Heightened Epstein-Barr virus immunity and potential cross-reactivities in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated. METHODS: Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens. RESULTS: EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP. DISCUSSION: Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.

Extract of Helicobacter pylori Ameliorates Parameters of Airway Inflammation and Goblet Cell Hyperplasia following Repeated Allergen Exposure.

BACKGROUND: An inverse relation between Helicobacter pylori infection and asthma has been shown in epidemiological studies. Infection with H. pylori, or application of an extract of it before or after sensitization, inhibits allergic airway disease in mice. OBJECTIVES: The aim of this study was to investigate the effect of an extract of H. pylori on allergic airway disease induced by repeated allergen exposure in mice that were sensitized and challenged prior to extract application. METHOD: C57BL/6 mice were intranasally (i.n.) sensitized and challenged with house dust mite (HDM). After a minimum of 4 weeks, mice received the H. pylori 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 H. pylori extract. RESULTS: Treatment with H. pylori 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 H. pylori-treated mice that were re-exposed to the H. pylori extract produced significantly more interferon gamma. CONCLUSIONS: An extract of H. pylori is effective in reducing mucus production and various features of inflammation in HDM rechallenged mice.

A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture.

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.

Therapeutic Application of an Extract of Helicobacter pylori Ameliorates the Development of Allergic Airway Disease.

Epidemiological and experimental studies have shown that exposure to the gastric bacterium Helicobacter pylori, 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 H. pylori in neonates prevents the development of airway inflammation and goblet cell metaplasia. In the current study, the effect of administration of an extract of H. pylori 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 H. pylori 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 H. pylori extract reduced the processing of allergen by dendritic cells in the lungs and mediastinal lymph node. Bone marrow-derived dendritic cells exposed to H. pylori extract were affected with regard to their ability to process Ag. These data show that application of H. pylori extract after sensitization effectively inhibits allergic airway disease.

The effect of tiotropium in combination with olodaterol on house dust mite-induced allergic airway disease.

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 ß-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.

Basal cells contribute to innate immunity of the airway epithelium through production of the antimicrobial protein RNase 7.

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.

Microorganism-induced suppression of allergic airway disease: novel therapies on the horizon?

Allergic airway disease is a major global health burden, and novel treatment options are urgently needed. Numerous epidemiological and experimental studies suggest that certain helminths and bacteria protect against respiratory allergies. These microorganisms are strong regulators of the immune system, and various potential regulatory mechanisms by which they protect against allergic airway inflammation have been proposed. Whereas early studies addressed the beneficial effect of natural infections, the focus now shifts toward identifying the dominant protective molecules and exploring their efficacy in models of allergic airway disease. In this article, we will review the evidence for microbe-mediated protection from allergic airway disease, the potential modes of action involved and discuss advances as well as limitations in the translation of this knowledge into novel treatment strategies against allergic airway disease.