The global population is rapidly aging. It is estimated that over the next thirty years, the number of individuals >60 years of age will increase by over a billion, and the number of individuals over age 80 may increase by 300 million. As humans age, our immune system becomes progressively weaker through a process called immune senescence. This age-related decrease in immune function increases susceptibility to infection and chronic diseases. Sepsis is a leading cause of death worldwide. Over the past two decades, there has been an increased incidence of sepsis which is due, in part, to our aging population and immune senescence. The gut microbiome, which plays an essential role in health and disease, is altered in aging and sepsis. Specifically, the commensal microorganisms of the gut microbiota are replaced with potentially pathogenic bacteria. This contributes to immune dysfunction and worsened outcomes in critical illness. The innate immune system can be “trained” to respond more effectively to pathogens. We examined trained immunity as an approach to modulating immunosenescence and microbiome diversity in aging. We investigated the effect of trained immunity on: i) immune cells from healthy aging subjects and sepsis patients and ii) the diversity of the microbiome in aging and sepsis. Our results indicate that trained immunity is effective in combatting age-related immunosenescence. We found that β-glucan induced trained immunity enhances monocyte metabolism, increases functionality as well as alters the transcriptome and epigenome in aging individuals and sepsis patients. We also found that trained immunity induced the expansion of a unique population of myeloid cells in sepsis. These cells are defined as FSChi, CD11b+, GR-1hi and express high levels of immunosuppressive PD-L1. In addition, we found that trained immunity reversed age-related changes to the microbiome and prevented alterations to the microbiome in septic mice. We found that the Firmicutes/Bacteroidetes ratio increased in aging; however, trained immunity reversed this increase and increased Clostridia in aged mice. In sepsis, trained immunity prevented expansion of Proteobacteria observed in control mice. Thus, our results indicate that trained immunity may be effective in modulating immune senescence and the microbiome in aging and sepsis.

No description available.

<p>Colorectal cancer is one of the leading causes of cancer deaths in the United States and multiple modifiable factors contribute to colorectal carcinogenesis. Gut microbiota are believed to play key roles in colon cancer development. Dietary factors may modulate gut microbiota composition, which may potentially have impact on carcinogenesis. Thus, it is reasonable to develop dietary interventions to effectively prevent colorectal cancer development through alteration of gut microbiota. In this thesis, the first objective is to evaluate the effect of vitamin E forms and metabolites, i.e., δ-tocotrienol (δTE), γ-tocotrienol (γTE) and δTE-13’-COOH (δTE-13’), respectively, on gut microbiota in mice. Healthy male balb/c mice were supplemented with a δTE/γTE mixture or δTE-13’ by gavage for two weeks, while control mice received soybean oil. We isolated DNAs from fecal samples and used 16S rRNA gene sequencing to evaluate the impact of these compounds on gut microbiota compositions. Further, we also examined the effect on short chain fatty acids (SCFAs). We observed that supplementation of δTE-13’ increased microbial richness using the Faith index. On the other hand, supplementation did not separate the microbial communities from the control group. But, these compounds managed to alter the relative abundances of several taxa that might present chemopreventive activities against colon cancer. Specifically, <i>Desulfovibrio</i>, a sulfur-reducing bacterium, was decreased after δTE/γTE supplementation. <i>Eubacterium coprostanoligenes</i> group, a group of microbes that can reduce circulating cholesterol, was increased after δTE/γTE supplementation. In addition, several members from the <i>Lachnospiraceae</i> family were elevated under δTE/γTE and δTE-13’ supplementation, and these microbes are known to produce SCFAs and maintain colonic health. However, the measurement of SCFAs showed that supplementation of δTE/γTE and δTE-13’ did not change SCFAs compared with controls. In the second project, I investigated anti-proliferative effects of combining δTE or δTE-13’ with sodium butyrate (NaBu) on human colorectal carcinoma HCT116 cells. Our data showed promising additive effects against cell growth. Collectively, these results indicate that δTE/γTE and δTE-13’ can modulate gut microbiota under healthy conditions, which provides insights into potential chemopreventive activities of these vitamin E forms. Our cell-based studies also showed additive anticancer effects of combining δTE or δTE-13’ with NaBu, which provides rationale to further develop combination of butyrate producers with vitamin E forms for cancer prevention.</p>

