Dorottya Nagy-Szakal

Genome‐wide peripheral blood leukocyte DNA methylation microarrays identified a single association with inflammatory bowel diseases

Background: Crohns disease (CD) and ulcerative colitis (UC) are common forms of inflammatory bowel disease (IBD). Monozygotic (MZ) twin discordance rates and epidemiologic data implicate that environmental changes and epigenetic factors may play a pathogenic role in IBD. DNA methylation (the methylation of cytosines within CpG dinucleotides) is an epigenetic modification, which can respond to environmental influences. We investigated whether DNA methylation might be connected with IBD in peripheral blood leukocyte (PBL) DNA by utilizing genome‐wide microarrays. Methods: Two different high‐throughput microarray‐based methods for genome‐wide DNA methylation analysis were employed. First, DNA isolated from MZ twin pairs concordant (CD: 4; UC: 3) and discordant (CD: 4; UC: 7) for IBD was interrogated by a custom‐made methylation‐specific amplification microarray (MSAM). Second, the recently developed Illumina Infinium HumanMethylation450 BeadChip arrays were used on 48 samples of PBL DNA from discordant MZ twin pairs (CD: 3; UC: 3) and treatment‐naive pediatric cases of IBD (CD: 14; UC: 8), as well as controls (n = 14). The microarrays were validated with bisulfite pyrosequencing. Results: The MSAMs did not yield significant IBD associations. The Methylation BeadChip approach identified a single DNA methylation association of IBD at TEPP (testis, prostate and placenta‐expressed protein) when DNA isolated selectively from peripheral blood mononuclear cells was analyzed (8.6% increase in methylation between CD and control, FDR = 0.0065). Conclusions: Microarray interrogation of IBD‐dependent DNA methylation from PBLs appears to have limited ability to detect significant disease associations. More detailed and/or selective approaches may be useful for the elucidation of connections between the DNA methylome and IBD in the future. (Inflamm Bowel Dis 2012;)

Transfer of Viral Communities between Human Individuals during Fecal Microbiota Transplantation

ABSTRACT Fecal microbiota transplantation (FMT) is a highly effective treatment for refractory Clostridium difficile infections. However, concerns persist about unwanted cotransfer of pathogenic microbes such as viruses. Here we studed FMT from a single healthy human donor to three pediatric ulcerative colitis patients, each of whom received a course of 22 to 30 FMT treatments. Viral particles were purified from donor and recipient stool samples and sequenced; the reads were then assembled into contigs corresponding to viral genomes or partial genomes. Transfer of selected viruses was confirmed by quantitative PCR. Viral contigs present in the donor could be readily detected in recipients, with up to 32 different donor viral contigs appearing in a recipient sample. Reassuringly, none of these were viruses are known to replicate on human cells. Instead, viral contigs either scored as bacteriophage or could not be attributed taxonomically, suggestive of unstudied phage. The two most frequently transferred gene types were associated with temperate-phage replication. In addition, members of Siphoviridae, the group of typically temperate phages that includes phage lambda, were found to be transferred with significantly greater efficiency than other groups. On the basis of these findings, we propose that the temperate-phage replication style may promote efficient phage transfer between human individuals. In summary, we documented transfer of multiple viral lineages between human individuals through FMT, but in this case series, none were from viral groups known to infect human cells. IMPORTANCE Transfer of whole communities of viruses between humans has rarely been studied but is of likely medical importance. Here we studied fecal microbiota transplantation (FMT), a highly successful treatment for relapsing Clostridium difficile infection and, potentially, other gastrointestinal (GI) diseases. We investigated the transfer of viral communities during FMT and documented transfer of multiple viral lineages between humans. None of these were viruses that replicated on animal cells or that are known to be pathogenic. We found that temperate bacteriophage, which form stable associations with their hosts, were significantly more likely to be transferred during FMT. This supports a model in which the viral temperate replication style may have evolved in part to support efficient viral transmission between environments. Transfer of whole communities of viruses between humans has rarely been studied but is of likely medical importance. Here we studied fecal microbiota transplantation (FMT), a highly successful treatment for relapsing Clostridium difficile infection and, potentially, other gastrointestinal (GI) diseases. We investigated the transfer of viral communities during FMT and documented transfer of multiple viral lineages between humans. None of these were viruses that replicated on animal cells or that are known to be pathogenic. We found that temperate bacteriophage, which form stable associations with their hosts, were significantly more likely to be transferred during FMT. This supports a model in which the viral temperate replication style may have evolved in part to support efficient viral transmission between environments.

