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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 [...]

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production., Introduction It is widely believed that the innate immune system mediates the acute response to an infectious agent, but recent work shows that this response can also translate acute infection [...]

The instruction of the immune system to be tolerant of self, thereby preventing autoimmunity, is facilitated by the education of T cells in a specialized organ, the thymus, in which self-reactive cells are either eliminated or differentiated into tolerogenic [Foxp3.sup.+] regulatory T ([T.sub.reg]) cells (1). However, it is unknown whether T cells are also educated to be tolerant of foreign antigens, such as those from commensal bacteria, to prevent immunopathology such as inflammatory bowel disease (2-4). Here we show that encounter with commensal microbiota results in the peripheral generation of [T.sub.reg] cells rather than pathogenic effectors. We observed that colonic [T.sub.reg] cells used T-cell antigen receptors (TCRs) different from those used by [T.sub.reg] cells in other locations, implying an important role for local antigens in shaping the colonic [T.sub.reg]-cell population. Many of the local antigens seemed to be derived from commensal bacteria, on the basis of the in vitro reactivity of common colon [T.sub.reg] TCRs. These TCRs did not facilitate thymic [T.sub.reg]-cell development, implying that many colonic [T.sub.reg] cells arise instead by means of antigen-driven peripheral [T.sub.reg]-cell development. Further analysis of two of these TCRs by the creation of retroviral bone marrow chimaeras and a TCR transgenic line revealed that microbiota indigenous to our mouse colony was required for the generation of colonic [T.sub.reg] cells from otherwise naive T cells. If T cells expressing these TCRs fail to undergo [T.sub.reg]-cell development and instead become effector cells, they have the potential to induce colitis, as evidenced by adoptive transfer studies. These results suggest that the efficient peripheral generation of antigen-specific populations of [T.sub.reg] cells in response to an individual's microbiota provides important post-thymic education of the immune system to foreign antigens, thereby providing tolerance to commensal microbiota., Although [T.sub.reg] cells are required for the maintainenance of gut tolerance (3), commensal bacteria are not necessary for colonic [T.sub.reg]-cell generation (5,6 ) (Supplementary Fig. 1). Moreover, [T.sub.reg] cells from [...]

The process of wound repair in epithelium-lined organs of mammals is complex and is influenced by numerous secreted factors including cytokines, growth factors, and chemokines. However, the cellular organizers of this process are still not understood. Recent studies of tissue regeneration in organisms with simpler development have uncovered details about the activity of stem cells in the mesenchyme (the blastema) during this process. These blastemal cells are well positioned to interpret cues from the environment and to execute decisions about the direction of wound repair. In mammalian wounds, stromal stem cells appear to be positioned to perform functions similar to those of blastemal cells, including communication with both the overlying epithelium and the inflammatory cells in the mesenchyme.

The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkuhn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear [beta]-catenin-positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle. CDC25 phosphatases | cell cycle | stem cells

The colonic epithelial lining undergoes constant replacement, driven by epithelial stem cells in crypts of Lieberkuhn. Stem cells lost because of damage or disease can be replaced by adjacent crypts that undergo fission. The close proximity of an extraordinary number of luminal microbes creates a challenge for this repair process; infection must be prevented while immune system activation and epithelial stem cell genetic damage must be minimized, To understand the factors that modulate crypt/stem cell replacement in the mouse colon, we developed an in vivo acute injury system analogous to punch biopsy of the skin. In contrast to epidermal stem cells, colonic epithelial progenitors did not migrate over the wound bed. Instead, their proliferative expansion was confined to crypts adjacent to wound beds and was delayed to the latter phase of healing. This increased epithelial proliferation was coincident with the infiltration of Trem2 expressing macrophages and increased expression of IL-4 and IL-13 in the wound bed. Interestingly, [Trem2.sup.-/-] mice displayed slow and incomplete wound healing of colonic mucosal injuries. We found the latter phase of healing in [Trem2.sup.-/-] mice showed a diminished burst of epithelial proliferation, increased expression of IFN-[gamma], and TNF-[alpha], diminished expression of IL-4 and IL-13, and increased markers of classical macrophage activation. Ablation of these cytokines in injured WT and [Trem2.sup.-/-] mice demonstrated that their expression ultimately determined the rate and nature of wound healing. These studies show that Trem2 signaling is an important pathway to promote healing of wounds in the colon where stem cell replacement is necessary. biopsy injury | epithelium | macrophage | stem cell | wound healing

Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci (1). One Crohn's disease risk allele is in ATG16L1, a gene homologous [...]