<p>Plant microbiomes are increasingly recognized for their potential to help plants with critical functions such as nutrient acquisition. Nitrogen is the most limiting nutrient in agriculture and growers apply substantial amounts to meet crop needs. Only 50% of N fertilizers are generally taken up by plants and the rest is subject to loss which negatively affects environmental quality. Organic fertilizers such as cover crops and animal manure can help reduce this loss, though these materials must mineralize via microbial mediated processes before they are available for plant uptake, which makes managing fertility using these sources difficult. Some plants can scavenge nutrients from organic materials by stimulating positive priming processes in soil. Carrot (<i>Daucus carota.</i> L) is known as an N scavenging crop, making it an ideal model crop to study these interactions. In a greenhouse trial, soils were amended with an isotopically labeled corn residue to track N movement, and planted with one of five carrot genotypes expected to differ in nitrogen use efficiency (NUE). Changes in soil b-glucosidase activity, ammonium (NH₄⁺-N) and nitrate (NO₃^--N) concentrations, soil bacterial community composition, weight and carbon and N concentrations, and total δ¹⁵N of above and below ground carrot biomass were determined. Results indicate that there are genetic differences in the ability of carrots to promote priming under N limited conditions, which could be exploited to enhance NUE in carrots. Soil microbial communities differed between genotypes, indicating that some of these microbes could play a role in the differential N scavenging responses observed, and/or contribute to other important functions such as resistance to pests. Endophytic microbes residing inside carrot taproots also have potential to contribute to NUE and other benefits, but are notoriously difficult to isolate and culture. New next generation sequencing technologies have revolutionized the study of microbiomes, though using these tools to study bacterial endophytes in plants is still difficult due to co-amplification of plant organelles. Consequently, a second study was conducted to determine if subjecting carrot tissues to hollow fiber microfiltration followed by enzymatic digestion could enhance recovery and amplification of bacterial endophytes. Carrot taproot digests were subject to amplification using a standard V3-V4 16S primer set, as well as two alternative (blocking and mismatch) primer sets that have prevented amplification of plastids/mitochondria in other plant species. Results indicate that the microfiltration/digestion procedure can increase the number of bacterial endophyte OTUs assigned and could be further optimized for use in carrots. The blocking and mismatch primer sets were not as effective in blocking co-amplification of plant products as they are in other studies, possibly due to the presence of a high number of chromoplasts in carrot tissues. Taxonomic assignment of bacterial endophytes differed significantly between the primer sets, indicating that multiple primer sets may be needed to fully characterize these communities in carrots. The enzymatic digestion procedure could artificially inflate certain taxa, which could be helpful if targeting specific taxa. These studies demonstrate that carrots are intimately connected with microbes residing in the soil and within their taproots, and further exploration of these plant-soil-microbial relationships could enhance the yield and sustainability of carrot production systems.</p>

Puberty is a critical period of development characterized by rapid physiological changes and significant brain reorganizing and remodeling. These rapid changes render the developing brain particularly vulnerable to stress and immune challenge. In mice, exposure to an immune challenge (lipopolysaccharide; LPS) during puberty causes enduring effects on stress reactivity, cognitive functioning, and depression- and anxiety-like behaviors later in life. However, the mechanisms underlying these effects are unknown. The gut microbiome can profoundly influence the immune system. There is also close bidirectional communication between the gut microbiome and the central nervous system (CNS) through neural, endocrine and immune signaling pathways, which can alter brain chemistry and emotional behaviour. Thus, we hypothesized that altering microbial composition during puberty could mitigate acute immune responses and prevent enduring outcomes later in life. The current thesis examined the effect of gut manipulation with probiotics during puberty on LPS-induced immune responses and enduring anxiety- and depression-like behaviours, and stress-reactivity in adulthood, in male and female CD1 mice (Article 1). Next, we examined age and sex differences in gut microbial composition before and after exposure to an immune challenge. We also examined the effects of consuming a single strain probiotic bacterium (Lactobacillus Reuteri) during puberty on the immune response and the long-term changes in memory, anxiety-like behavior, and stress reactivity in adulthood (Article 2). Lastly, we examined how microbial colonization between pubertal and adult mice can alter acute peripheral and central inflammatory responses to LPS (Article 3). The current dissertation has addressed sex-specific vulnerabilities to an immune challenge during pubertal development and the moderating influence of the gut microbiome. These studies have demonstrated that manipulating the gut microbiome during puberty can mitigate acute immune responses and prevent enduring neurobehavioural outcomes later in life.

