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Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.

Inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, affect several million individuals worldwide. Crohn's disease and ulcerative colitis are complex diseases that are heterogeneous at the clinical, immunological, molecular, genetic, and microbial levels. Individual contributing factors have been the focus of extensive research. As part of the Integrative Human Microbiome Project (HMP2 or iHMP), we followed 132 subjects for one year each to generate integrated longitudinal molecular profiles of host and microbial activity during disease (up to 24 time points each; in total 2,965 stool, biopsy, and blood specimens). Here we present the results, which provide a comprehensive view of functional dysbiosis in the gut microbiome during inflammatory bowel disease activity. We demonstrate a characteristic increase in facultative anaerobes at the expense of obligate anaerobes, as well as molecular disruptions in microbial transcription (for example, among clostridia), metabolite pools (acylcarnitines, bile acids, and short-chain fatty acids), and levels of antibodies in host serum. Periods of disease activity were also marked by increases in temporal variability, with characteristic taxonomic, functional, and biochemical shifts. Finally, integrative analysis identified microbial, biochemical, and host factors central to this dysregulation. The study's infrastructure resources, results, and data, which are available through the Inflammatory Bowel Disease Multi'omics Database ( http://ibdmdb.org ), provide the most comprehensive description to date of host and microbial activities in inflammatory bowel diseases.

Macrophages are specialized phagocytic cells involved in clearing invading pathogens. Previously we reported that engulfment and cell motility protein 1 (ELMO1) in macrophages mediates bacterial internalization and intestinal inflammation. Here we studied the role of ELMO1 in the fate of internalized targets. ELMO1 is present in the intracellular vesicles and enhances accumulation of the protein LC3B following engulfment of Salmonella or treatment with autophagy-inducing rapamycin. The protein ATG5 and the kinase ULK1 are involved in classical autophagy, while LC3-associated phagocytosis is ULK1 independent. ATG5 but not ULK1 cooperated with ELMO1 in LC3 accumulation after infection, suggesting the ELMO1 preferentially regulated LC3-associated phagocytosis. Because LC3-associated phagocytosis delivers cargo for degradation, the contribution of ELMO1 to the lysosome degradation pathways was evaluated by studying pH and cathepsin B activity. ELMO1-depleted macrophages showed a time-dependent increase in pH and a decrease in cathepsin B activity associated with bacterial survival. Together, ELMO1 regulates LC3B accumulation and antimicrobial responses involved in the clearance of enteric pathogens. This paper investigated how innate immune pathways involving ELMO1 work in a coordinated fashion to eliminate bacterial threats. ELMO1 is present in the phagosome and enhances bacterial clearance by differential regulation of lysosomal acidification and enzymatic activity.

To model how interactions among enteropathogens and gut microbial community members contribute to undernutrition, we colonized gnotobiotic mice fed representative Bangladeshi diets with sequenced bacterial strains cultured from the fecal microbiota of two 24-month-old Bangladeshi children: one healthy and the other underweight. The undernourished donor's bacterial collection contained an enterotoxigenic Bacteroides fragilis strain (ETBF), whereas the healthy donor's bacterial collection contained two nontoxigenic strains of B. fragilis (NTBF). Analyses of mice harboring either the unmanipulated culture collections or systematically manipulated versions revealed that ETBF was causally related to weight loss in the context of its native community but not when introduced into the healthy donor's community. This phenotype was transmissible from the dams to their offspring and was associated with derangements in host energy metabolism manifested by impaired tricarboxylic acid cycle activity and decreased acyl-coenzyme A utilization. NTBF reduced ETBF's expression of its enterotoxin and mitigated the effects of ETBF on the transcriptomes of other healthy donor community members. These results illustrate how intraspecific (ETBF-NTBF) and interspecific interactions influence the effects of harboring B. fragilis.

To gain insights into the interrelationships among childhood undernutrition, the gut microbiota, and gut mucosal immune/barrier function, we purified bacterial strains targeted by immunoglobulin A (IgA) from the fecal microbiota of two cohorts of Malawian infants and children. IgA responses to several bacterial taxa, including Enterobacteriaceae, correlated with anthropometric measurements of nutritional status in longitudinal studies. The relationship between IgA responses and growth was further explained by enteropathogen burden. Gnotobiotic mouse recipients of an IgA(+) bacterial consortium purified from the gut microbiota of undernourished children exhibited a diet-dependent enteropathy characterized by rapid disruption of the small intestinal and colonic epithelial barrier, weight loss, and sepsis that could be prevented by administering two IgA-targeted bacterial species from a healthy microbiota. Dissection of a culture collection of 11 IgA-targeted strains from an undernourished donor, sufficient to transmit these phenotypes, disclosed that Enterobacteriaceae interacted with other consortium members to produce enteropathy. These findings indicate that bacterial targets of IgA responses have etiologic, diagnostic, and therapeutic implications for childhood undernutrition.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Mammals are defined by their metagenome, a combination of host and microbiome genes. This knowledge presents opportunities to further basic biology with translation to human diseases. However, the now-documented influence of the metagenome on experimental results and the reproducibility of in vivo mammalian models present new challenges. Here we provide the scientific basis for calling on all investigators, editors and funding agencies to embrace changes that will enhance reproducible and interpretable experiments by accounting for metagenomic effects. Implementation of new reporting and experimental design principles will improve experimental work, speed discovery and translation, and properly use substantial investments in biomedical research., Recent studies highlight that differences in human genetics explain only a fraction of observed human phenotypic variation (1,2). Another well-recognized source of variation is the microbiome, which is the collection [...]

Mesenchymal stem cell (MSC) therapy is an emerging field of regenerative medicine; however, it is often unclear how these cells mediate repair. Here, we investigated the use of MSCs in the treatment of intestinal disease and modeled abnormal repair by creating focal wounds in the colonic mucosa of prostaglandin- deficient mice. These wounds developed into ulcers that infiltrated the outer intestinal wall. We determined that penetrating ulcer formation in this model resulted from increased hypoxia and smooth muscle wall necrosis. Prostaglandin [I.sub.2] ([PGI.sub.2]) stimulated VEGF-dependent angiogenesis to prevent penetrating ulcers. Treatment of mucosally injured WT mice with a VEGFR inhibitor resulted in the development of penetrating ulcers, further demonstrating that VEGF is critical for mucosal repair. We next used this model to address the role of transplanted colonic MSCs (cMSCs) in intestinal repair. Compared with intravenously injected cMSCs, mucosally injected cMSCs more effectively prevented the development of penetrating ulcers, as they were more efficiently recruited to colonic wounds. Importantly, mucosally injected cMSCs stimulated angiogenesis in a VEGF-dependent manner. Together, our results reveal that penetrating ulcer formation results from a reduction of local angiogenesis and targeted injection of MSCs can optimize transplantation therapy. Moreover, local MSC injection has potential for treating diseases with features of abnormal angiogenesis and repair., Introduction Mesenchymal stem cells (MSCs) are an enticing potential therapeutic agent for a variety of inflammatory conditions, including those that occur in the gastrointestinal tract. MSCs are currently being tested [...]

The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control (1-5). In many cases, the microbiota is the presumed cause of the variation. Current solutions to account for phenotypic variability include littermate and maternal controls or defined microbial consortia in gnotobiotic mice (6,7). In conventionally raised mice, the microbiome is transmitted from the dam (2,8,9). Here we show that microbially driven dichotomous faecal immunoglobulinA (IgA) levels in wild-type mice within the same facility mimic the effects of chromosomal mutations. We observe in multiple facilities that vertically transmissible bacteria in IgA-low mice dominantly lower faecal IgA levels in IgA-high mice after co-housing or faecal transplantation. In response to injury, IgA-low mice show increased damage that is transferable by faecal transplantation and driven by faecal IgA differences. We find that bacteria from IgA-low mice degrade the secretory component of secretory IgA as well as IgA itself. These data indicate that phenotypic comparisons between mice must take into account the non-chromosomal hereditary variation between different breeders. We propose faecal IgA as one marker of microbial variability and conclude that co-housing and/or faecal transplantation enables analysis of progeny from different dams., We chose to study the role of secretory immunoglobulin A (SIgA), which is a critical intersection between the host immune system and the microbiota (10). While interrogating baseline intestinal IgA [...]