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Ampicillin treatment casues dysbiosis that persists until study endpoint [RNA-seq]

ABSTRACT: The development of neutralizing FVIII antibodies is the most serious complication of hemophilia A treatment. The currently known patient- and treatment-related risk factors for inhibitor development do not accurately predict this adverse event in all patients. The composition of the gut microbiota has been shown to influence immune mediated diseases at distant anatomical sites (e.g. lungs, brain and joints). We demonstrate that a disrupted gut microbiome can be created in a mouse model of hemophilia A using a broad-spectrum antibiotic. Under controlled conditions, this sustained dysbiosis was associated with an enhanced anti-FVIII immune response as evidenced by increased splenic B cells and the development of higher titre FVIII specific IgG antibodies following FVIII challenge. Splenic and mesenteric lymph node cytokines, T cells and dendritic cells were unaffected prior to FVIII administration. However, the immune transcriptome of both aforementioned secondary lymphoid organs was significantly modified. Short chain fatty acids (SCFA), which are microbial metabolites, were depleted in cecal contents of the dysbiotic mice. Furthermore, supplementation of the drinking water with the most immunologically active SCFA, butyrate, successfully achieved attenuation of the FVIII immune response. Collectively, our data suggest that the composition of the gut microbiome alters the FVIII immune response via the action of specific microbial metabolites on the immune cell transcriptome and that oral butyrate supplementation effectively reduces the FVIII immune response. Mice treated with one week of ampicillin at 3 weeks of age have a dysbiotic gut microbiome at 6 weeks of age and at the study endpoint. The immune response to FVII was compared between the two cohorts. The dysbiotic cohort had a greater immune response to FVIII

ORGANISM(S): mouse gut metagenome

PROVIDER: GSE138330 | GEO | 2019/10/04

REPOSITORIES: GEO

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Project description:Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). In addition to genetic mutations in the gene encoding FVIII (F8), there is evidence that other molecular mechanisms may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them, hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-M-CM- -vis the role of nc-RNAs in the regulation of F8 expression and the development of anti-drug antibodies to FVIII infusions used to treat HA. Whole blood samples from nine patients with hemophilia A and five controls were obtained. Among those patients with hemophilia A, three of them were patients who developed neutralizing-anti-FVIII antibodies (inhibitors) and the others are patients without FVIII inhibitors. RNA was isolated and purified from all samples using the RiboPure Blood Kit (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol with modifications to enhance the enrichment of low molecular weight RNAs. All RNA samples were poly (A)-tailed and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). After labeling, the enzyme linked oligosorbent assays (ELOSA) were performed to confirm that the biotin labeling processes were successful. Biotin-labeled RNA samples (500 ng/sample) were hybridized on GeneChip miRNA 3.0 microarrays (Affymetrix). Microarray data were analyzed to identify ncRNAs that were differentially expressed in hemophilia A groups compared to controls.

Project description:Differences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established, the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiome bacteria on humoral immune activation. Sham-operated and gonadectomized male and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotypes. Ex vivo studies investigated the functional role of Kdm6a, an X-linked epigenetic regulatory gene of interest, in mitogenic B cell activation. We further evaluated whether gut microbiome communities, or their metabolites, differentially influence immune cell activation in a sex chromosome-dependent manner. Endogenous gut microbiomes were antibiotically depleted and reconstituted with select short-chain fatty acid (SCFA)-producing bacteria prior to HKSP immunization and immune responses assessed. XX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice, regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells, inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion, indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria increased humoral responses in XX, but not XY, FCG mice. This XX-dependent enhancement appears to be independent of SCFA production in males, while female XX-dependent responses relied on SCFAs. FCG mice have been used to assess sex hormones and sex chromosome complements influences on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner, suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphisms.

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Ampicillin treatment casues dysbiosis that persists until study endpoint [RNA-seq]

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