Alterations in composition and function of human gut microbiota can affect physiological processes and are known to be associated with many diseases including inflammatory bowel disease, hypertension, asthma and colon cancer. Complex interactions between gut microbiota, environmental toxins, nutrients and host genetics may result in an increased permeability of the gut, which is closely linked to the presence of adverse metabolic conditions. As a consequence, translocation of bacterial DNA into the blood circulation increases in patients with obesity. Obesity is a growing health problem worldwide and often paired with severe secondary complications, such as type 2 diabetes or cardiovascular problems. A main feature of disease progression is a chronic low-grade inflammation of adipose tissue which contributes to the development and aggravation of insulin resistance and many of the underlying mechanisms are still unknown. Although data from mice studies suggest that the presence of bacterial components in adipose tissue can support these processes, human studies on this subject are lacking. In my thesis entitled ``Bacterial translocation to adipose tissue in metabolic diseas'' I provide evidence supporting the initial hypothesis, namely that bacterial DNA is present in adipose tissue, even after stringent controlling for contaminants. To this end, I established a wet lab routine protocol to eliminate contamination as well as a bioinformatics pipeline accounting for contamination by subtracting negative controls. Briefly, this included the use of lab ware and reagents UV-treated for at least 90 minutes, the use of breath protection, extra-long gloves and single-use lab coats as well as working under a sterile laminar flood hood in a clean lab free of any PCR products. The bioinformatics pipeline employed commonly used 16S rRNA gene analysis tools including qiime2, phyloseq and DESeq2 as well as decontam, a novel tool to extract negative controls. Observed quantity of bacterial DNA was in the range of 1 to 10 pg/µg total isolated genomic DNA, which is equivalent to about 0.01 to 0.7% of bacterial cells per human cell. The highest quantity was present in subcutaneous adipose tissue, followed by mesenteric adipose tissue. Bacterial amount correlated with adipose tissue macrophages and PPARG expression in omental and with IL1B and TNF expression in subcutaneous adipose tissue. Mesenteric adipose tissue showed the highest diversity of the observed genera. The most commonly observed phyla in all tissues were Proteobacteria and Firmicutes, which is in line with previously published data on blood bacterial DNA. Still, many genera were predominantly found in specific tissues, e.g. Enterobacter in subcutaneous and Acinetobacter in omental adipose tissue. I further showed that the distribution of observed features could partially be explained by markers of insulin resistance (HOMA-IR, HbA1c) and inflammation (IL-6, TNFa, macrophages) and that certain genera, such as Rhodoferax or Lactobacillus are associated with type 2 diabetes status. In first functional approaches I demonstrated that concentrations of bacterial DNA in the observed range are sufficient to stimulate an inflammatory response in immortalized subcutaneous adipocytes derived from a healthy donor. The effect was most prominent after four hours of treatment and increased in a dose-dependent matter. One of the aims in the present study was to determine levels of gut leakage by measuring zonulin, the most commonly used biomarker for intestinal permeability in humans, and analyze possible associations with adipose tissue bacterial signature. As there are few well-conducted studies on circulating zonulin levels in patients with metabolic diseases, I first performed a correlation study in the available and metabolically well-characterized Sorbs cohort. Circulating zonulin correlated significantly positive with BMI, fasting glucose, triglycerides and cholesterol and negatively with HOMA-IS, high density lipoprotein and circulating adiponectin levels. Albeit these strong correlations with markers of glucose and lipid metabolism supported previously reported findings, the results pointed to some inconsistencies. As zonulin is reported to be pre-haptoglobin 2 (preHP2), and about 15% percent of a typical western European population are homozygous for haptoglobin 1, they should not express zonulin at all. I confirmed in the Sorbs cohort previously reported distributions of haptoglobin genotypes and showed that the target of the only commercially available zonulin ELISA kit was not related to haptoglobin genotype, therefore presumably not measuring zonulin/ preHP2. Subsequently, I identified properdin as a possible target by employing mass spectrometry approaches. Properdin is structurally related to haptoglobin, as both proteins belong to the mannose-associated serine protease family, however further experiments are needed to validate a possible functional resemblance. In regard to bacterial translocation two adipose tissue depots were of notable interest due to their close proximity to the gastrointestinal tract. Mesenteric adipose tissue is located around the small intestine and the adipose tissue of the appendices epiploicae, small chunks of fat also called epiploic adipose tissue, are directly attached to the colon. After a thorough literature research I could also assert that both adipose tissues were rarely analyzed in the context of obesity. Therefore they were extensively investigated by measuring gene expression of adipo(cyto)kines, circulating inflammatory markers and analyzing adipocyte size and adipose tissue macrophages. Furthermore, a ``multiomics'' characterization was conducted and by analyzing transcriptome and methylome data I could identify epiploic adipose tissue as a tissue of interest in regard to type 2 diabetes and insulin resistance, which was further confirmed by untargeted proteomics data. Contrary to initial assumptions, I observed only a slight increase in translocation of bacterial DNA and no increased inflammation, as measured by tissue specific TNF and IL6 expression as well as adipose tissue macrophage infiltration. However, both transcriptome and proteome profiles allowed a clear discrimination of patients with and without insulin resistance which was most distinct in epiploic adipose tissue. Compared to other fat depots, epiploic adipose tissue exhibited a discriminable metabolic profile whereas mesenteric adipose tissue was more similar to omental-visceral adipose tissue. Most strikingly, epiploic adipose tissue showed a strong increase in leptin expression and, in general, the upregulation of various metabolic pathways involved in sugar, amino acid or sphingolipid metabolism. In accord with the leaky gut hypothesis high expression of lipopolysaccharide binding protein and various pathways involved in chemokine signaling were observed. In summary, I did not observe an increase in bacterial DNA or adipose tissue macrophages, but demonstrated elevated inflammatory signals such as increased chemokine or IL-8 signaling which are linked with an overall increase of metabolic processes and an increased expression of various adipokines. Epiploic adipose tissue might have a watch dog function by being the first adipose tissue sensing and forwarding certain (microbial) stimuli from the large intestine to the host. In the last part of my thesis I addressed a possible role of the HLA genomic region on the development of type 2 diabetes. The influence of HLA genetics on type 2 diabetes has been under debate for several decades, since HLA was recognized to largely contribute to type 1 diabetes heritability. However, studies remained inconclusive due to lacking cohorts with sample sizes providing sufficient statistical power for association analyses. More recently, animal studies suggested MHC class II proteins as crucial factors mediating adipose tissue inflammation and insulin resistance. The sample size of the leaky gut cohort was insufficient to determine any correlation between HLA class II genotypes and the presence or type of bacteria in adipose tissue due to the high variability in the observed genomic region. Yet, I had access to three large population-based cohorts which allowed me to analyze associations between HLA class II alleles and type 2 diabetes. Therefore HLA genotypes of the LIFE-Adult (N=4649), LIFE-Heart (N=4815) and Sorbs (N=949) cohort were imputed from SNP genotyping data and analyzed for association with type 2 diabetes. In a meta-analysis including all three cohorts, I identified a protective effect for the well-established type 1 diabetes protective haplotype DRB1*15:01~DQA1*01:02~DQB1*06:02 and confirmed DRB1*07:01~DQA1*02:01~DQB1*03:03 as a risk haplotype in non-insulin treated diabetes. These results suggest that the genetic foundation of both type 1 and 2 diabetes shares common elements involving the HLA class II locus. In conclusion, to the best of my knowledge, I provide in my work the first contaminant-aware identification of bacterial DNA in human adipose tissue and highlight the importance of analyzing novel adipose tissue depots by showing that fat of the appendices epiploicae, previously only considered to have a cushioning function, is metabolically active and possibly involved in the development of insulin resistance.

