammatory Responses after a Challenge with the Mycotoxin Deoxynivalenol in Human Caco-2 Cell Monolayers and B6C3F1 Mice

BACKGROUND The integrity of the epithelial layer in the gastrointestinal tract protects organisms from exposure to luminal antigens, which are considered the primary cause of chronic intestinal inflammation and allergic responses. The common wheat-associated fungal toxin deoxynivalenol acts as a specific disruptor of the intestinal tight junction network and hence might contribute to the pathogenesis of inflammatory bowel diseases. OBJECTIVE The aim of the current study was to assess whether defined galacto-oligosaccharides (GOSs) can prevent deoxynivalenol-induced epithelial dysfunction. METHODS Human epithelial intestinal Caco-2 cells, pretreated with different concentrations of GOSs (0.5%, 1%, and 2%) for 24 h, were stimulated with 4.2-μM deoxynivalenol (24 h), and 6/7-wk-old male B6C3F1 mice were fed a diet supplemented with 1% GOSs for 2 wk before being orally exposed to deoxynivalenol (25 mg/kg body weight, 6 h). Barrier integrity was determined by measuring transepithelial electrical resistance (TEER) and intestinal permeability to marker molecules. A calcium switch assay was conducted to study the assembly of epithelial tight junction proteins. Alterations in tight junction and cytokine expression were assessed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, or ELISA, and their localization was visualized by immunofluorescence microscopy. Sections of the proximal and distal small intestine were stained with hematoxylin/eosin for histomorphometric analysis. RESULTS The in vitro data showed that medium supplemented with 2% GOSs improved tight junction assembly reaching an acceleration of 85% after 6 h (P < 0.05). In turn, GOSs prevented the deoxynivalenol-induced loss of epithelial barrier function as measured by TEER (114% of control), and paracellular flux of Lucifer yellow (82.7% of prechallenge values, P < 0.05). Moreover, GOSs stabilized the expression and cellular distribution of claudin3 and suppressed by >50% the deoxynivalenol-induced synthesis and release of interleukin-8 [IL8/chemokine CXC motif ligand (CXCL8)] (P < 0.05). In mice, GOSs prevented the deoxynivalenol-induced mRNA overexpression of claudin3 (P = 0.022) and CXCL8 homolog keratinocyte hemoattractant (Kc) (Cxcl1) (P = 0.06) as well as the deoxynivalenol-induced morphologic defects. CONCLUSIONS The results demonstrate that GOSs stimulate the tight junction assembly and in turn mitigate the deleterious effects of deoxynivalenol on the intestinal barrier of Caco-2 cells and on villus architecture of B6C3F1 mice.

A comparison of fixation methods on lung morphology in a murine model of emphysema.

Emphysema is characterized by enlargement of the alveoli, which is the most important parameter to assess the presence and severity of this disease. Alveolar enlargement is primarily defined on morphological criteria; therefore, characterization of this disease with morphological parameters is a prerequisite to study the pathogenesis. For this purpose, different methods of lung fixation were evaluated in a murine model of LPS-induced lung emphysema. Five different methods of lung fixation were evaluated: intratracheal instillation of fixatives, in situ fixation, fixed-volume fixation, vascular whole body perfusion, and vacuum inflation. In addition, the effects of three different fixatives (10% formalin, Carnoys, and agarose/10% formalin solution) and two embedding methods (paraffin and plastic) were investigated on the murine lung morphology. Mice received intranasal administration of LPS to induce alveolar wall destruction. Quantification of air space enlargement was determined by mean linear intercept analysis, and the histological sections were analyzed for the most optimal fixation method. Additionally, routine immunohistological staining was performed on lung tissue of PBS-treated mice. Intratracheal instillation of formalin or agarose/formalin solution, in situ fixation, and fixed-volume fixation provided a normal lung architecture, in contrast to the lungs fixed via whole body perfusion and vacuum inflation. Formalin-fixed lungs resulted in the most optimal lung morphology for lung emphysema analysis when embedded in paraffin, while for Carnoys fixed lungs, plastic embedding was preferred. The histological findings, the mean linear intercept measurement, and the immunohistochemistry data demonstrated that fixation by intratracheal instillation of 10% formalin or in situ fixation with 10% formalin are the two most optimal methods to fix lungs for alveolar enlargement analysis to study lung emphysema.

