Petra Ina Pfefferle

Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter lwoffii F78

The pre- and postnatal environment may represent a window of opportunity for allergy and asthma prevention, and the hygiene hypothesis implies that microbial agents may play an important role in this regard. Using the cowshed-derived bacterium Acinetobacter lwoffii F78 together with a mouse model of experimental allergic airway inflammation, this study investigated the hygiene hypothesis, maternal (prenatal) microbial exposure, and the involvement of Toll-like receptor (TLR) signaling in prenatal protection from asthma. Maternal intranasal exposure to A. lwoffii F78 protected against the development of experimental asthma in the progeny. Maternally, A. lwoffii F78 exposure resulted in a transient increase in lung and serum proinflammatory cytokine production and up-regulation of lung TLR messenger RNA. Conversely, suppression of TLRs was observed in placental tissue. To investigate further, the functional relevance of maternal TLR signaling was tested in TLR2/3/4/7/9−/− knockout mice. The asthma-preventive effect was completely abolished in heterozygous offspring from A. lwoffii F78–treated TLR2/3/4/7/9−/− homozygous mother mice. Furthermore, the mild local and systemic inflammatory response was also absent in these A. lwoffii F78–exposed mothers. These data establish a direct relationship between maternal bacterial exposures, functional maternal TLR signaling, and asthma protection in the progeny.

Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes

BACKGROUND Bronchial asthma is a chronic inflammatory disease resulting from complex gene-environment interactions. Natural microbial exposure has been identified as an important environmental condition that provides asthma protection in a prenatal window of opportunity. Epigenetic regulation is an important mechanism by which environmental factors might interact with genes involved in allergy and asthma development. OBJECTIVE This study was designed to test whether epigenetic mechanisms might contribute to asthma protection conferred by early microbial exposure. METHODS Pregnant maternal mice were exposed to the farm-derived gram-negative bacterium Acinetobacter lwoffii F78. Epigenetic modifications in the offspring were analyzed in T(H)1- and T(H)2-relevant genes of CD4(+) T cells. RESULTS Prenatal administration of A lwoffii F78 prevented the development of an asthmatic phenotype in the progeny, and this effect was IFN-γ dependent. Furthermore, the IFNG promoter of CD4(+) T cells in the offspring revealed a significant protection against loss of histone 4 (H4) acetylation, which was closely associated with IFN-γ expression. Pharmacologic inhibition of H4 acetylation in the offspring abolished the asthma-protective phenotype. Regarding T(H)2-relevant genes only at the IL4 promoter, a decrease could be detected for H4 acetylation but not at the IL5 promoter or the intergenic T(H)2 regulatory region conserved noncoding sequence 1 (CNS1). CONCLUSION These data support the hygiene concept and indicate that microbes operate by means of epigenetic mechanisms. This provides a new mechanism in the understanding of gene-environment interactions in the context of allergy protection.

Cord blood cytokines are modulated by maternal farming activities and consumption of farm dairy products during pregnancy: The PASTURE Study

BACKGROUND Traditional farming represents a unique model situation to investigate the relationship of early-life farm-related exposure and allergy protection. OBJECTIVES To investigate associations between maternal farm exposures and cytokine production in cord blood (CB) mononuclear cells in a prospective multinational birth cohort of 299 farm and 326 nonfarm children and their families. METHODS Supernatants from phorbol 12-myristate 13-acetate/ionomycin-stimulated CB mononuclear cells were assessed for the production of IFN-gamma, TNF-alpha, IL-5, IL-10, and IL-12. RESULTS Significantly higher levels of IFN-gamma and TNF-alpha in farm compared with nonfarm children were found, whereas IL-5, IL-10, and IL-12 levels did not differ between study groups. Maternal contact with different farm animal species and barns and consumption of farm-produced butter during pregnancy enhanced the production of proinflammatory CB cytokines, whereas maternal consumption of farm-produced yogurt resulted in significant lower levels of IFN-gamma and TNF-alpha in umbilical blood. CONCLUSION Maternal exposure to farming activities and farm dairy products during pregnancy modulated cytokine production patterns of offspring at birth.

Increased regulatory T-cell numbers are associated with farm milk exposure and lower atopic sensitization and asthma in childhood

BACKGROUND European cross-sectional studies have suggested that prenatal and postnatal farm exposure decreases the risk of allergic diseases in childhood. Underlying immunologic mechanisms are still not understood but might be modulated by immune-regulatory cells early in life, such as regulatory T (Treg) cells. OBJECTIVE We sought to assess whether Treg cells from 4.5-year-old children from the Protection against Allergy: Study in Rural Environments birth cohort study are critical in the atopy and asthma-protective effect of farm exposure and which specific exposures might be relevant. METHODS From 1133 children, 298 children were included in this study (149 farm and 149 reference children). Detailed questionnaires until 4 years of age assessed farming exposures over time. Treg cells were characterized as upper 20% CD4(+)CD25(+) forkhead box protein 3 (FOXP3)(+) (intracellular) in PBMCs before and after stimulation (with phorbol 12-myristate 13-acetate/ionomycin or LPS), and FOXP3 demethylation was assessed. Atopic sensitization was defined by specific IgE measurements; asthma was defined by a doctors diagnosis. RESULTS Treg cells were significantly increased in farm-exposed children after phorbol 12-myristate 13-acetate/ionomycin and LPS stimulation. Exposure to farm milk was defined as a relevant independent farm-related exposure supported by higher FOXP3 demethylation. Treg cell (upper 20% CD4(+)CD25(+), FOXP3(+) T cells) numbers were significantly negatively associated with doctor-diagnosed asthma (LPS stimulated: adjusted odds ratio, 0.26; 95% CI, 0.08-0.88) and perennial IgE (unstimulated: adjusted odds ratio, 0.21; 95% CI, 0.08-0.59). Protection against asthma by farm milk exposure was partially mediated by Treg cells. CONCLUSIONS Farm milk exposure was associated with increased Treg cell numbers on stimulation in 4.5-year-old children and might induce a regulatory phenotype early in life, potentially contributing to a protective effect for the development of childhood allergic diseases.

