Dörthe A. Kesper

The gut microbiota and inflammatory noncommunicable diseases : associations and potentials for gut microbiota therapies.

Rapid environmental transition and modern lifestyles are likely driving changes in the biodiversity of the human gut microbiota. With clear effects on physiologic, immunologic, and metabolic processes in human health, aberrations in the gut microbiome and intestinal homeostasis have the capacity for multisystem effects. Changes in microbial composition are implicated in the increasing propensity for a broad range of inflammatory diseases, such as allergic disease, asthma, inflammatory bowel disease (IBD), obesity, and associated noncommunicable diseases (NCDs). There are also suggestive implications for neurodevelopment and mental health. These diverse multisystem influences have sparked interest in strategies that might favorably modulate the gut microbiota to reduce the risk of many NCDs. For example, specific prebiotics promote favorable intestinal colonization, and their fermented products have anti-inflammatory properties. Specific probiotics also have immunomodulatory and metabolic effects. However, when evaluated in clinical trials, the effects are variable, preliminary, or limited in magnitude. Fecal microbiota transplantation is another emerging therapy that regulates inflammation in experimental models. In human subjects it has been successfully used in cases of Clostridium difficile infection and IBD, although controlled trials are lacking for IBD. Here we discuss relationships between gut colonization and inflammatory NCDs and gut microbiota modulation strategies for their treatment and prevention.

Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS)

During myogenesis in Drosophila embryos, a prominent adhesive structure is formed between precursor cells and fusion‐competent myoblasts (fcms). Here, we show that Duf/Kirre and its interaction partners Rols7 (found in founder myoblasts and growing myotubes) and Sns (found in fcms) are organized in a ring‐structure at the contact points of fcms with precursor cells, while cytoskeletal components like F‐actin and Titin are centered in this ring in both cell types. The cytoplasmic protein Blow colocalizes with the actin plugs in fcms after cell adhesion. Furthermore, the requirement of additional as yet unidentified components was demonstrated by using mammalian C2C12 myoblasts. In this study, we propose that the fusion‐restricted myogenic‐adhesive structure (FuRMAS) is pivotal in linking cell adhesion as well as local F‐actin assembly and dynamics to downstream events that ultimately lead to plasma membrane fusion. Moreover, we suggest that the FuRMAS may restrict the area of membrane breakdown. Developmental Dynamics 236:404–415, 2007.

WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion

Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. Here, we show the identification and characterization of schwächling – a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findings we derived a new model for actin regulation during myoblast fusion.

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

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.

In Drosophila melanogaster, the Rolling pebbles isoform 6 (Rols6) is essential for proper Malpighian tubule morphology

During myoblast fusion, cell-cell recognition along with cell migration and adhesion are essential biological processes. The factors involved in these processes include members of the immunoglobulin superfamily like Sticks and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling pebbles (Rols). For rolling pebbles, two differentially expressed transcripts have been defined (rols7 and rols6). However, to date, only a muscle fusion phenotype has been described and assigned to the lack of the mesoderm-specific expressed rols7 transcript. Here, we show that a loss of the second rolling pebbles transcript, rols6, which is expressed from the early bud to later embryonic stages during Malpighian tubule (MpT) development, leads to an abnormal MpT morphology that is not due to defects in cell determination or proliferation but to aberrant morphogenesis. In addition, when Myoblast city or Rac are knocked out, a similar phenotype is observed. Myoblast city and Rac are essentially involved in the development of the somatic muscles and were proposed to be interaction partners of Rols7. Because of the predicted structural similarities of the Rols7 and Rols6 proteins, we argue that genetic interaction of rols6, mbc and rac might lead to proper MpT morphology. We also propose that these interactions result in stable cell connections due to rearrangement of the cytoskeleton.

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.

Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion

BackgroundThe visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei.ResultsUsing fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion.ConclusionsOur results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.