Abstract Purpose: The oral microbiome has been related to numerous extra oral diseases. Recent studies detected a high abundance of oral bacteria in inflamed appendices in pediatric patients. To elucidate the role of oral bacteria in acute pediatric appendicitis, we studied the oral and appendiceal microbiome of affected children compared to healthy controls. Methods: Between 1-6/2015, 21 children undergoing appendectomy for acute appendicitis and 28 healthy controls were prospectively enrolled in the study. All individuals underwent thorough dental examination as well as laboratory for CRP, neutrophil and leukocyte count. Samples of inflamed appendices and the gingival sulcus were taken for 16S rDNA sequencing. Additionally, RT-qPCR of Fusobacterium nucleatum, Peptostreptococcus stomatis and Eikenella corrodens was performed and their viability was tested under acidic conditions to mimic gastric transfer. Results: In appendices, Bacteriodetes and Porphyromonas were discovered as dominant phylum and genus. In sulcus samples, Firmicutes and Streptococcus were detected predominantly. Peptostreptococcus stomatis, Eikenella corrodens and Fusobacterium nucleatum were identified in each sample group. In contrast to controls, viable amounts of Peptostreptococcus stomatis were increased in appendices and correlated with leukocyte count and CRP levels. Postprandial viability could be demonstrated for all tested bacteria. Conclusion: In children with acute appendicitis, we identified several oral bacterial pathogens. Based on postprandial viability of selected species, a viable migration from the oral cavity through the stomach to the appendix seems possible. Thus, the oral cavity could be a relevant reservoir for acute appendicitis.

