Dental Biofilm and Saliva Microbiome and Its Interplay with Pediatric Allergies

Little is known about the interplay and contribution of oral microorganisms to allergic diseases, especially in children. The aim of the clinical study was to associate saliva and dental biofilm microbiome with allergic disease, in particular with allergic asthma. In a single-center study, allergic/asthmatic children (n = 15; AA-Chd; age 10.7 ± 2.9), atopic/allergic children (n = 16; AT/AL-Chd; 11.3 ± 2.9), and healthy controls (n = 15; CON-Chd; age 9.9 ± 2.2) were recruited. After removing adhering biofilms from teeth and collecting saliva, microbiome was analyzed by using a 16s-rRNA gene-based next-generation sequencing in these two mediums. Microbiome structure differed significantly between saliva and dental biofilms (β-diversity). Within the groups, the dental biofilm microbiome of AA-Chd and AT/AL-Chd showed a similar microbial fingerprint characterized by only a small number of taxa that were enriched or depleted (4) compared to the CON-Chd, while both diseased groups showed a stronger microbial shift compared to CON-Chd, revealing 14 taxa in AA-Chd and 15 taxa in AT/AL-Chd that were different. This could be the first note to the contribution of dental biofilm and its metabolic activity to allergic health or disease

Identification of underlying molecular mechanisms of obesity-associated asthma

In the evolution of personalized medicine and stratified therapies, comprehensive understanding of the molecular mechanisms underlying different disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Biological pathways and functions of differentially expressed genes and miRNAs can play an essential role in the development and severity of obesity-related asthma. To fulfill this purpose, a preliminary work was initiated to establish optimal experimental conditions by investigating the effects related to different timings of CD4+ T cells processing from peripheral blood. Thus, 2 peripheral blood samples were drawn from each of 3 atopic patients and 3 healthy donors (12 samples), CD4+ T cells isolation was conducted from the first blood sample within two hours (Immediate) and from the second blood sample after 24 hours (Delayed), transcriptomic profiles of CD4+ T cells were examined using RNA-Seq analysis and readouts were verified on the epigenetic level through the H3K27ac ChIP-Seq analysis. After a successful establishment of ideal experimental conditions, peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90), 10 non-obese, non- atopic asthmatic adults with normal BMI (23.88 ± 2.73), and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to have a low type- 2 asthma phenotype according to blood eosinophils counts, FeNO and IgE levels criteria. Peripheral blood CD4+ T cells were isolated, mRNA sequencing was conducted. Moreover, plasma was also taken from the same blood samples of the same previous individuals, EVs were isolated, EVs RNA was extracted and small/microRNA-Seq was performed. The transcriptomic profiles of delayed processed CD4+ T cells showed only 3 differentially expressed genes at FDR < 0.1 when comparing atopic with healthy individuals. CD4+ T cells of healthy donors were not harshly affected by the delayed ex vivo blood incubation, while a drastic change has been shown in CD4+ T cells of atopic patients following the delayed blood processing accompanying by downregulation atopy-related biological pathways such as IL-2 and IL-17 signaling pathways. Concordant results were observed on the epigenetic level through H3K27ac profiles. IFN signaling pathways dominated the CD4+ T cells responses solely in low type-2 obese asthmatics represented by upregulation of different ISGs such as IFITM3, IFIT3, OAS2, OAS3, EIF2AK2, MX1, USP18, GBP3 genes, which correlated positively with lung function parameters including FEV1, FVC, VC max, TIFF, IC and PEF and negatively with the airway inflammation marker; FeNO. Viral infection pathways were also enriched for low type-2 obese asthmatics augmented by upregulation of different toll-like receptor genes such as TLR1, TLR-2, TLR-3, TLR-4, TLR-6, and TLR-8. Furthermore, obesity gene markers like IL15 and SOCS3 were also up-regulated in CD4+ T cells from obese asthmatics compared to both non-obese asthmatics and healthy controls. On the other hand, gap junction and GPCR ligand binding pathways were enriched in both low type-2 asthma groups. EVs miRNA clusters such as miR- 2329 and miR-106b seemed to be assigned to IFN signaling and viral infection pathways, respectively, in low type-2 obese asthmatics. In addition, single Plasma EV downregulated miRNAs including miR-665, miR-4419b, miR-4769-3p, miR-6893-5p, miR-4743-3p, miR-6721-5p, miR-1207-5p, miR-6132, miR-4700-3p, miR-4731-5p, miR-5089-5p, miR-502-5p, miR-6088, miR-148a-5p and miR-373-3p seemed to target most of the enriched ISGs of IFN signaling pathway in CD4+ T cells. In conclusion, the dominance of the IFN signaling pathways and their association with viral infection pathways in CD4+ T cells response could underpin the underlying molecular mechanism of low type-2 obesity-associated asthma. The IFN signaling pathway enriched ISGs, their associated miRNAs and other miRNA clusters might be a target for biological and stratified therapies for this unique asthma phenotype

Effects of obesity on asthma: immunometabolic links.

Asthma is a widespread chronic inflammatory disease, which has a highly heterogeneous etiopathogenesis, with predominance of either T‑helper cell type 2 (Th2; type 2) or non-Th2 (non-type 2) mechanisms. Together with cardiovascular or autoimmune diseases, obesity, and others, asthma belongs to so called noncommunicable diseases, a group of disorders with immunometabolic links as underlying mechanisms. So far, obesity and asthma have been considered mostly independently, but there are clear signs of relevant interactions. First, obese patients are at increased risk of asthma or asthma‑like symptoms. Second, asthma accompanied by obesity is more severe and more difficult to treat. A specific phenotype called obesity‑associated asthma has been also described, which is late‑onset, rather severe, non-type 2‑driven disease, present mostly in women. In addition, obesity can coincide with asthma also in children, and, although obesity generally skews the Th1/Th2 balance towards Th1, it can also accompany type 2‑driven asthma. However, those combinations represent less precisely defined disease entities. Despite a substantial increase in our knowledge on the mechanisms mediating the effects of obesity on the development of asthma in several recent years, still much needs to be done, especially on the molecular level

Differential Regulation of Interferon Signaling Pathways in CD4+ T Cells of the Low Type-2 Obesity-Associated Asthma Phenotype

In the era of personalized medicine, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90); 10 non-obese, non-atopic asthmatic adults with normal BMI (23.88 ± 2.73); and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to represent a low type-2 asthma phenotype according to selective clinical parameters. RNA sequencing (RNA-Seq) was conducted on peripheral blood CD4+ T cells. Thousands of differentially expressed genes were identified in both asthma groups compared with heathy controls. The expression of interferon (IFN)-stimulated genes associated with IFN-related signaling pathways was specifically affected in obese asthmatics, while the gap junction and G protein-coupled receptor (GPCR) ligand binding pathways were enriched in both asthma groups. Furthermore, obesity gene markers were also upregulated in CD4+ T cells from obese asthmatics compared with the two other groups. Additionally, the enriched genes of the three abovementioned pathways showed a unique correlation pattern with various laboratory and clinical parameters. The specific activation of IFN-related signaling and viral infection pathways might provide a novel view of the molecular mechanisms associated with the development of the low type-2 obesity-associated asthma phenotype, which is a step ahead in the development of new stratified therapeutic approaches

Identification of extracellular vesicle microRNA signatures specifically linked to inflammatory and metabolic mechanisms in obesity-associated low type-2 asthma

International audienceRationale and Objective Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma. Methods Obese low type-2 asthma (OA) and non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV-associated small RNAs were extracted, sequenced and bioinformatically analysed. Results Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17–92 and miR-106a–363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C-reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood-derived CD4+ T-cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways. Conclusion The identified plasma EV miRNA signatures and particularly the miR-17–92 and -106a–363 clusters were capable to disentangle specific mechanisms of the obesity-associated low type-2 asthma phenotype, which may serve as basis for stratified treatment development

Histone modifications and their role in epigenetics of atopy and allergic diseases

This review covers basic aspects of histone modification and the role of posttranslational histone modifications in the development of allergic diseases, including the immune mechanisms underlying this development. Together with DNA methylation, histone modifications (including histone acetylation, methylation, phosphorylation, ubiquitination, etc.) represent the classical epigenetic mechanisms. However, much less attention has been given to histone modifications than to DNA methylation in the context of allergy. A systematic review of the literature was undertaken to provide an unbiased and comprehensive update on the involvement of histone modifications in allergy and the mechanisms underlying this development. In addition to covering the growing interest in the contribution of histone modifications in regulating the development of allergic diseases, this review summarizes some of the evidence supporting this contribution. There are at least two levels at which the role of histone modifications is manifested. One is the regulation of cells that contribute to the allergic inflammation (T cells and macrophages) and those that participate in airway remodeling [(myo-) fibroblasts]. The other is the direct association between histone modifications and allergic phenotypes. Inhibitors of histone-modifying enzymes may potentially be used as anti-allergic drugs. Furthermore, epigenetic patterns may provide novel tools in the diagnosis of allergic disorders

In vitro induction of human embryonal carcinoma differentiation by a crude extract of Rhazya stricta

