Chitinase-Induced Airway Hyperreactivity and Inflammation in a Mouse Model of Nonallergic Asthma.

INTRODUCTION Environmental exposure to mites and fungi has been proposed to critically contribute to the development of IgE-mediated asthma. A common denominator of such organisms is chitin. Human chitinases have been reported to be upregulated by interleukin-13 secreted in the context of Th2-type immune responses and to induce asthma. We assessed whether chitin-containing components induced chitinases in an innate immune-dependent way and whether this results in bronchial hyperresponsiveness. MATERIALS AND METHODS Monocyte/macrophage cell lines were stimulated with chitin-containing or bacterial components in vitro. Chitinase activity in the supernatant and the expression of the chitotriosidase gene were measured by enzyme assay and quantitative PCR, respectively. Non-sensitized mice were stimulated with chitin-containing components intranasally, and a chitinase inhibitor was administered intraperitoneally. As markers for inflammation leukocytes were counted in the bronchoalveolar lavage (BAL) fluid, and airway hyperresponsiveness was assessed via methacholine challenge. RESULTS We found both whole chitin-containing dust mites as well as the fungal cell wall component zymosan A but not endotoxin-induced chitinase activity and chitotriosidase gene expression in vitro. The intranasal application of zymosan A into mice led to the induction of chitinase activity in the BAL fluid and to bronchial hyperresponsiveness, which could be reduced by applying the chitinase inhibitor allosamidin. DISCUSSION We propose that environmental exposure to mites and fungi leads to the induction of chitinase, which in turn favors the development of bronchial hyperreactivity in an IgE-independent manner

The influence of early exposition to enviromental microbes on receptors of the innate immunity and their protective effects against allergic diseases

Die Prävalenz für eine Sensibilisierung gegenüber gewöhnlichen Allergenen, sowie die Häufigkeit allergischer Krankheiten, wie Asthma, Rhinitis, atopischer Dermatitis und Lebensmittelallergien hauptsächlich in den ersten drei Lebensjahren, ist im Laufe der letzten 50 Jahre markant gestiegen. Allergische Reaktionen sind gekennzeichnet durch ihre Produktion von IgE Antikörpern, Mastzellendegranulation und Sekretion proinflammatorischer Moleküle. Die Entstehung von Allergien ist zu einem gewissen Teil genetisch veranlagt. Es konnte jedoch bereits gezeigt werden, dass die Exposition verschiedener Umweltfaktoren, wie Mikroben oder Nahrungsmittelbestandteilen Kinder vor Allergien schützt, vor allem dann, wenn die Exposition schon während der Schwangerschaft oder früh im Leben stattfand. Im ersten Teil dieser Dissertation untersuchten wir den Einfluss früher pre- und postnataler Expositionen diverser bäuerlichen Umweltfaktoren auf die Genexpression verschiedener Rezeptoren des angeborenen Immunsystems (Toll-like Rezeptoren und CD14). Toll-like Rezeptoren (TLRs) und CD14 erkennen konservierte Moleküle von Mikroorganismen und lösen die Aktivierung des adaptiven Immunsystems aus. Wir konnten dabei zeigen, dass bei Kindern, deren Mütter während der Schwangerschaft auf einem Bauernhof arbeiteten, verschiedene TLRs im Nabelschnurblut teilweise signifikant stärker exprimiert waren als bei Kontrollkindern. Die TLR-Genexpression nach einem Jahr war assoziiert mit Milchkonsum vom Bauernhof, besonders mit ungekochter Milch. Insgesamt weisen die Resultate darauf hin, dass sowohl die Exposition während der Schwangerschaft, als auch im ersten Lebensjahr die Expression von TLRs beeinflusst. Im Weiteren analysierten wir den Zusammenhang zwischen TLR-Expression und der Entstehung atopischer Dermatitis in den ersten zwei Lebensjahren. Bei Kindern, die in dieser Zeitspanne an einer atopischen Dermatitis erkrankten, konnte eine signifikant tiefere Expression von TLR5 und TLR9 im Nabelschnurblut nachgewiesen werden. Schützend wirkte sich mütterlicher Kontakt während der Schwangerschaft gegenüber Bauernhofexpositionen wie z.B. Tierkontakt aus, was möglicherweise auf eine Gen-Umwelt- Interaktion schliessen lässt. Zu guter letzt fanden wir heraus, dass eine ergänzende mütterliche Vitamin D Gabe während der Schwangerschaft in Zusammenhang mit einer erhöhten Genexpression der immunglobulin-like transcripts (ILT)3 und ILT4 steht, beides Rezeptoren für tolerogene dentritische Zellen, welche den Transkriptionsfaktor (NF)-κB und die durch ihn induzierte Entzündungsantwort hemmen. Diese Resultate deuten auf eine frühe Induktion einer toleranzbildenden Immunantwort durch Vitamin D hin. Der zweite Teil dieser Arbeit beschäftigt sich mit der Problematik Lebensmittelallergien zu behandeln, da die strikte Vermeidung allergieauslösender Lebensmittel immer noch die beste und einzige Methode ist um ernste anaphylaktische Reaktionen zu unterbinden. Unsere Arbeit fokussierten wir dabei auf die Desensibilisierung, eine Methode bei welcher die Menge des Allergens in steigenden Konzentrationen über eine Zeitspanne verabreicht wird und die bereits in der Behandlung von Heuschnupfen und Insektenstichallergien Anwendung findet. Wir verwendeten attenuierte, intrazellulär lebende Bakterien (Salmonella enterica serovar Typhimurium) als Vektoren für die Freisetzung des Allergens im Darm. Die Allergenproduktion mittels verschiedener Bakterienstämme und Regulationsmechanismen untersuchten wir in vitro, während wir die Wirksamkeit unseres Konstruktes prophylaktisch und therapeutisch in einem murinen Allergensensibilisierungsmodel austesteten. Nach kombinierter Einnahme von Mikroorganismen und Ausschüttung geringer Allergenmengen konnten abgeschwächte anaphylaktische Symptome und erhöhte antigenspezifische IgA-Werte sowie reduzierte Genexpressionen verschiedener TLRs festgestellt werden, was auf eine mögliche Eignung dieser Methode zur Prävention und/oder Behandlung von Lebensmittelallergien hindeutet. Die genauen molekularen Mechanismen bleiben allerdings weiterhin unklar und es benötigt zusätzliche Untersuchungen in diesem Bereich. Over the last half century, the prevalence of sensitization to common allergens has increased markedly as well as the incidence of allergic diseases like asthma, rhinitis, atopic dermatitis and food allergies, mainly during the first three years of life. Allergic reactions are characterized through production of IgE antibodies, mast cell degranulation and secretion of proinflammatory molecules. The development of allergies is at least partly influenced by genetical predispositions. However, it was already shown, that the exposition to various environmental factors such as microbial or dietary components protect children against allergic diseases, mainly if the exposure occurs during pregnancy or early in life. In the first part of this thesis we investigated various farming exposition factors during pregnancy and first year of age on the expression of receptors of the innate immunity (Toll- like receptors and CD14). Toll-like receptors (TLRs) and CD14 recognize conserved molecules of microorganisms and trigger the induction of the adaptive immune response. We were able to demonstrate that the expression of different TLRs in cord blood of children whose mothers worked on a farm during pregnancy were partly significantly higher compared to non-farming newborns. The TLRs expression at one year of age was associated with farm milk consumption, especially unboiled farm milk. These results indicate that both, the pre- and early postnatal farming exposure influence the expression of TLRs in children. Next, we analyzed the association between the gene expression of TLRs and CD14 and the development of atopic dermatitis in the 2 first years of life. Children, suffering from atopic dermatitis in the first two years of life have a significantly decreased expression of TLR5 and TLR9 in cord blood. Maternal farming exposure such as contact to farm animals during pregnancy has a protective effect on atopic dermatitis in the two first years of life, which may indicate a gene-environment interaction. Finally, we found that maternal vitamin D supplementation during pregnancy was related to an increase in the gene expression of immunglobulin-like transcripts (ILT)3 and ILT4, both receptors of tolerogenic dentritic cells which inhibit the transcription nuclear factor (NF)-κB and inflammatory responses. These results may point towards an early induction of tolerogenic immune responses by vitamin D. The second part of this thesis concerns the current major problem to treat food allergy, since strict avoidance of allergenic food is still the best method to prevent severe anaphylactic reactions. We focused our work on desensitization, a method where the amount of allergen is administrated increasingly over a period of time and which finds appliance to treat hay fever or insect sting allergies. We used attenuated intracellular living bacteria (Salmonella enterica serovar Typhimurium) as vector for allergen delivery in the gut. The allergen production by different bacterial strains and regulation mechanisms was investigated in vitro and the efficacy of our construct was tested prophylactically as well as therapeutically in a murine allergen sensitization model. It could be demonstrated that the combined intake of microorganisms and the release of low amounts of allergen reduced anaphylactic symptoms combined with increased antigen- specific immunoglobulin A (IgA) titres and decreased gene expression of TLRs. This approach may be a possible method to prevent or treat food allergies. However, the exact molecular mechanisms are still unclear and further analyses have to be done

Prenatal animal contact and gene expression of innate immunity receptors at birth are associated with atopic dermatitis

BACKGROUND: Cross-sectional studies have suggested that prenatal farm exposures might protect against allergic disease and increase the expression of receptors of the innate immune system. However, epidemiologic evidence supporting the association with atopic dermatitis remains inconsistent. OBJECTIVE: To study the association between prenatal farm-related exposures and atopic dermatitis in a prospective study. We further analyzed the association between the expression of innate immune genes at birth and atopic dermatitis. METHODS: A total of 1063 children who participated in a birth cohort study, Protection against Allergy-Study in Rural Environments, were included in this study. Doctor diagnosis of atopic dermatitis was reported by the parents from 1 to 2 years of age by questionnaire. Gene expression of Toll-like receptors (TLRs) and CD14 was assessed in cord blood leukocytes by quantitative PCR. RESULTS: Maternal contact with farm animals and cats during pregnancy had a significantly protective effect on atopic dermatitis in the first 2 years of life. The risk of atopic dermatitis was reduced by more than half among children with mothers having contact with 3 or more farm animal species during pregnancy compared with children with mothers without contact (adjusted odds ratio, 0.43; 95% CI, 0.19-0.97). Elevated expression of TLR5 and TLR9 in cord blood was associated with decreased doctor diagnosis of atopic dermatitis. A significant interaction between polymorphism in TLR2 and prenatal cat exposure was observed in atopic dermatitis. CONCLUSION: Maternal contact with farm animals and cats during pregnancy has a protective effect on the development of atopic dermatitis in early life, which is associated with a lower expression of innate immune receptors at birt

Prenatal and early-life exposures alter expression of innate immunity genes: the PASTURE cohort study

BACKGROUND: There is evidence that gene expression of innate immunity receptors is upregulated by farming-related exposures. OBJECTIVE: We sought to determine environmental and nutritional exposures associated with the gene expression of innate immunity receptors during pregnancy and the first year of a child's life. METHODS: For the Protection Against Allergy: Study in Rural Environments (PASTURE) birth cohort study, 1133 pregnant women were recruited in rural areas of Austria, Finland, France, Germany, and Switzerland. mRNA expression of the Toll-like receptor (TLR) 1 through TLR9 and CD14 was assessed in blood samples at birth (n= 938) and year 1 (n= 752). Environmental exposures, as assessed by using questionnaires and a diary kept during year 1, and polymorphisms in innate receptor genes were related to gene expression of innate immunity receptors by using ANOVA and multivariate regression analysis. RESULTS: Gene expression of innate immunity receptors in cord blood was overall higher in neonates of farmers (P for multifactorial multivariate ANOVA= .041), significantly so for TLR7 (adjusted geometric means ratio [aGMR], 1.15; 95% CI, 1.02-1.30) and TLR8 (aGMR, 1.15; 95% CI, 1.04-1.26). Unboiled farm milk consumption during the first year of life showed the strongest association with mRNA expression at year 1, taking the diversity of other foods introduced during that period into account: TLR4 (aGMR, 1.22; 95% CI, 1.03-1.45), TLR5 (aGMR, 1.19; 95% CI, 1.01-1.41), and TLR6 (aGMR, 1.20; 95% CI, 1.04-1.38). A previously described modification of the association between farm milk consumption and CD14 gene expression by the single nucleotide polymorphism CD14/C-1721T was not found. CONCLUSION: Farming-related exposures, such as raw farm milk consumption, that were previously reported to decrease the risk for allergic outcomes were associated with a change in gene expression of innate immunity receptors in early life