Intestinal microbiota is involved in metabolic processes and the pathophysiology of various gastrointestinal disorders. We aimed to characterize the microbiome of the appendix in acute pediatric appendicitis comparing extraluminal and intraluminal samples. Between January and June 2015, 29 children (3–17 years, mean age 10.7±3.4 years, sex M:F=2.6:1) undergoing laparoscopic appendectomy for acute appendicitis were prospectively included in the study. Samples for bacterial cultures (n=29) and 16S ribosomal desoxyribonucleic acid (rDNA) sequencing (randomly chosen n=16/29) were taken intracorporeally from the appendiceal surface before preparation (“extraluminal”) and from the appendiceal lumen after removal (“intraluminal”). The degree of inflammation was histologically classified into catarrhal, phlegmonous, and gangrenous appendicitis. Seventeen bacterial species were cultivated in 28 of 29 intraluminal samples and 4 species were cultivated in 2 of 29 extraluminal samples. Using 16S rDNA sequencing, 267 species were detected in intraluminal but none in extraluminal samples. Abundance and diversity of detected species differed significantly between histological groups of acute appendicitis in bacterial cultures (P=.001), but not after 16S rDNA sequencing. The appendiceal microbiome showed a high diversity in acute pediatric appendicitis. The intraluminal microbial composition differed significantly depending on the degree of inflammation. As bacteria were rarely found extraluminally by culture and not at all by sequencing, the inflammation in acute appendicitis may start inside the appendix and spread transmurally.

Thesis advisor: Catherine Y. Read / Background. Clostridium difficile infections (CDI) account for 20-30% of healthcare-acquired infections, resulting in serious patient and economic burdens. CDI incidence has grown rapidly due to overuse of antibiotics and an aging population, posing a significant public health threat. Fecal microbiota transplantation (FMT) using donor stool has demonstrated clinical efficacy rates up to 94% and long-term restoration of a healthy intestinal microbiome. Challenges with donor screening, lack of research about optimal stool donor characteristics and intestinal microbiome composition, and a poorly fit screening model, create barriers to the availability of FMT. Purpose. This study aimed to generate essential information about FMT donor characteristics predictive of passing the screening and donor intestinal microbiome compositions associated with FMT clinical efficacy. The primary aims were to 1) identify previously unstudied characteristics of prospective FMT donors that are predictive of passing a stool bank’s screening process; and 2) determine whether donor intestinal microbial diversity is related to FMT clinical efficacy in preventing recurrent CDI. Methods. This study was conducted as a secondary analysis on a cohort of previously screened donors (n=770). Aim 1 was tested through a logistic regression of donor characteristics (gender, age, body mass index, frequency of bowel movements, diet, tobacco and alcohol use, and seasonality) with screening outcomes. Aim 2 was tested through a simple regression evaluating donor intestinal microbial diversity and rates of FMT clinical efficacy. Results. One donor characteristic in the logistic regression, frequency of bowel movements (p = 0.018), was significantly predictive of whether a donor passed the screening. Specifically, donors who had fewer than two bowel movements per day were more likely to pass. All other characteristics were not predictive. Similarly, the linear regression evaluating alpha diversity and FMT clinical efficacy was not significantly predictive of clinical efficacy (p = 0.140). Conclusion. Findings were used to support recommendations for improving prospective donor screening that nurses and other clinicians can implement to decrease challenging logistics, reduce costs and barriers, and potentially increase FMT clinical efficacy. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

No description available.