Background The constellation of human inflammatory bowel disease (1131D) includes ulcerative colitis and Crohn's disease, which both display a wide spectrum in the severity of pathology. One theory is that multiple genetic hits to the host immune system may contribute to the susceptibility and severity of 1131D. However, experimental proof of this concept is still lacking. Several genetic mouse models that each recapitulate some aspects of human 1131D have utilized a single gene defect to induce colitis. However, none have produced pathology clearly distinguishable as either ulcerative colitis or Crohn's disease, in part because none of them reproduce the most severe forms of disease that are observed in human patients. This lack of severe 1131D models has posed a challenge for research into pathogenic mechanisms and development of new treatments. We hypothesized that multiple genetic hits to the regulatory machinery that normally inhibits immune activation in the intestine would generate more severe, reproducible pathology that would mimic either ulcerative colitis or Crohn's disease. Methods and Findings We generated a novel mouse line (dnKO) that possessed defects in both TGFβRII and IL-10R2 signaling. These mice rapidly and reproducibly developed a disease resembling fulminant human ulcerative colitis that was quite distinct from the much longer and more variable course of pathology observed previously in mice possessing only single defects. Pathogenesis was driven by uncontrolled production of proinflarnmatory cytokines resulting in large part from T cell activation. The disease process could be significantly ameliorated by administration of antibodies against IFNγ and TNFα and was completely inhibited by a combination of broad-spectrum antibiotics. Conclusions Here, we develop to our knowledge the first mouse model of fulminant ulcerative colitis by combining multiple genetic hits in immune regulation and demonstrate that the resulting disease is sensitive to both anticytokine therapy and broad-spectrum antibiotics. These findings indicated the IL-10 and TGFβ pathways synergize to inhibit microbially induced production of proinflarnmatory cytokines, including IFNγ and TNFα, which are known to play a role in the pathogenesis of human ulcerative colitis. Our findings also provide evidence that broad-spectrum antibiotics may have an application in the treatment of patients with ulcerative colitis. This model system will be useful in the future to explore the microbial factors that induce immune activation and characterize how these interactions produce disease. The Editors' Summary of this article follows the references., Introduction Inflammatory bowel disease JBD) is an idiopathic disease of the intestinal tract with the hallmark features of mucosal inflammation and loss of barrier function. Although the exact mechanisms for [...]

We identified cellular and molecular mechanisms within the stem cell niche that control the activity of colonic epithelial progenitors (ColEPs) during injury. Here, we show that while WT mice maintained [...]

Discs-large homolog 1 (DLGH1) is a mouse ortholog of the Drosophila discs-large (DLG) tumor suppressor protein, a founding member of the PDZ and MAGUK protein families. DLG proteins play important roles in regulating cell proliferation, epithelial cell polarity, and synapse formation and function. Here, we generated a null allele of Dlgh1 and studied its role in urogenital development. [Dlgh1.sup.-/-] mice developed severe urinary tract abnormalities, including congenital hydronephrosis, which is the leading cause of renal failure in infants and children. DLGH1 is expressed in the developing ureter; in its absence, the stromal cells that normally lie between the urothelial and smooth muscle layers were missing. Moreover, in ureteric smooth muscle, the circular smooth muscle cells were misaligned in a longitudinal orientation. These abnormalities in the ureter led to severely impaired ureteric peristalsis. Similar smooth muscle defects are observed frequently in patients with ureteropelvic junction obstruction, a common form of hydronephrosis. Our results suggest that (i) besides its well documented role in regulating epithelial polarity, Dlgh1 also regulates smooth muscle orientation, and (ii) human DLG1 mutations may contribute to hereditary forms of hydronephrosis. SAP97 | kidney | postsynaptic density-95/discs-large/zonula occludens-1 urogenital | sonic hedgehog

We have identified cellular and molecular features of the stem cell niche required for marked amplification of mouse colonic epithelial progenitors (ColEPs) that occurs in response to wounding of the epithelium with dextran sodium sulfate. This regenerative response in areas adjacent to breaches in the epithelial barrier depends on the gut microbiota because ColEP proliferation is markedly diminished in germ-free animals. Analysis of conventionally raised C57BL/6 (B6) knockout mice lacking the Toll-like receptor signal transduction pathway component Myd88 and wild-type animals transplanted with Myd[88.sup.-/-] bone marrow, revealed that Myd88-mediated signaling through mesenchymal cells is also required for the ColEP response. Studies of B6 Csf[1.sup.op/op] (lacking macrophages) mice, Rag[1.sup.-/-] mice, and wild-type mice treated with neutrophil-specific Gr1 mAbs, disclosed that macrophages but not lymphocytes or neutrophils are necessary. GeneChip analysis of laser-capture-microdissected mesenchymal cells coupled with immunohistochemical and electron microscopic studies showed that, during the regenerative response, macrophages in the pericryptal stem cell niche express genes associated with their activation and extend processes to directly contact ColEPs near the crypt base. GeneChip analysis also identified a number of potential molecular mediators of regeneration expressed in the pericryptal progenitor niche, including secreted factors that stimulate epithelial proliferation and proteins involved in extracellular matrix and basement membrane function, stability, and growth factor binding. Together, these studies indicate that the colonic epithelial progenitor niche is a dynamic structure in which macrophages function as mobile 'cellular transceivers' that coordinate inputs from luminal microbes and injured epithelium and transmit regenerative signals to neighboring ColEPs. laser capture microdissection | epithelial-mesenchymal cross-talk | macrophage activation | stem cell | stem cell niche

The adult mouse small intestinal epithelium undergoes perpetual regeneration, fueled by a population of multipotential stem cells and oligopotential daughters located at the base of crypts of Lieberkuhn. Although the morphologic features of small intestinal epithelial progenitors (SiEPs) are known, their molecular features are poorly defined. Previous impediments to purification and molecular characterization of SiEPs include lack of ex vivo clonigenic assays and the difficulty of physically retrieving them from their niche where they are interspersed between their numerous differentiated Paneth cell daughters. To overcome these obstacles, we used germ-free transgenic mice lacking Paneth cells to obtain a consolidated population of SiEPs with normal proliferative activity. These cells were harvested by laser capture microdissection. Functional genomics analysis identified 163 transcripts enriched in SiEPs compared with Paneth cell-dominated normal crypt base epithelium. The dataset was validated by (i) correlation with the organellar composition of SiEPs versus Paneth cells, (ii) similarities to databases generated from recent mouse hematopoietic and neural stem cell genome anatomy projects, and (iii) laser capture microdissection/real-time quantitative RT-PCR studies of progenitor cell-containing populations retrieved from the small intestines, colons, and stomachs of conventionally raised mice. The SiEP profile has prominent representation of genes involved in c-myc signaling and in the processing, localization, and translation of mRNAs. This dataset, together with our recent analysis of gene expression in the gastric stem cell niche, discloses a set of molecular features shared by adult mouse gut epithelial progenitors. stern cells | Paneth cells | c-myc | RNA binding proteins | laser capture microdissection

The adult mouse intestine contains an intricate vascular network. The factors that control development of this network are poorly understood. Quantitative three-dimensional imaging studies revealed that a plexus of branched interconnected vessels developed in small intestinal villi during the period of postnatal development that coincides with assembly of a complex society of indigenous gut microorganisms (microbiota). To investigate the impact of this environmental transition on vascular development, we compared the capillary networks of germ-free mice with those of ex-germ-free animals colonized during or after completion of postnatal gut development. Adult germ-free mice had arrested capillary network formation. The developmental program can be restarted and completed within 10 days after colonization with a complete microbiota harvested from conventionally raised mice, or with Bacteroides thetaiotaomicron, a prominent inhabitant of the normal mouse/human gut. Paneth cells in the intestinal epithelium secrete antibacterial peptides that affect luminal microbial ecology. Comparisons of germ-free and B. thetaiotaomicron-colonized transgenic mice lacking Paneth cells established that microbial regulation of angiogenesis depends on this lineage. These findings reveal a previously unappreciated mechanism of postnatal animal development, where microbes colonizing a mucosal surface are assigned responsibility for regulating elaboration of the underlying microvasculature by signaling through a bacteria-sensing epithelial cell. small intestine | gnotobiotics | ecology | symbiosis

The adult mouse gastric epithelium undergoes continuous renewal in discrete anatomic units. Lineage tracing studies have previously disclosed the morphologic features of gastric epithelial lineage progenitors (GEPs), including those of the presumptive multipotent stem cell. However, their molecular features have not been defined. Here, we present the results of an analysis of genes and pathways expressed in these cells. One hundred forty-seven transcripts enriched in GEPs were identified using an approach that did not require physical disruption of the stem cell niche. Real-time quantitative RT-PCR studies of laser capture microdissected cells retrieved from this niche confirmed enriched expression of a selected set of genes from the GEP list. An algorithm that allows quantitative comparisons of the functional relatedness of automatically annotated expression profiles showed that the GEP profile is similar to a dataset of genes that defines mouse hematopoietic stem cells, and distinct from the profiles of two differentiated GEP descendant lineages (parietal and zymogenic cell). Overall, our analysis revealed that growth factor response pathways are prominent in GEPs, with insulin-like growth factor appearing to play a key role. A substantial fraction of GEP transcripts encode products required for mRNA processing and cytoplasmic localization, including numerous homologs of Drosophila genes (e.g., Y14, staufen, mago nashi) needed for axis formation during oogenesis, mRNA targeting proteins may help these epithelial progenitors establish differential communications with neighboring cells in their niche. stem cells | growth factor signaling | protein turnover | mRNA localization | bioinformatics

The activity of full-length desmoplakin I (DP I) in ectopically expressed cultured cells is examined. Functional sequences in the carboxyl ending of DP I is divided. Alignment with and the hindrance of keratin IF, and not vimentin IF, need a sequence at the carboxyl ending of DP. Keratin and vimentin IF network react with DP in a differential manner.

The function of desmoplakins I and II (DP I and DP II) in the combination of desmosomal plaque with cytoplasmic intermediate filaments (IF) was examined using a molecular genetic technique which employs an array of mammalian expression vectors. Light and electron microscopy revealed the colocalization of DP I and DP II with IF which disrupts the latter. Immunogold techniques revealed that the IF subunits were confined in mutant proteins around the nucleus. Results of further studies imply the involvement of the carboxyl terminus of DP in the attachment of IF to desmosomes.

Defining molecular interactions that occur at the interface between 'normal' and 'abnormal' cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to 'control' crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts. mouse intestinal stem cells | genetic mosaic analysis