The prevalence of allergic asthma and other atopic diseases has reached epidemic proportions in large parts of the developed world. The gradual loss of the human indigenous microbiota has been held responsible for this trend. The bacterial pathogen Helicobacter pylori is a constituent of the normal gastric microbiota whose presence has been inversely linked to allergy and asthma in humans and experimental models. Here we show that oral or i.p. tolerization with H. pylori extract prevents the airway hyperresponsiveness, bronchoalveolar eosinophilia, pulmonary inflammation, and Th2 cytokine production that are hallmarks of allergen-induced asthma in mice. Asthma protection is not conferred by extracts from other enteropathogens and requires a heat-sensitive H. pylori component and the DC-intrinsic production of IL-10. The basic leucine zipper ATF-like 3 (BATF3)-dependent CD103 + CD11b − dendritic cell lineage is enriched in the lungs of protected mice and strictly required for protection. Two H. pylori persistence determinants, the γ-glutamyl-transpeptidase GGT and the vacuolating cytotoxin VacA, are required and sufficient for asthma protection and can be administered in purified form to prevent asthma. In conclusion, we provide preclinical evidence for the concept that the immunomodulatory properties of H. pylori can be exploited for tolerization strategies aiming to prevent allergen-induced asthma.bacterial immunomodulation | allergy and asthma prevention | tolerogenic dendritic cells | bacterial persistence determinants

Basal cells play a critical role in the response of the airway epithelium to injury and are recently recognized to also contribute to epithelial immunity. Antimicrobial proteins and peptides are essential effector molecules in this airway epithelial innate immunity. However, little is known about the specific role of basal cells in antimicrobial protein and peptide production and about the regulation of the ubiquitous antimicrobial protein RNase 7. In this study, we report that basal cells are the principal cell type producing RNase 7 in cultured primary bronchial epithelial cells (PBEC). Exposure of submerged cultured PBEC (primarily consisting of basal cells) to the respiratory pathogen nontypeable Haemophilus influenzae resulted in a marked increase in expression of RNase 7, although this was not observed in differentiated air–liquid interface cultured PBEC. However, transient epithelial injury in air–liquid interface–cultured PBEC induced by cigarette smoke exposure led to epidermal growth factor receptor–mediated expression of RNase 7 in remaining basal cells. The selective induction of RNase 7 in basal cells by cigarette smoke was demonstrated using confocal microscopy and by examining isolated luminal and basal cell fractions. Taken together, these findings demonstrate a phenotype-specific innate immune activity of airway epithelial basal cells, which serves as a second line of airway epithelial defense that is induced by airway epithelial injury.

Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells, but current methods require large numbers of animals which is unwanted in view of the 3R principle and introduces variation. Moreover, stringent breeding schemes are frequently needed to generate sufficient numbers of genetically modified animals. Current protocols do not incorporate expansion of MTEC, and therefore we developed a protocol to expand MTEC while maintaining their differentiation capacity. MTEC were isolated and expanded using the ROCK inhibitor Y-27632 in presence or absence of the γ-secretase inhibitor DAPT, a Notch pathway inhibitor. Whereas MTEC proliferated without DAPT, growth rate and cell morphology improved in presence of DAPT. ALI-induced differentiation of expanded MTEC resulted in an altered capacity of basal cells to differentiate into ciliated cells, whereas IL-13-induced goblet cell differentiation remained unaffected. Ciliated cell differentiation improved by prolonging the ALI differentiation or by adding DAPT, suggesting that basal cells retain their ability to differentiate. This technique using expansion of MTEC and subsequent ALI differentiation drastically reduces animal numbers and costs for in vitro experiments, and will reduce biological variation. Additionally, we provide novel insights in the dynamics of basal cell populations in vitro.

The BBB endothelial cells play the predominant role in actively transporting nutrients to the brain and limit the entrance of potentially harmful blood components, including cells of the immune system. This is essential for brain homeostasis and reliable functioning of the neuronal environment. A common feature of diverse neurological diseases, including MS, vascular dementia and stroke, is the typical loss of the specialized function of the BBB which leads to unstable brain homeostasis, inflammation, and neuronal damage (de Vries et al. 1997;Zlokovic 2008). In the past, we and others showed that loss of BBB function is an early and typical phenomenon in the neuro-inflammatory disease MS and models thereof (Floris et al. 2004;Vos et al. 2005). In MS, infiltrated monocyte-derived macrophages form the major cell type in perivascular infiltrates and are key mediators of demyelination and axonal damage, two characteristic features of MS (Bruck et al. 1996). To travel into the brain and exert their detrimental effects, monocytes have to cross the BBB. This requires the active participation of brain endothelial cells, and we and others have demonstrated the strong contribution of soluble mediators, extracellular proteases and intracellular signaling events (Walters et al. 2002;Reijerkerk et al. 2006Reijerkerk et al. , 2008Reijerkerk et al. , 2010Schreibelt et al. 2007).Endothelin-1 is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases (Khimji and Rockey 2010 AbstractHomeostasis of the brain is dependent on the blood-brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent proinflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ET B ) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.

SummaryChronic helminth infection with Schistosoma (S.) mansoni protects against allergic airway inflammation (AAI) in mice and is associated with reduced Th2 responses to inhaled allergens in humans, despite the presence of schistosome‐specific Th2 immunity. Schistosome eggs strongly induce type 2 immunity and allow to study the dynamics of Th2 versus regulatory responses in the absence of worms. Treatment with isolated S. mansoni eggs by i.p. injection prior to induction of AAI to ovalbumin (OVA)/alum led to significantly reduced AAI as assessed by less BAL and lung eosinophilia, less cellular influx into lung tissue, less OVA‐specific Th2 cytokines in lungs and lung‐draining mediastinal lymph nodes and less circulating allergen‐specific IgG1 and IgE antibodies. While OVA‐specific Th2 responses were inhibited, treatment induced a strong systemic Th2 response to the eggs. The protective effect of S. mansoni eggs was unaltered in μMT mice lacking mature (B2) B cells and unaffected by Treg cell depletion using anti‐CD25 blocking antibodies during egg treatment and allergic sensitization. Notably, prophylactic egg treatment resulted in a reduced influx of pro‐inflammatory, monocyte‐derived dendritic cells into lung tissue of allergic mice following challenge. Altogether, S. mansoni eggs can protect against the development of AAI, despite strong egg‐specific Th2 responses.

Epidemiological and experimental studies have shown that exposure to the gastric bacterium , especially in early life, prevents the development of asthma. Recent mouse studies have shown that this protective effect does not require live bacteria and that treatment with an extract of in neonates prevents the development of airway inflammation and goblet cell metaplasia. In the current study, the effect of administration of an extract of was assessed in a therapeutic study design with application of the extract just prior to allergen challenge. C57BL/6 mice were sensitized and challenged with OVA or house dust mite. Treatment with extract just prior to the challenge significantly reduced airway inflammation, as assessed in bronchoalveolar lavage fluid and lung tissue, and reduced airway remodeling, as assessed by goblet cell quantification. These effects were apparent in the OVA model and in the house dust mite model. Injection of extract reduced the processing of allergen by dendritic cells in the lungs and mediastinal lymph node. Bone marrow-derived dendritic cells exposed to extract were affected with regard to their ability to process Ag. These data show that application of extract after sensitization effectively inhibits allergic airway disease.

<b><i>Background:</i></b> An inverse relation between <i>Helicobacter pylori</i> infection and asthma has been shown in epidemiological studies. Infection with <i>H. pylori</i>, or application of an extract of it before or after sensitization, inhibits allergic airway disease in mice. <b><i>Objectives:</i></b> The aim of this study was to investigate the effect of an extract of <i>H. pylori</i> on allergic airway disease induced by repeated allergen exposure in mice that were sensitized and challenged prior to extract application. <b><i>Method:</i></b> C57BL/6 mice were intranasally (i.n.) sensitized and challenged with house dust mite (HDM). After a minimum of 4 weeks, mice received the <i>H. pylori</i> extract intraperitoneally and were rechallenged i.n. with HDM. Allergen-specific antibodies were measured by ELISA. Cells present in the bronchoalveolar lavage fluid and dendritic cell (DC) subsets in the lung tissue were analyzed by flow cytometry. Tissue inflammation and goblet cell hyperplasia were assessed by histology. Cells of the mediastinal lymph node (mLN) were isolated and in vitro restimulated with HDM or <i>H. pylori</i> extract. <b><i>Results:</i></b> Treatment with <i>H. pylori</i> extract before rechallenge reduced allergen-specific IgE, the DC numbers in the tissue, and goblet cell hyperplasia. Cells isolated from mLN of mice treated with the extract produced significantly more IL-10 and IL-17 after in vitro restimulation with HDM. mLN cells of <i>H. pylori-</i>treated mice that were re-exposed to the <i>H. pylori</i> extract produced significantly more interferon gamma. <b><i>Conclusions:</i></b> An extract of <i>H. pylori</i> is effective in reducing mucus production and various features of inflammation in HDM rechallenged mice.

One of the major goals of asthma therapy is to maintain asthma control and prevent acute exacerbations. Long-acting bronchodilators are regularly used for the treatment of asthma patients and in clinical studies the anti-cholinergic tiotropium has recently been shown to reduce exacerbations in patients with asthma. So far it is unclear how tiotropium exerts this effect. For this purpose, we designed an allergen-driven rechallenge model of allergic airway inflammation in mice, to assess the effectiveness of tiotropium and the long-acting β-2 adrenoceptor agonist olodaterol on allergen-induced exacerbations of airway disease. Female C57BL/6J mice were sensitized intranasally (i.n.) with 1 μg of house dust mite (HDM) extract followed by a challenge regime (5 consecutive days 10 μg HDM extract i.n.) after one week. Mice were exposed to a secondary challenge five weeks after sensitization and were treated i.n. with different concentrations of tiotropium or olodaterol (1, 10 and 100 μg/kg) or a combination thereof (10 μg/kg each) prior to and during the secondary challenge period. Three days after the last challenge, bronchoalveolar lavage (BAL) fluid and lung tissue were collected for flow cytometry and histologic analysis, respectively. Secondary challenge with HDM extract strongly induced allergic airway disease reflected by inflammatory cell infiltration and goblet cell metaplasia. Treatment with tiotropium, but not with olodaterol reduced tissue inflammation and goblet cell metaplasia in a dose-dependent manner. The combination of tiotropium and olodaterol was more effective in significantly reducing tissue inflammation compared to tiotropium treatment alone, and also led to a decrease in BAL cell counts. These data suggest that in a model of relapsing allergic airway disease tiotropium directly prevents exacerbations by reducing inflammation and mucus production in the airways. In addition, the combination of tiotropium and olodaterol exerts synergistic effects.