The development of allergic inflammation in a murine house dust mite asthma model is suppressed by synbiotic mixtures of non-digestible oligosaccharides and Bifidobacterium breve M-16V

AbstractPurpose The incidence and severity of allergic asthma is rising, and novel strategies to prevent or treat this disease are needed. This study investigated the effects of different mixtures of non-digestible oligosaccharides combined with Bifidobacterium breve M-16V (BB) on the development of allergic airway inflammation in an animal model for house dust mite (HDM)-induced allergic asthma.MethodsBALB/c mice were sensitized intranasally (i.n.) with HDM and subsequently challenged (i.n.) with PBS or HDM while being fed diets containing different oligosaccharide mixtures in combination with BB or an isocaloric identical control diet. Bronchoalveolar lavage fluid (BALF) inflammatory cell influx, chemokine and cytokine concentrations in lung homogenates and supernatants of ex vivo HDM-restimulated lung cells were analyzed.ResultsThe HDM-induced influx of eosinophils and lymphocytes was reduced by the diet containing the short-chain and long-chain fructo-oligosaccharides and BB (FFBB). In addition to the HDM-induced cell influx, concentrations of IL-33, CCL17, CCL22, IL-6, IL-13 and IL-5 were increased in supernatants of lung homogenates or BALF and IL-4, IFN-γ and IL-10 were increased in restimulated lung cell suspensions of HDM-allergic mice. The diet containing FFBB reduced IL-6, IFN-γ, IL-4 and IL-10 concentrations, whereas the combination of galacto-oligosaccharides and long-chain fructo-oligosaccharides with BB was less potent in this model.ConclusionThese findings show that synbiotic dietary supplementation can affect respiratory allergic inflammation induced by HDM. The combination of FFBB was most effective in the prevention of HDM-induced airway inflammation in mice.

Measurement of airway function using invasive and non-invasive methods in mild and severe models for allergic airway inflammation in mice

In this study a direct comparison was made between non-invasive and non-ventilated unrestrained whole body plethysmography (Penh) (conscious animals) and the invasive ventilated lung resistance (RL) method (anesthetized animals) in both mild and severe allergic airway inflammation models. Mild inflammation was induced by intraperitoneal sensitization and aerosols of ovalbumin. Severe inflammation was induced by intraperitoneal sensitization using trinitrophenyl-ovalbumin, followed by intranasal challenges with IgE-allergen complexes. A significant increase in airway responsiveness to methacholine was observed in the mild inflammation group when RL was measured. Significant changes in both RL and Penh were observed in the severe inflammation groups. There was a significant increase in the number of inflammatory cells in the Broncho-Alveolar Lavage Fluid (BALF) in both the mild and severe inflammation animals. The enforced ventilation of the animals during the RL measurement further increased the number of cells in the BALF. IL-2 and RANTES levels in the BALF were higher in the severe inflammation groups compared to the mild inflammation groups. Penh gave only reliable measurements during severe airway inflammation. Measuring RL gave consistent results in both mild and severe allergic airway inflammation models however, ventilation induced an additional cell influx into the airways.

Differential Regulation of Inflammation and Immunity in Mild and Severe Experimental Asthma

This study aimed at exploring innate and adaptive immunity in allergic asthma by investigation of mRNA expression of pattern recognition receptors, T-cell-specific cytokines, and transcription factors. Mouse models for mild and severe asthma, with similar pathological characteristics observed in humans, were used to study the involved inflammatory markers as a first step in the development of phenotype-directed treatment approaches. In the mild model, mice were sensitized to ovalbumin-Imject Alum and challenged with ovalbumin. In the severe model, mice were sensitized to trinitrophenyl-conjugated ovalbumin and challenged with trinitrophenyl-ovalbumin/IgE immune complex. Pulmonary airway inflammation and mRNA expression of Toll-like receptors (TLRs), NOD-like receptors (NLRs), T cell cytokines, and transcription factors in lung tissue were examined. Different mRNA expression profiles of TLRs, NLRs, T cell cytokines, and transcription factors were observed. In the mild model, Il10 showed the largest increase in expression, whereas in the severe model, it was Inf γ with the largest increase. Expression of Tbet was also significantly increased in the severe model. Inflammation and immunity are differentially regulated in mild and severe experimental asthma. This preclinical data may help in directing clinical research towards a better understanding and therapy in mild and severe asthmatic patients.