Abstract Background Rhazya stricta Decne. is a medicinal plant that is widespread in Saudi Arabia and desert areas of the Arabian Peninsula. Its extract contains alkaloids, tannins, and flavonoids that are involved in different biological activities. The study aim was to evaluate the effects of Rhazya stricta plant extracts on the proliferation and differentiation of NTERA-2 (NT2) pluripotent embryonal carcinoma cells. Methods Soxhlet extraction was carried out using different solvents to extract stems, leaves and fruit parts of this plant. Cytotoxicity was evaluated by an MTS cell viability assay. The ability of the plant extract to induce cell differentiation was examined phenotypically using an inverted light microscope. The expression of pluripotency markers was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Phytochemical screening of chloroform stem extracts was carried out and a chromatographic fingerprint was generated using gas chromatography – mass spectrometry (GC-MS). Results Chloroform stem extract induced differentiation of NT2 cells at 5 μg/ml, and the differentiated cells exhibited neurite formation. Following induction of differentiation, there was significant down-regulation of the pluripotency marker genes Oct4 and Sox2. In addition, the surface antigen pluripotency marker, TRA-1-60, was strongly down-regulated. Phytochemical analysis of the extract showed the presence of alkaloids and saponins. The chromatogram revealed the presence of fifteen compounds with different retention times. Conclusion Our results demonstrate for the first time that chloroform stem extract of R. stricta can induce neuronal differentiation of stem cells at an early stage and may contain potential therapeutic agent that can be used in neurodegenerative diseases

Raw Cow's Milk Reduces Allergic Symptoms in a Murine Model for Food Allergy-A Potential Role For Epigenetic Modifications

Epidemiological studies identified raw cow's milk consumption as an important environmental exposure that prevents allergic diseases. In the present study, we investigated whether raw cow's milk has the capacity to induce tolerance to an unrelated, non-milk, food allergen. Histone acetylation of T cell genes was investigated to assess potential epigenetic regulation. Female C3H/HeOuJ mice were sensitized and challenged to ovalbumin. Prior to sensitization, the mice were treated with raw milk, processed milk, or phosphate-buffered saline for eight days. Allergic symptoms were assessed after challenge and histone modifications in T cell-related genes of splenocyte-derived CD4+ T cells and the mesenteric lymph nodes were analyzed after milk exposure and after challenge. Unlike processed milk, raw milk decreased allergic symptoms. After raw milk exposure, histone acetylation of Th1-, Th2-, and regulatory T cell-related genes of splenocyte-derived CD4+ T cells was higher than after processed milk exposure. After allergy induction, this general immune stimulation was resolved and histone acetylation of Th2 genes was lower when compared to processed milk. Raw milk reduces allergic symptoms to an unrelated, non-milk, food allergen in a murine model for food allergy. The activation of T cell-related genes could be responsible for the observed tolerance induction, which suggested that epigenetic modifications contribute to the allergy-protective effect of raw milk

Intranasal administration of Acinetobacter lwoffii in a murine model of asthma induces IL-6-mediated protection associated with cecal microbiota changes

International audienceBackground Early-life exposure to certain environmental bacteria including Acinetobacter lwoffii (AL) has been implicated in protection from chronic inflammatory diseases including asthma later in life. However, the underlying mechanisms at the immune-microbe interface remain largely unknown. Methods The effects of repeated intranasal AL exposure on local and systemic innate immune responses were investigated in wild-type and Il6−/−, Il10−/−, and Il17−/− mice exposed to ovalbumin-induced allergic airway inflammation. Those investigations were expanded by microbiome analyses. To assess for AL-associated changes in gene expression, the picture arising from animal data was supplemented by in vitro experiments of macrophage and T-cell responses, yielding expression and epigenetic data. Results The asthma preventive effect of AL was confirmed in the lung. Repeated intranasal AL administration triggered a proinflammatory immune response particularly characterized by elevated levels of IL-6, and consequently, IL-6 induced IL-10 production in CD4+ T-cells. Both IL-6 and IL-10, but not IL-17, were required for asthma protection. AL had a profound impact on the gene regulatory landscape of CD4+ T-cells which could be largely recapitulated by recombinant IL-6. AL administration also induced marked changes in the gastrointestinal microbiome but not in the lung microbiome. By comparing the effects on the microbiota according to mouse genotype and AL-treatment status, we have identified microbial taxa that were associated with either disease protection or activity. Conclusion These experiments provide a novel mechanism of Acinetobacter lwoffii-induced asthma protection operating through IL-6-mediated epigenetic activation of IL-10 production and with associated effects on the intestinal microbiome

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses

BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials