Hani Harb

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.

Update on epigenetics in allergic disease

Chronic inflammatory diseases, including allergies and asthma, are the result of complex gene-environment interactions. One of the most challenging questions in this regard relates to the biochemical mechanism of how exogenous environmental trigger factors modulate and modify gene expression, subsequently leading to the development of chronic inflammatory conditions. Epigenetics comprises the umbrella of biochemical reactions and mechanisms, such as DNA methylation and chromatin modifications on histones and other structures. Recently, several lifestyle and environmental factors have been investigated in terms of such biochemical interactions with the gene expression-regulating machinery: allergens; microbes and microbial compounds; dietary factors, including vitamin B12, folic acid, and fish oil; obesity; and stress. This article aims to update recent developments in this context with an emphasis on allergy and asthma research.

Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma

Polymorphisms in the interleukin-4 receptor α chain (IL-4Rα) have been linked to asthma incidence and severity, but a causal relationship has remained uncertain. In particular, a glutamine to arginine substitution at position 576 (Q576R) of IL-4Rα has been associated with severe asthma, especially in African Americans. We show that mice carrying the Q576R polymorphism exhibited intense allergen-induced airway inflammation and remodeling. The Q576R polymorphism did not affect proximal signal transducer and activator of transcription (STAT) 6 activation, but synergized with STAT6 in a gene target– and tissue-specific manner to mediate heightened expression of a subset of IL-4– and IL-13–responsive genes involved in allergic inflammation. Our findings indicate that the Q576R polymorphism directly promotes asthma in carrier populations by selectively augmenting IL-4Rα–dependent signaling.

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.

Genome-wide DNA methylation profiling identifies a folate-sensitive region of differential methylation upstream of ZFP57-imprinting regulator in humans

Folate intake during pregnancy may affect the regulation of DNA methylation during fetal development. The genomic regions in the offspring that may be sensitive to folate exposure during in utero development have not been characterized. Using genome‐scale profiling, we investigated DNA methylation in 2 immune cell types (CD4+ and antigen‐presenting cells) isolated from neonatal cord blood, selected on the basis of in utero folate exposure. High‐folate (HF; n=11) and low‐folate (LF; n=12) groups were selected from opposite extremes of maternal serum folate levels measured in the last trimester of pregnancy. A comparison of these groups revealed differential methylation at 7 regions across the genome. By far, the biggest effect observed was hypomethylation of a 923 bp region 3 kb upstream of the ZFP57 transcript, a regulator of DNA methylation during development, observed in both cell types. Levels of H3/H4 acetylation at ZFP57 promoter and ZFP57 mRNA expression were higher in CD4+ cells in the HF group relative to the LF group. Hypomethylation at this region was replicated in an independent sample set. These data suggest that exposure to folate has effects on the regulation of DNA methylation during fetal development, and this may be important for health and disease.—Amarasekera, M., Martino, D., Ashley, S., Harb, H., Kesper, D., Strickland, D., Saffery, R., Prescott, S. L. Genome‐wide DNA methylation profiling identifies a folate‐sensitive region of differential methylation upstream of ZFP57‐imprinting regulator in humans. FASEB J. 28, 4068‐4076 (2014). www.fasebj.org

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.

Epigenetics and allergy: from basic mechanisms to clinical applications

Allergic diseases are on the rise in the Western world and well-known allergy-protecting and -driving factors such as microbial and dietary exposure, pollution and smoking mediate their influence through alterations of the epigenetic landscape. Here, we review key facts on the involvement of epigenetic modifications in allergic diseases and summarize and critically evaluate the lessons learned from epigenome-wide association studies. We show the potential of epigenetic changes for various clinical applications: as diagnostic tools, to assess tolerance following immunotherapy or possibly predict the success of therapy at an early time point. Furthermore, new technological advances such as epigenome editing and DNAzymes will allow targeted alterations of the epigenome in the future and provide novel therapeutic tools.

Epigenetics in immune development and in allergic and autoimmune diseases

Epigenetic mechanisms such as DNA methylation, histone modification, and micro RNA signaling regulate the activity of the genome. Virtually all aspects of immunity involve some level of epigenetic regulation, whether it be host defense or in mediating tolerance. These processes are critically important in mediating dynamic responses to the environment over the life course of the individual, yet we are only just beginning to understand how dysregulation in these pathways may play a role in immune disease. Here, we give a brief chronological overview of epigenetic processes during immune development in health and disease.