Human metastable epiallele candidates link to common disorders

Metastable epialleles (MEs) are mammalian genomic loci where epigenetic patterning occurs before gastrulation in a stochastic fashion leading to systematic interindividual variation within one species. Importantly, periconceptual nutritional influences may modulate the establishment of epigenetic changes, such as DNA methylation at MEs. Based on these characteristics, we exploited Infinium HumanMethylation450 BeadChip kits in a 2-tissue parallel screen on peripheral blood leukocyte and colonic mucosal DNA from 10 children without identifiable large intestinal disease. This approach led to the delineation of 1776 CpG sites meeting our criteria for MEs, which associated with 1013 genes. The list of ME candidates exhibited overlaps with recently identified human genes (including CYP2E1 and MGMT, where methylation has been associated with Parkinson disease and glioblastoma, respectively) in which perinatal DNA methylation levels where linked to maternal periconceptual nutrition. One hundred 18 (11.6%) of the ME candidates overlapped with genes where DNA methylation correlated (r > 0.871; p < 0.055) with expression in the colon mucosa of 5 independent control children. Genes involved in homophilic cell adhesion (including cadherin-associated genes) and developmental processes were significantly overrepresented in association with MEs. Additional filtering of gene expression-correlated MEs defined 35 genes, associated with 2 or more CpG sites within a 10 kb genomic region, fulfilling the ME criteria. DNA methylation changes at a number of these genes have been linked to various forms of human disease, including cancers, such as asthma and acute myeloid leukemia (ALOX12), gastric cancer (EBF3), breast cancer (NAV1), colon cancer and acute lymphoid leukemia (KCNK15), Wilms tumor (protocadherin gene cluster; PCDHAs) and colorectal cancer (TCERG1L), suggesting a potential etiologic role for MEs in tumorigenesis and underscoring the possible developmental origins of these malignancies. The presented compendium of ME candidates may accelerate our understanding of the epigenetic origins of common human disorders.

Serial Fecal Microbiota Transplantation Alters Mucosal Gene Expression in Pediatric Ulcerative Colitis

Serial Fecal Microbiota Transplantation Alters Mucosal Gene Expression in Pediatric Ulcerative Colitis

DNA methylation-associated colonic mucosal immune and defense responses in treatment-naïve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.

Cellulose Supplementation Early in Life Ameliorates Colitis in Adult Mice

Decreased consumption of dietary fibers, such as cellulose, has been proposed to promote the emergence of inflammatory bowel diseases (IBD: Crohn disease [CD] and ulcerative colitis [UC]) where intestinal microbes are recognized to play an etiologic role. However, it is not known if transient fiber consumption during critical developmental periods may prevent consecutive intestinal inflammation. The incidence of IBD peaks in young adulthood indicating that pediatric environmental exposures may be important in the etiology of this disease group. We studied the effects of transient dietary cellulose supplementation on dextran sulfate sodium (DSS) colitis susceptibility during the pediatric period in mice. Cellulose supplementation stimulated substantial shifts in the colonic mucosal microbiome. Several bacterial taxa decreased in relative abundance (e.g., Coriobacteriaceae [p = 0.001]), and other taxa increased in abundance (e.g., Peptostreptococcaceae [p = 0.008] and Clostridiaceae [p = 0.048]). Some of these shifts persisted for 10 days following the cessation of cellulose supplementation. The changes in the gut microbiome were associated with transient trophic and anticolitic effects 10 days following the cessation of a cellulose-enriched diet, but these changes diminished by 40 days following reversal to a low cellulose diet. These findings emphasize the transient protective effect of dietary cellulose in the mammalian large bowel and highlight the potential role of dietary fibers in amelioration of intestinal inflammation.

Microbiota separation and C-reactive protein elevation in treatment-naive pediatric granulomatous Crohn disease.

