Browsing by Person "Fricke, Florian W."

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Characterization of dietary and genetic influences on the gastrointestinal microbiota
(2023) Bubeck, Alena Marie; Fricke, Florian W.
Although the gut microbiota is known to contribute fundamentally to human health, e.g. by promoting the maturation of the immune system and intestinal homeostasis, the factors shaping its composition are only poorly understood. Extrinsic and intrinsic influences can disturb the tightly controlled equilibrium between the microbiome and the host and induce dysbiosis, which has been linked to diverse health conditions such as obesity, atherosclerotic cardiovascular disease (ACVD) and inflammatory bowel disease (IBD). Therefore, understanding events leading to microbial perturbations and the prediction of associated health outcomes could aid in the prevention and treatment of these conditions. In this work, the impact of dietary and genetic factors on gastrointestinal microbiota compositions were determined, with the diet serving as an exemplary extrinsic, modifiable microbiota-relevant factor and with a genetic deficiency in a mouse model for intestinal inflammation serving as an exemplary intrinsic, non-modifiable microbiota-relevant factor. In both studies, microbial communities obtained from either a human or a murine cohort, respectively, were taxonomically characterized by 16S rRNA gene amplicon sequencing and analyzed in the context of metabolic and inflammatory implications for the host. In ACVD, the reduction of excess blood cholesterol, which is a main risk factor, is tackled by clinical interventions aiming to reduce cholesterol uptake from exogenous, dietary sources or by inhibiting endogenous cholesterol biosynthesis. Cholesterol-to-coprostanol conversion by the intestinal microbiota has also been suggested to reduce intestinal and serum cholesterol availability, but the dependencies of cholesterol conversion on specific bacterial taxa and dietary habits, as well as its association with serum lipid levels remain largely unknown. To study microbiota contributions to human cholesterol metabolism under varying conditions, fecal microbiota and lipid profiles, as well as serum lipid biomarkers, were determined in two independent human cohorts, including individuals with (CARBFUNC study) and without obesity (KETO study) on very low-carbohydrate high-fat diets (LCHF) for three to six months and six weeks, respectively. Across these two geographically independent studies, conserved distributions of cholesterol high and low-converter types were measured. Also, cholesterol conversion was most dominantly linked to the relative abundance of the cholesterol-converting bacterial species Eubacterium coprostanoligenes, which was further increased in low-converters by LCHF diets, shifting them towards a high-conversion state. Lean cholesterol high-converters, which were characterized by adverse serum lipid profiles even before the LCHF diet, responded to the intervention with increased LDL-C, independently of fat, cholesterol and saturated fatty acid intake. These findings identify the cholesterol high-converter type as a potential predictive biomarker for an increased LDL-C response to LCHF diet in metabolically healthy lean individuals. Although the etiology of IBD has not been fully resolved, an interplay between the intestinal microbiota, environmental factors and an individual’s genetic susceptibility is thought to trigger chronic inflammation by a dysregulation of the immune response in the gut. To identify colitis-associated microbiota alterations throughout the development of spontaneous colitis, mice with a genetic deficiency of the anti-inflammatory cytokine Interleukin-10 (IL-10) from different litters were co-housed with wild-type mice and monitored for 20 weeks. The scoring of mice based on their phenotype and stool consistency mirrored the state of mucosal inflammation as assessed based on histopathological examinations and cytokine expression profiles. Also, the state of colitis was characterized by global microbiota alterations and susceptibility to colitis was dependent on litter-specific microbiome compositions that mice adopted early on in their lives. Colitis development was further associated with the presence of the bacterial genus Akkermansia in mature mice shortly before symptoms manifested. This genus was also a good predictor of colitis-related mice withdrawal, suggesting the potential of Akkermansia to serve as an early onset, subclinical colitis marker. In summary, fecal microbiota characterizations in response to LCHF diets in humans and throughout the development of intestinal inflammation in a colitis mouse model highlight the potential of personalized microbiome-based patient classifications to predict clinical outcomes and improve treatment approaches.
Strain-resolved analysis of the human intestinal microbiota
(2022) Podlesny, Daniel; Fricke, Florian W.
The gut microbiota is ascribed a crucial role in human health, particularly in regulating immune and inflammatory responses, which is why it is being associated with a wide range of diseases, including obesity, diabetes, and cancer. Nonetheless, fundamental ecological questions of microbiome establishment, stability and resilience, as well as its transmission across hosts and generations remain incompletely understood, partly due to the lack of methods for high-resolution microbiome profiling. New insights in this field can therefore directly contribute to the development of bacterial and microbiota-based therapies. This work introduces SameStr, a novel bioinformatic program for strain-resolved metagenomics that allows for the specific tracking of microbes across samples, enabling the detection and quantification of microbial transmission and persistence, as well as the observation of direct strain competition. Deployed across cohorts to process over 4200 metagenomes, SameStr enabled analysis of the microbiome with unprecedented phylogenetic resolution. The data included both publicly available metagenomes and sequence data generated in collaboration with our research partners, and was examined using multivariate statistics and machine learning frameworks. First, the establishment and development of the neonatal microbiota was studied, revealing a birth mode-dependent vertical transmission of the maternal microbiota. The microbiota of neonates born by cesarean section was characterized by increased relative abundance of oxygen-tolerant and atypical organisms and showed signs of a delayed establishment of a strictly anaerobic gut environment in these children. Such birth mode-dependent differences diminished over time, yet were measurable within the first two years of life. Furthermore, strain analysis verified the transmission and colonization of parental microbes, which indicated a possible lifelong colonization by microbes from selected species. The temporal persistence of microbes was also characterized in healthy adults, revealing similar taxonomy-dependent patterns of stability. For some species, persistence has been demonstrated both in children and in adults over a period of at least two years. These species are known for their capability to metabolize host-derived glycans found both in breastmilk and intestinal mucus, pointing to a potential strategy for effective cross-generational microbiota transmission, and warranting additional research to assess the implications of their disturbed transfer for long-term health. Since their specificity allows assignment to individual hosts, fingerprints of individual microbial strains offer the potential to be used in forensics and data quality control applications. Finally, to gain new insights into the microbiota dynamics during Fecal Microbiota Transplantation (FMT), microbial strain transmission was analyzed in the context of a diverse set of patient, microbiome, and clinical conditions. In the analyzed studies, FMT was used for the experimental treatment of a variety of diseases, including colonization with drug-resistant and pathogenic microbes, metabolic and inflammatory bowel diseases, and as an adjunct to the immunotherapeutic treatment of cancer. Analyses uncovered what appear to be the universal drivers of post-FMT microbiota assembly, including clinical and ecological factors that are important for successful transplantation of donor strains. In particular, the relevance of the microbiota dysbiosis of the recipient was emphasized, which was inducible by pre-treating the patient with antibiotics or laxatives. Presumably, this can open up ecological niches in the patients intestines, which favors colonization with donor strains. Colonization rates did not play a role for the treatment success of recurrent C. difficile infections and inflammatory bowel disease, but indicated a trend associated with an improved immune response in cancer patients. Concerningly, the transfer of an atypical and potentially pro-inflammatory microbial community from one donor was also observed, calling for further investigations into the immediate and long-term clinical consequences of FMT. These analyses demonstrate the advantages of a strain-based microbiome analysis. Due to the achieved methodological accuracy, strain-resolved microbial dynamics could be precisely disentangled when comparing longitudinal samples from healthy adults as well as parent-child and patient-donor pairs. This revealed taxonomic, clinical, and ecological factors that are critical to microbiome assembly, including microbial transmission, persistence, and competition. Together, these findings lay the groundwork for future developments of precision personalized microbiota modulation therapies.
Toll-like Receptor 9 (TLR9) activation and the innate immune response to microbial and human DNA
(2023) Hsu, Emily; Fricke, Florian W.
The human Toll-like Receptor 9 (TLR9) is an endosomal Pattern Recognition Receptor (PRR) that recognizes DNA sequences containing the unmethylated Cytosine-Guanine (CpG) dimers, which are present in greater abundance in most bacterial genomes compared to those of vertebrates. Specific CpG-containing sequences are strongly stimulatory of human TLR9, as shown in published studies using synthetic oligonucleotides (ODN) and DNA from bacterial species of varying genomic CpG concentration. Human TLR9 activation was experimentally examined in this thesis using DNA extracted from different bacterial sources, human DNA from Caco-2 cells, known immunostimulatory ODN, and short ODN. In vitro assays using fragment length-standardized microbial genomic DNA on HEK-Dual TLR9 Cells and human peripheral blood mononuclear cells (PBMCs) revealed that TLR9 activation strongly correlated to CpG concentration of the input DNA, with an additional influence of CpG-containing 5-mer TCGTT concentration. When DNA of varying origins and fragment lengths were used together, however, complex dynamics of TLR9 activation, co-activation, and repression were observed, which were less predictable than expected from genomic CpG concentration alone. DNase I-treated microbial DNA fragments of less than 15 bp of length were non-activating on their own, but co-activated human TLR9 together with ODN-2006 in Ramos Blue (B) cells. Similarly, human DNA fragments at the length of 50-200 bp co-activated human TLR9 with both ODN-2006 and Escherichia coli DNA in HEK-dual TLR9 cells. In contrast, large human DNA fragments at over 10000 bp of length repressed TLR9 activation by ODN-2006 in Ramos Blue cells. Finally, a preliminary study was conducted in HT-29 cells on the effect of TLR9 activation on the invasion of Fusobacterium nucleatum, an opportunistic gut pathogen with a very low genomic CpG concentration at 0.296%, using ODN-2006 and human DNA as TLR9 activators. While increased presence of intracellular Fusobacterium nucleatum upon treatment with both ODN-2006 and human DNA was noted, more studies are needed to confirm TLR9 activation as a cause of greater bacterial invasion. The human colon is the location of the largest microbial population of the human body, which provides a rich source of non-human DNA in contact with human TLR9 present in intestinal epithelial cells, plasmacytoid dendritic cells (pDCs), and B lymphocytes. Additionally, the daily mass shedding and death of human intestinal epithelial cells provide large amounts of human DNA, which when combined with microbial DNA could result in co-activation and possible autoimmunity. The thesis thus provided an in vitro model of TLR9 activation by complex DNA of varying origins and fragment lengths likely to present in the human gut environment, and prepared a working basis for future studies of TLR9 activation by human fecal metagenomic DNA.