Childhood allergic asthma is associated with increased IL-13 and FOXP3 histone acetylation

reactivity in the BAL of Der p 2.1–vaccinated mice, confirming previous reports demonstrating the lack of IgE reactivity toward this recombinant hypoallergenic derivative (Fig 2, E). However, contrary to recent results, we did not observe an increase in the inhibitory Der p 2–specific IgG1 (Fig 2, E). 8 Furthermore, we observed no influence of Der p 2.1 vaccination on the production of Der p 2–specific IgG2a. This result could be explained by the fact that B cells require IL-4 to produce both IgG1 and IgE. Consequently, by decreasing IL-4–producing T cells, vaccination with Der p 2.1 also reduced the production of TH2-related immunoglobulins. Given their structural homology, Der p and Der f antigens demonstrate an important IgE cross-reactivity. On the basis of this cross-reactivity, we investigated whether vaccination with Der p 2.1 peptide could control the development of Der f–induced airway hyperresponsiveness. Vaccination with Der p 2.1 inhibited bronchial hyperresponsiveness in Der f–induced asthma to the same extent as in the Der p–induced asthma model (Fig 2, F). Collectively, these data demonstrate the protective effect of Der p 2.1 vaccinations given before and duringHDM sensitization. By inhibiting T-cell–mediated IL-4 production, Der p 2.1 vaccination is believed to dampen IgE production, leading to a global attenuation on airway inflammation and hyperresponsiveness. Lower levels of IgE lead to fewer allergen-IgE pathogenic complexes and therefore to less activation of innate cells such as eosinophils, mastocytes, and basophils. The fact that IgG1 was also decreased is intriguing. The decrease in IgG1 may be explained by the fact that IL-4 is also involved in the production of IgG1 in mice. Der p 2.1 vaccination could also promote the expansion of immunosuppressive cells, such as regulatory T cells or induced-regulatory B cells. Indeed, many reports demonstrate that specific immunotherapy promotes the emergence of IL-10–producing T and B cells in allergic patients. Another interesting finding is the fact that vaccination also dampens the frequencyofTH17 cells in lungs.Given thepotent role of these cells in severe asthma, this therapeutic strategy could represent an interesting alternative in some cases of severe allergic asthma.Most interestingly, we demonstrated that vaccination with Der p 2.1 could be effective inDer f asthmaticmice. Consequently, this vaccine may reduce IgE-mediated as well as T-cell–mediated allergic inflammation irrespective of the HDM species. Finally, the protective role provided by vaccination with a hypoallergenic peptide could be of interest clinically, especially in sensitized children in whom allergic processes are sequential from sensitization to asthma. For these individuals, peptide immunotherapy may offer an alternative to block the allergic process and prevent its progression to allergic asthma.

Epigenetic Regulation in Early Childhood: A Miniaturized and Validated Method to Assess Histone Acetylation.

Introduction: Chronic inflammatory diseases including allergies and asthma are the result of complex interactions between genes and environmental factors. Epigenetic mechanisms comprise a set of biochemical reactions that regulate gene expression. In order to understand the cause-effect relationship between environmental exposures and disease development, methods capable of assessing epigenetic regulation (also) in large cohorts are needed. Methods: For this purpose, we developed and evaluated a miniaturized chromatin immunoprecipitation (ChIP) assay allowing for a cost-effective assessment of histone acetylation of candidate genes in a quantitative fashion. This method was then applied to assess H3 and H4 histone acetylation changes in cord blood (CB) samples from an established cohort of Australian children exposed in the fetal period to either very low or very high levels of maternal folate. Results: Our ChIP assay was validated for a minimum requirement of 1 × 105 target cells (e.g. CD4+ T cells). Very high levels of maternal folate were significantly associated with increased H3/H4 acetylation at GATA3 and/or IL9 promoter regions in CD4+ T cells in CB. Conclusion: We developed a ChIP method allowing reliable assessment of H3/H4 acetylation using 1 × 105 cells only. Practical application of this assay demonstrated an association between high maternal folate exposure and increased histone acetylation, corresponding to a more transcriptionally permissive chromatin status in the promoter regions of some Th2-related genes.