Increased food diversity in the first year of life is inversely associated with allergic diseases

BACKGROUND The role of dietary factors in the development of allergies is a topic of debate, especially the potential associations between infant feeding practices and allergic diseases. Previously, we reported that increased food diversity introduced during the first year of life reduced the risk of atopic dermatitis. OBJECTIVE In this study we investigated the association between the introduction of food during the first year of life and the development of asthma, allergic rhinitis, food allergy, or atopic sensitization, taking precautions to address reverse causality. We further analyzed the association between food diversity and gene expression of T-cell markers and of Cε germline transcript, reflecting antibody isotype switching to IgE, measured at 6 years of age. METHODS Eight hundred fifty-six children who participated in a birth cohort study, Protection Against Allergy Study in Rural Environments/EFRAIM, were included. Feeding practices were reported by parents in monthly diaries during the first year of life. Data on environmental factors and allergic diseases were collected from questionnaires administered from birth up to 6 years of age. RESULTS An increased diversity of complementary food introduced in the first year of life was inversely associated with asthma with a dose-response effect (adjusted odds ratio with each additional food item introduced, 0.74 [95% CI, 0.61-0.89]). A similar effect was observed for food allergy and food sensitization. Furthermore, increased food diversity was significantly associated with an increased expression of forkhead box protein 3 and a decreased expression of Cε germline transcript. CONCLUSION An increased diversity of food within the first year of life might have a protective effect on asthma, food allergy, and food sensitization and is associated with increased expression of a marker for regulatory T cells.

Clinical and epidemiologic phenotypes of childhood asthma.

RATIONALE Clinical and epidemiologic approaches have identified two distinct sets of classifications for asthma and wheeze phenotypes. OBJECTIVES To compare epidemiologic phenotype definitions identified by latent class analysis (LCA) with clinical phenotypes based on patient histories, diagnostic work-up, and treatment responses. To relate phenotypes to genetic and environmental determinants as well as diagnostic and treatment-related parameters. METHODS LCA was performed in an international multicenter birth cohort based on yearly questions about current wheeze until age 6 years. Associations of wheeze classes and clinical phenotypes with asthma-related characteristics such as atopy, lung function, fraction of exhaled nitric oxide, and medication use were calculated using regression models. MEASUREMENTS AND MAIN RESULTS LCA identified five classes, which verified the clinically defined wheeze phenotypes with high sensitivity and specificity; the respective receiver operating characteristics curves displayed an area under the curve ranging from 84% (frequent wheeze) to 85% (asthma diagnosis) and 87% (unremitting wheeze) to 97% (recurrent unremitting wheeze). Recurrent unremitting wheeze was the most specific and unremitting wheeze at least once the most sensitive definition. The latter identified a subgroup of children with decreased lung function, increased genetic risk, and in utero smoke exposure (ODDS RATIO, 2.03; 95% CONFIDENCE INTERVAL, 1.12-3.68; P = 0.0191), but without established asthma diagnosis and treatment. CONCLUSIONS Clinical phenotypes were well supported by LCA analysis. The hypothesis-free LCA phenotypes were a useful reference for comparing clinical phenotypes. Thereby, we identified children with clinically conspicuous but undiagnosed disease. Because of their high area under the curve values, clinical phenotypes such as (recurrent) unremitting wheeze emerged as promising alternative asthma definitions for epidemiologic studies.

The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells

Robust cytotoxic CD8+ T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8+ T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4−/−) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenes-specific CD8+ T cells with impaired effector phenotype and function. Transfer of wild-type CD8+ T cells into Irf4−/− mice improved bacterial clearance, suggesting an intrinsic defect of CD8+ T cells in Irf4−/− mice. Following transfer into wild-type recipients, Irf4−/− CD8+ T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4−/− CD8+ T cells rescued the defect. During acute infection, Irf4−/− CD8+ T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4−/− CD8+ T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4−/− CD8+ T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4+ T-cell differentiation, the identification of its decisive role in peripheral CD8+ T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.

Microbial influence on tolerance and opportunities for intervention with prebiotics/probiotics and bacterial lysates

Epidemiologic studies indicate that microbes and microbial components are associated with protection against chronic inflammatory disease. Consequently, a plethora of clinical approaches have been used to investigate the benefits of a range of microbial products on inflammatory conditions in human trials. Centered particularly on the use of prebiotics, probiotic bacteria, and bacterial lysates in early life, this review provides an overview on clinical approaches aimed at reducing the global burden of allergic disease through primary prevention. Microbial interventions beginning before birth and in early infancy are discussed in the context of underlying mechanisms of oral tolerance and the establishment of gut colonization as a critical early homeostatic influence. We explore both the findings and challenges faced in existing studies with a view toward improving future clinical studies of the application of microbial compounds for the prevention of allergic disease and other inflammatory diseases.

Soluble immunoglobulin A in breast milk is inversely associated with atopic dermatitis at early age: the PASTURE cohort study

The role of breastfeeding for the development of atopic diseases in childhood is contradictory. This might be due to differences in the composition of breast milk and levels of antimicrobial and anti‐inflammatory components.

Farm-derived Gram-positive bacterium Staphylococcus sciuri W620 prevents asthma phenotype in HDM- and OVA-exposed mice.

Farm‐derived dust samples have been screened for bacteria with potential allergo‐protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation.