SUMMARY Regulation of intestinal dietary fat absorption is critical to maintaining energy balance. While intestinal microbiota clearly impact the host’s energy balance, their role in intestinal absorption and extra-intestinal metabolism of dietary fat is less clear. Using in vivo imaging of fluorescent fatty acid (FA) analogs delivered to gnotobiotic zebrafish hosts, we reveal that microbiota stimulate FA uptake and lipid droplet (LD) formation in the intestinal epithelium and liver. Microbiota increase epithelial LD number in a diet-dependent manner. The presence of food led to the intestinal enrichment of bacteria from the phylum Firmicutes. Diet-enriched Firmicutes and their products were sufficient to increase epithelial LD number, whereas LD size was increased by other bacterial types. Thus, different members of the intestinal microbiota promote FA absorption via distinct mechanisms. Diet-induced alterations in microbiota composition might influence fat absorption, providing mechanistic insight into how microbiota-diet interactions regulate host energy balance.

Background & Aims The nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) transcription factor pathway is activated in response to diverse microbial stimuli to regulate expression of genes involved in immune responses and tissue homeostasis. However, the temporal and spatial activation of NF-κB in response to microbial signals have not been determined in whole living organisms, and the molecular and cellular details of these responses are not well understood. We used in vivo imaging and molecular approaches to analyze NF-κB activation in response to the commensal microbiota in transparent gnotobiotic zebrafish. Methods We used DNA microarrays, in situ hybridization, and quantitative reverse transcription PCR analyses to study the effects of the commensal microbiota on gene expression in gnotobiotic zebrafish. Zebrafish PAC2 and ZFL cells were used to study the NF-κB signaling pathway in response to bacterial stimuli. We generated transgenic zebrafish that express enhanced green fluorescent protein under transcriptional control of NF-κB, and used them to study patterns of NF-κB activation during development and microbial colonization. Results Bacterial stimulation induced canonical activation of the NF-κB pathway in zebrafish cells. Colonization of germ-free transgenic zebrafish with a commensal microbiota activated NF-κB and led to up-regulation of its target genes in intestinal and extra-intestinal tissues of the digestive tract. Colonization with the bacterium Pseudomonas aeruginosa was sufficient to activate NF-κB, and this activation required a functional flagellar apparatus. Conclusions In zebrafish, transcriptional activity of NF-κB is spatially and temporally regulated by specific microbial factors. The observed patterns of NF-κB-dependent responses to microbial colonization indicate that cells in the gastrointestinal tract respond robustly to the microbial environment.

BACKGROUND & AIMS Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. METHODS Germ-free, wild-type, and Il10−/− mice and germ free zebrafish embryos were colonized with pathogen-free (SPF). Germ-free or SPF-raised wild-type and Il10−/− mice were given intraperitoneal injections of cobalt protoporphyrin (CoPP), which upregulates HO-1, the CO releasing molecule ALF186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with S. typhimurium. RESULTS In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of Nrf2–, IL-10–, and toll-like receptor–dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10−/− mice. Administration of CoPP to Il10−/− mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10−/− mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes (MLN). In mice with S. typhimurium-induced enterocolitis, CoPP reduced the numbers of live S. typhimurium recovered from the lamina propria, MLN, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E. coli, E. faecalis, and S. typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. CONCLUSIONS Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.

Epigenetic enzymes regulate higher-order chromatin architecture and cell-type specific gene expression. The ATPase BRG1 and the SWI/SNF chromatin remodeling complex are epigenetic enzymes that regulate chromatin accessibility during steady and transitional cell states. Experiments in mice show that the loss of BRG1 inhibits cellular reprogramming, while studies using human cells demonstrate that the overexpression of BRG1 enhances reprogramming. We hypothesized that the variation of SWI/SNF subunit expression in the human population would contribute to variability in the efficiency of induced pluripotent stem cells (iPSC) reprogramming. To examine the impact of an individual's sex, ancestry, and age on iPSC reprogramming, we created a novel sex and ancestry balanced cohort of 240 iPSC lines derived from human dermal fibroblasts (DF) from 80 heathy donors. We methodically assessed the reprogramming efficiency of each DF line and then quantified the individual and demographic-specific variations in SWI/SNF chromatin remodeling proteins and mRNA expression. We identified BRG1, BAF155, and BAF60a expression as strongly correlating with iPSC reprogramming efficiency. Additionally, we discovered that high efficiency iPSC reprograming is negatively correlated with donor age, positively correlated with African American descent, and uncorrelated with donor sex. These results show the variations in chromatin remodeling protein expression have a strong impact on iPSC reprogramming. Additionally, our cohort is unique in its large size, diversity, and focus on healthy donors. Consequently, this cohort can be a vital tool for researchers seeking to validate observational results from human population studies and perform detailed mechanistic studies in a controlled cell culture environment. Stem Cells 2018;36:1697-1708.

Induced pluripotent stem cells (iPSCs) can be derived from differentiated cells, enabling the generation of personalized disease models by differentiating patient-derived iPSCs into disease-relevant cell lines. While genetic variability between different iPSC lines affects differentiation potential, how this variability in somatic cells affects pluripotent potential is less understood. We generated and compared transcriptomic data from 72 dermal fibroblast–iPSC pairs with consistent variation in reprogramming efficiency. By considering equal numbers of samples from self-reported African Americans and White Americans, we identified both ancestry-dependent and ancestry-independent transcripts associated with reprogramming efficiency, suggesting that transcriptomic heterogeneity can substantially affect reprogramming. Moreover, reprogramming efficiency–associated genes are involved in diverse dynamic biological processes, including cancer and wound healing, and are predictive of 5-year breast cancer survival in an independent cohort. Candidate genes may provide insight into mechanisms of ancestry-dependent regulation of cell fate transitions and motivate additional studies for improvement of reprogramming.