Objectives: In patients with inflammatory bowel diseases (IBDs), the presence of noncaseating mucosal granuloma is sufficient for diagnosing Crohn disease (CD) and may represent a specific immune response or microbial-host interaction. The cause of granulomas in CD is unknown and their association with the intestinal microbiota has not been addressed with high-throughput methodologies. Methods: The mucosal microbiota from 3 different pediatric centers was studied with 454 pyrosequencing of the bacterial 16S rRNA gene and the fungal small subunit (SSU) ribosomal region in transverse colonic biopsy specimens from 26 controls and 15 treatment-naïve pediatric CD cases. Mycobacterium avium subspecies paratuberculosis (MAP) was tested with real-time polymerase chain reaction. The correlation of granulomatous inflammation with C-reactive protein was expanded to 86 treatment-naïve CD cases. Results: The CD microbiota separated from controls by distance-based redundancy analysis (P = 0.035). Mucosal granulomata found in any portion of the intestinal tract associated with an augmented colonic bacterial microbiota divergence (P = 0.013). The granuloma-based microbiota separation persisted even when research center bias was eliminated (P = 0.04). Decreased Roseburia and Ruminococcus in granulomatous CD were important in this separation; however, principal coordinates analysis did not reveal partitioning of the groups. CRP levels >1 mg/dL predicted the presence of mucosal granulomata (odds ratio 28 [6–134.32]; 73% sensitivity, 91% specificity). Conclusions: Granulomatous CD associates with microbiota separation and C-reactive protein elevation in treatment-naïve children; however, overall dysbiosis in pediatric CD appears rather limited. Geographical/center bias should be accounted for in future multicenter microbiota studies.

Composition and function of the pediatric colonic mucosal microbiome in untreated patients with ulcerative colitis

ABSTRACT Inflammatory bowel diseases (IBD) are chronic intestinal inflammatory disorders characterized by a complex disruption of the physiologic interaction between the host immune system and intestinal microbes precipitated by environmental factors. Numerous observations indicate the altered composition and function of the intestinal microbiome of patients with ulcerative colitis (UC), a subtype of IBD. The accuracy of these results may be limited by confounding factors, such as concurrent medication use. To address these limitations, we examined the colonic mucosal microbiome of pediatric patients with UC prior to initiating treatment. Based on bacterial 16S rRNA gene sequencing, we identified a significant decrease in the phylum Verrucomicrobia in patients with UC. At the genus level, we observed a significant decrease in the short chain fatty acid producer Roseburia. Despite these compositional changes, we did not identify inferred gene content differences between the UC and control groups. To determine if microbial taxa may be associated with clinical outcomes, we retrospectively assessed the clinical course of the UC patients. Despite similar metrics of OTU richness and diversity, multiple OTU differences were observed between patients who responded to therapy and those who did not. Our observations regarding the mucosal microbiome and the associations with differential clinical outcomes support the contributions of gut microbes to disease onset and modulation.

Maternal micronutrients can modify colonic mucosal microbiota maturation in murine offspring

Epidemiologic data suggest that early nutritional exposures may inflict persistent changes in the developing mammalian “super-organism” (i.e., the host and its residing microbiota). Such persistent modifications could predispose young adults to inflammatory bowel diseases (IBD). We recently observed that the dietary supplementation of four micronutrients to dams augmented colitis susceptibility in murine offspring in association with mucosal microbiota composition changes. In this study the effects of the four micronutrients on the microbiota of dams and female mice was examined. Additionally, age dependent microbiota composition shifts during pediatric development were delineated from the previous offspring data sets. Maternal and adult female microbiota did not separate secondary to the nutritional intervention. Significant microbiota composition changes occurred from postnatal day 30 (P30) to P90 at the level of 1 phylum and 15 genera. Most of these changes were absent or opposite in the maternally supplemented offspring. Nutritionally induced alterations in mucosal microbiota maturation may be contributors to colitis susceptibility in mammals.

Monotonous diets protect against acute colitis in mice: Epidemiologic and therapeutic implications

Objectives: Multiple characteristics of industrialization have been proposed to contribute to the global emergence of inflammatory bowel diseases (IBDs: Crohn disease and ulcerative colitis). Major changes in eating habits during the last decades and the effectiveness of exclusive enteral nutrition in the treatment of Crohn disease indicate the etiologic importance of dietary intake in IBDs. A uniform characteristic of nutrition in developing countries (where the incidence of IBD is low) and exclusive enteral nutrition is their consistent nature for prolonged periods; however, the potentially beneficial effect of dietary monotony in respect to mammalian intestinal inflammation has not been examined. Methods: The association between alternating (2 different complete chows) and persistent regular diets, and dextran sulfate sodium colitis susceptibility in C57BL/6J mice was studied. Colonic mucosal microbiota changes were investigated by high-throughput pyrosequencing of the 16S rRNA gene. Results: The severity of colitis increased upon dietary alternation compared with consistent control feeding. The microbiota of the alternating nutritional group clustered discretely from both control groups. Conclusions: Our findings highlight that monotonous dietary intake may decrease mammalian vulnerability against colitis in association with microbiota separation. The epidemiologic and therapeutic implications of our results are also discussed.