applying-advanced-vitro-culturing-technology-to-study-human-gut

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

Children's Hospital of Philadelphia

Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.

Optimizing methods and dodging pitfalls in microbiome research.

The human cervicovaginal microbiota resides at an interface between the host and the environment and may affect susceptibility to disease. Puerto Rican women have high human papillomavirus (HPV) infection and cervical cancer rates. We hypothesized that the population structure of the cervicovaginal bacterial and fungal biota changed with cervical squamous intraepithelial lesions and HPV infections. DNA was extracted from cervix, introitus, and anal sites of 62 patients attending high-risk San Juan clinics. The 16S rRNA V4 region and ITS-2 fungal regions were amplified and sequenced using Illumina technology. HPV genotyping was determined by reverse hybridization with the HPV SPF10-LiPA25 kit. HPV prevalence was 84% of which ∼44% subjects were infected with high-risk HPV, ∼35% were co-infected with as many as 9 HPV types and ∼5% were infected with exclusively low-risk HPV types. HPV diversity did not change with cervical dysplasia. Cervical bacteria were more diverse in patients with CIN3 pre-cancerous lesions. We found enrichment of  and  in patients with CIN3 lesions. We found no significant bacterial biomarkers associated with HPV infections. Fungal diversity was significantly higher in cervical samples with high-risk HPV and introitus samples of patients with Atypical Squamous Cells of Undetermined Significance (ASCUS). Fungal biomarker signatures for vagina and cervix include Sporidiobolaceae and  for ASCUS, and  for high-risk HPV infections. Our combined data suggests that specific cervicovaginal bacterial and fungal populations are related to the host epithelial microenvironment, and could play roles in cervical dysplasia.

Cervicovaginal Fungi and Bacteria Associated With Cervical Intraepithelial Neoplasia and High-Risk Human Papillomavirus Infections in a Hispanic Population.

Host-commensal interactions are critical for the generation of robust inflammatory responses, yet the mechanisms leading to this effect remain poorly understood. Using a murine model of cytokine storm, we identified that host microbiota are required to sustain systemic TLR-driven immune responses. Mice treated with broad-spectrum antibiotics or raised in germ-free conditions responded normally to an initial TLR signal but failed to sustain production of proinflammatory cytokines following administration of repeated TLR signals in vivo. Mechanistically, host microbiota primed JAK signaling in myeloid progenitors to promote TLR-enhanced myelopoiesis, which is required for the accumulation of TLR-responsive monocytes. In the absence of TLR-enhanced monocytopoiesis, antibiotic-treated mice lost their ability to respond to repeated TLR stimuli and were protected from cytokine storm-induced immunopathology. These data reveal priming of TLR-enhanced myelopoiesis as a microbiota-dependent mechanism that regulates systemic inflammatory responses and highlight a role for host commensals in the pathogenesis of cytokine storm syndromes.

Kyle Bittinger

Division of Gastroenterology,
Hepatology,
and Nutrition,
Division of Gastroenterology, Hepatology, and Nutrition

Children's Hospital of Philadelphia

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

Kyle Bittinger

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Gastroenterology,Hepatology,and Nutrition,Division of Gastroenterology, Hepatology, and Nutrition

Children's Hospital of Philadelphia

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

Division of Gastroenterology,
Hepatology,
and Nutrition,
Division of Gastroenterology, Hepatology, and Nutrition