Inflammation-Induced Expression of the Alarmin Interleukin 33 Can Be Suppressed by Galacto-Oligosaccharides

Background: The alarmin interleukin 33 (IL-33) and its receptor ST2 play an important role in mucosal barrier tissues, and seem to be crucial for Th2-cell mediated host defense. Galacto-oligosaccharides (GOS), used in infant formulas, exhibit gut and immune modulatory effects. To enhance our understanding of the immunomodulatory capacity of GOS, this study investigated the impact of dietary GOS intervention on IL-33 and ST2 expression related to intestinal barrier dysfunction and asthma. Methods: B6C3F1 and BALB/c mice were fed a control diet with or without 1% GOS. To simulate intestinal barrier dysfunction, B6C3F1 mice received a gavage with the mycotoxin deoxynivalenol (DON). To mimic asthma-like inflammatory airway responses, BALB/c mice were sensitized on day 0 and challenged on days 7-11 with house-dust mite (HDM) allergen. Samples from the intestines and lungs were collected for IL-33 and ST2 analysis by qRT-PCR, immunoblotting and immunohistochemistry. Results: Dietary GOS counteracted the DON-induced IL-33 mRNA expression and changed the IL-33 distribution pattern in the mouse small intestine. The IL-33 mRNA expression was positively correlated to the intestinal permeability. A strong positive correlation was also observed between IL-33 mRNA expression in the lung and the number of bronchoalveolar fluid cells. Reduced levels of IL-33 protein, altered IL-33 distribution and reduced ST2 mRNA expression were observed in the lungs of HDM-allergic mice after GOS intervention. Conclusions: Dietary GOS mitigated IL-33 at the mucosal surfaces in a murine model for intestinal barrier dysfunction and HDM-induced asthma. This promising effect may open up new avenues to use GOS not only as a prebiotic in infant nutrition, but also as a functional ingredient that targets inflammatory processes and allergies associated with IL-33 expression.

Raw Cow's Milk Prevents the Development of Airway Inflammation in a Murine House Dust Mite-Induced Asthma Model.

Epidemiological studies show an inverse relation between raw cow’s milk consumption and the development of asthma. This protective effect seems to be abolished by milk processing. However, evidence for a causal relationship is lacking, and direct comparisons between raw and processed milk are hardly studied. Therefore, this study investigated the preventive capacity of raw and heated raw milk on the development of house dust mite (HDM)-induced allergic asthma in mice. Six- to seven-week-old male BALB/c mice were intranasally (i.n.) sensitized with 1 µg HDM or PBS on day 0, followed by an i.n. challenge with 10 µg HDM or PBS on days 7–11. In addition, mice were fed 0.5 mL raw cow’s milk, heated raw cow’s milk, or PBS three times a week throughout the study, starting 1 day before sensitization. On day 14, airway hyperresponsiveness (AHR) in response to increasing doses of methacholine was measured to assess lung function. Bronchoalveolar lavage fluid (BALF) and lungs were furthermore collected to study the extent of airway inflammation. Raw milk prevented both HDM-induced AHR and pulmonary eosinophilic inflammation, whereas heated raw milk did not. Both milk types suppressed the Th2-polarizing chemokine CCL17 in lung homogenates and reduced lung Th2 and Th17 cell frequency. IL-4 and IL-13 production after ex vivo restimulation of lung T cells with HDM was also reduced by both milk types. However, local IL-5 and IL-13 concentrations were only suppressed by raw milk. These findings support the asthma-protective capacity of raw cow’s milk and show the importance of reduced local type 2 cytokine levels. Heated raw milk did not show an asthma-protective effect, which indicates the involvement of heat-sensitive components. Besides causal evidence, this study provides the basis for further mechanistic studies.

The Combination Therapy of Dietary Galacto-Oligosaccharides With Budesonide Reduces Pulmonary Th2 Driving Mediators and Mast Cell Degranulation in a Murine Model of House Dust Mite Induced Asthma

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective. Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice. Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined. Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice. Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation.