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Associations between the dynamic community of microbes (the microbiota) and the host they colonize appear to be vital for ensuring host health. Microbe-host communication is actively maintained across physiological barriers of various body sites and is mediated by a range of bidirectional secreted proteins and small molecules. So far, a range of “omics” methods have succeeded in revealing the multiplicity of associations between members of a microbiota and a wide range of host processes and diseases. Although these advances point to possibilities for treating disease, there has not been much translational success thus far. We know little about which organisms are key contributors to host health, the importance of strain differences, and the activities of much of the chemical “soup” that is produced by the microbiota. Adding to this complexity are emerging hints of the role of interkingdom interactions between bacteria, phages, protozoa, and/or fungi in regulating the microbiota-host interactions. Functional approaches, although experimentally challenging, could be the next step to unlocking the power of the microbiota. [ABSTRACT FROM AUTHOR]

• Microbial metabolites are increasingly recognized as a functional output of the microbiome that impacts both host physiology and immunity. • A broad array of components of the innate immune system are targeted by microbial metabolites including the intestinal epithelium. • Microbial metabolites effect on innate immunity is to modulate inflammation and infection. Numerous scientific disciplines, including immunology, are now positioned to fully realize the potential of the intestinal microbiome to modulate a wide array of basic processes. Increasingly, microbiota-derived metabolites are being recognized for mediating these effects. Coupled with advances in large scale sequencing and mass spectrometry, research into the microbiota and their metabolites has entered into an era of rapid discovery. Here, we review recent studies that have shown how-specific metabolic products of the microbiome alter properties of the innate immune system that in turn modulate response to infection and immunity. [ABSTRACT FROM AUTHOR]

A potential role for viral and bacterial–viral interactions in the pathogenesis of autoimmune disease has been long recognized. Recently, intensive investigation has begun to decipher interactions between specific microbes with the host that contribute toward autoimmunity. This work has primarily focused on known viral and bacterial pathogens. A major challenge is to determine the role of bacteria that are typically considered as commensals as well as chronic viruses. Furthermore, equally challenging is to prove causality given the potential complexity of microbe–microbe interactions. Important initial contributions to this field have shown that specific interactions of microbes with hosts that contain a background of genetic susceptibility can play a role in autoimmune pathogenesis. In this review, we describe principles of immune tolerance with a focus on its breakdown during pathogenic as well as commensal relationships between the host and the microbial world. [ABSTRACT FROM AUTHOR]

Tremendous advances have been made in mapping the complexity of the human gut microbiota in both health and disease states. These analyses have revealed that, rather than a constellation of individual species, a healthy microbiota comprises an interdependent network of microbes. The microbial and host interactions that shape both this network and the gastrointestinal environment are areas of intense investigation. Here we review emerging concepts of how microbial metabolic processes control commensal composition, invading pathogens, immune activation, and intestinal barrier function. We posit that all of these factors are critical for the maintenance of homeostasis and avoidance of overt inflammatory disease. A greater understanding of the underlying mechanisms will shed light on the pathogenesis of many diseases and guide new therapeutic interventions. [ABSTRACT FROM AUTHOR]

Highlights: [•] Germ-free mice demonstrate hypoplastic secondary immune organs, reduced T and B cells, and decreased innate responses. [•] Germ free mice demonstrate exaggerated responses to immunogens. [•] Specific commensal bacteria can stimulate Th1, Th17, and Treg responses. [•] Systemic and mucosal responses to bacteria and bacterial antigens are not necessarily the same. [•] Associations have been made between altered intestinal microbiota and risk of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Nutrient absorption is the basic function that drives mammalian intestinal biology. To facilitate nutrient uptake, the host's epithelial barrier is composed of a single layer of cells. This constraint is problematic, as a design of this type can be easily disrupted. The solution during the course of evolution was to add numerous host defense mechanisms that can help prevent local and systemic infection. These mechanisms include specialized epithelial cells that produce a physiochemical barrier overlying the cellular barrier, robust and organized adaptive and innate immune cells, and the ability to mount an inflammatory response that is commensurate with a specific threat level. The autophagy pathway is a critical cellular process that strongly influences all these functions. Therefore, a fundamental understanding of the components of this pathway and their influence on inflammation, immunity, and barrier function will facilitate our understanding of homeostasis in the gastrointestinal tract. INSET: SUMMARY POINTS. [ABSTRACT FROM AUTHOR]

This review explores the recent advances that have been made in our understanding of host viral interactions in the intestine. Technical advances have allowed the initial definition of intestinal viromes in a number of species including humans. Important advances in our knowledge of the host response to viral infection have shown that interferon lambda has a role that is unique from type I interferons in the intestine. Lastly, our understanding of virally induced phenotypes has expanded through new studies that show bacteria can play an important role in the outcome of viral infection in the intestine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have sequenced 36,641 expressed sequence tags from laser capture microdissected adult mouse gastric and small intestinal epithelial progenitors, obtaining 4031 and 3324 unique transcripts, respectively. Using Gene Ontology (GO) terms, each data set was compared with cDNA libraries from intact adult stomach and small intestine. Genes in GO categories enriched in progenitors were filtered against genes in GO categories represented in hematopoietic, neural, and embryonic stem cell transcriptomes and mapped onto transcription factor networks, plus canonical signal transduction and metabolic pathways. Wnt/β-catenin, phosphoinositide-3/Akt kinase, insulin-like growth factor-1, vascular endothelial growth factor, integrin, and γ-aminobutyric acid receptor signaling cascades, plus glycerolipid, fatty acid, and amino acid metabolic pathways are among those prominently represented in adult gut progenitors. The results reveal shared as well as distinctive features of adult gut stem cells when compared with other stem cell populations. [ABSTRACT FROM AUTHOR]

Although angiogenins have been implicated in tumor-associated angiogenesis, their normal physiologic function remains unclear. We show that a previously uncharacterized angiogenin, Ang4, is produced by mouse Paneth cells, is secreted into the gut lumen and has bactericidal activity against intestinal microbes. Ang4 expression is induced by Bacteroides thetaiotaomicron, a predominant member of the gut microflora, revealing a mechanism whereby intestinal commensal bacteria influence gut microbial ecology and shape innate immunity. Furthermore, mouse Angl and human angiogenin, circulating proteins induced during inflammation, exhibit microbicidal activity against systemic bacterial and fungal pathogens, suggesting that they contribute to systemic responses to infection. These results establish angiogenins as a family of endogenous antimicrobial proteins. [ABSTRACT FROM AUTHOR]

The adult mouse small intestinal epithelium undergoes perpetual regeneration, fueled by a population of multipotentiai stem cells and oligopotential daughters located at the base of crypts of Lieberkühn. 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 ce!l-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. [ABSTRACT FROM AUTHOR]

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-germfree 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. [ABSTRACT FROM AUTHOR]

Microbially derived metabolites in the intestine regulate host immunity and impact disease pathophysiology in various organs. Sun et al. (2015) suggest a direct effect of microbial metabolites on pancreatic endocrine cells in regulating type 1 diabetes pathophysiology. [ABSTRACT FROM AUTHOR]

Investigates the behavior of ectopically expressed full-length desmoplakin I (DP I) in cultured cells. Assembly of cDNA encoding full-length DP I from overlapping phage clones; Necessity of sequences in the amino terminus of DP for colocalization with desmosomes; Importance of carboxyl terminus of DP for desmosomes and intermediate filament.

The importance of the gut microbiota has been recognized since the days of Pasteur. What makes today different from yesterday, and tomorrow so exciting, is that we now have the tools to identify the molecular mechanisms that regulate assembly of the microbiota and determine how its components affect postnatal mammalian development and adult physiology. [Copyright &y& Elsevier]

The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations (the crypts of Lieberkühn) that are continuous with evaginations (villi) into the lumen. The colon also contains crypts of Lieberkühn, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the laminin family. The basement membranes of the small intestine and colon contain distinct laminin subtypes; notably, the villus basement membrane is rich in laminin {alpha}5. Here, we show that the diminution of laminin {alpha}5 in a mouse model led to a compensatory deposition of colonic laminins, which resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1 - a cell-cycle regulator - and altered intestinal epithelial cell proliferation, migration and differentiation. Our results suggest that laminin {alpha}5 has a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (FGF) signaling mediates reciprocal mesenchymal-epithelial cell interactions in the developing mouse lung and limb. In the gastrointestinal (GI) tract, FGF10 is expressed in the cecal mesenchyme and signals to an epithelial splice form of FGF receptor (FGFR) 2 to regulate epithelial budding. Here, we identify FGF9 as a reciprocal epithelial-mesenchymal signal required for cecal morphogenesis. Fgf9 null (Fgf9-/-) mouse embryos have agenesis of the embryonic cecum, lacking both mesenchymal expansion and an epithelial bud. In the cecal region of Fgf9-/- embryos, mesenchymal expression of Fgf10 and Bmp4 is notably absent, whereas the expression of epithelial markers, such as sonic hedgehog, is not affected. Using epithelial and whole explant cultures, we show that FGF9 signals to mesenchymal FGFRs and that FGF10 signals to epithelial FGFRs. Taken together, these data show that an epithelial FGF9 signal is necessary for the expansion of cecal mesenchyme and the expression of mesenchymal genes that are required for epithelial budding. Thus, these data add to our understanding of FGF-mediated reciprocal epithelial-mesenchymal signaling. [ABSTRACT FROM AUTHOR]

Loss of differentiated cells to tissue damage is a hallmark of many diseases. In slow-turnover tissues, long-lived differentiated cells can re-enter the cell cycle or transdifferentiate to another cell type to promote repair. Here, we show that in a high-turnover tissue, severe damage to the differentiated compartment induces progenitors to transiently acquire a unique transcriptional and morphological postmitotic state. We highlight this in an acute villus injury model in the mouse intestine, where we identified a population of progenitor-derived cells that covered injured villi. These atrophy-induced villus epithelial cells (aVECs) were enriched for fetal markers but were differentiated and lineage committed. We further established a role for aVECs in maintaining barrier integrity through the activation of yes-associated protein (YAP). Notably, loss of YAP activity led to impaired villus regeneration. Thus, we define a key repair mechanism involving the activation of a fetal-like program during injury-induced differentiation, a process we term "adaptive differentiation." [Display omitted] • Epithelial dynamics in human enteropathy captured in an acute gastroenteritis model • Villus injury triggers TA cells to differentiate and acquire a fetal-like state • Adaptive epithelial differentiation restores barrier integrity • Proper adaptive differentiation is required for rebuilding damaged villi Ohara et al. demonstrate that, following injury, intestinal villus cells transiently adopt a unique differentiated state that incorporates a fetal program. Cells in this differentiated state can quickly re-establish the epithelial barrier to help restore the villus architecture. [ABSTRACT FROM AUTHOR]

Complex microbial populations are organized in relation to their environment. In the intestine, the inner lining (mucosa) is a potential focal point for such organization. The proximal murine colon contains mucosal folds that are known to be associated with morphologically distinct microbes. To identify these microbes, we used the technique of laser capture microdissection (LCM) to sample microbes associated with these folds (interfold region) and within the central lumen (digesta region). Using 16S rRNA gene tag pyrosequencing, we found that microbes in the interfold region were highly enriched for the phylum Firmicutes and, more specifically, for the families Lachnospiraceae and Ruminococcaceae. Other families such as Bacteroidaceae, Enterococcaceae and Lactobacillaceae were all enriched in the digesta region. This high-resolution system to capture and examine spatial organization of intestinal microbes should facilitate microbial analysis in other mouse models, furthering our understanding of host-microbial interactions. [ABSTRACT FROM AUTHOR]

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 ± 100 cal mol[sup -1] K[sup -1] and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 °C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol[sup -1] more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitots. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics. [ABSTRACT FROM AUTHOR]

Alterations of the mycobiota composition associated with Crohn’s disease (CD) are challenging to link to defining elements of pathophysiology, such as poor injury repair. Using culture-dependent and -independent methods, we discovered that Debaryomyces hansenii preferentially localized to and was abundant within incompletely healed intestinal wounds of mice and inflamed mucosal tissues of CD human subjects. D. hansenii cultures from injured mice and inflamed CD tissues impaired colonic healing when introduced into injured conventionally raised or gnotobiotic mice. We reisolated D. hansenii from injured areas of these mice, fulfilling Koch’s postulates. Mechanistically, D. hansenii impaired mucosal healing through the myeloid cell–specific type 1 interferon–CCL5 axis. Taken together, we have identified a fungus that inhabits inflamed CD tissue and can lead to dysregulated mucosal healing. [ABSTRACT FROM AUTHOR]

The transcription factor BHLHE40 is an emerging regulator of the immune system. Recent studies suggest that BHLHE40 regulates type 2 immunity, but this has not been demonstrated in vivo. We found that BHLHE40 is required in T cells for a protective TH2 cell response in mice infected with the helminth Heligmosomoides polygyrus bakeri. H. polygyrus elicited changes in gene and cytokine expression by lamina propria CD4+ T cells, many of which were BHLHE40 dependent, including production of the common b (CSF2RB) chain family cytokines GM-CSF and IL-5. In contrast to deficiency in GM-CSF or IL-5 alone, loss of both GM-CSF and IL-5 signaling impaired protection against H. polygyrus. Overall, we show that BHLHE40 regulates the TH2 cell transcriptional program during helminth infection to support normal expression of Csf2, Il5, and other genes required for protection and reveal unexpected redundancy of common β chain-dependent cytokines previously thought to possess substantially divergent functions. [ABSTRACT FROM AUTHOR]

Bacteria express multiple diverse capsular polysaccharides (CPSs) for protection against environmental and host factors, including the host immune system. Using a mouse TCR transgenic CD4+ T cell, BuOM, that is specific for B. thetaiotaomicron and a complete set of single CPS-expressing B. thetaiotaomicron strains, we ask whether CPSs can modify the immune responses to specific bacterial Ags. Acapsular B. thetaiotaomicron, which lacks all B. thetaiotaomicron CPSs, stimulated BuOM T cells more strongly than wild-type B. thetaiotaomicron. Despite similar levels of BuOM Ag expression, many single CPS-expressing B. thetaiotaomicron strains were antistimulatory and weakly activated BuOM T cells, but a few strains were prostimulatory and strongly activated BuOM T cells just as well or better than an acapsular strain. B. thetaiotaomicron strains that expressed an antistimulatory CPS blocked Ag delivery to the immune system, which could be rescued by Fc receptor-dependent Ab opsonization. All single CPS- expressing B. thetaiotaomicron strains stimulated the innate immune system to skew toward M1 macrophages and release inflammatory cytokines in an MyD88-dependent manner, with antistimulatory CPS activating the innate immune system in a weaker manner than prostimulatory CPS. The expression of antistimulatory versus prostimulatory CPSs on outer membrane vesicles also regulated immune responses. Moreover, antistimulatory and prostimulatory single CPS-expressing B. thetaiotaomicron strains regulated the activation of Ag-specific and polyclonal T cells as well as clearance of dominant Ag in vivo. These studies establish that the immune responses to specific bacterial Ags can be modulated by a diverse set of CPSs. [ABSTRACT FROM AUTHOR]

We recently demonstrated that cellular responses to butyrate depend on the differentiation status of the colonic epithelium. Here, we apply the implications of these findings to cancer biology and discuss discrepancies in the effects of butyrate on cancer progression. [ABSTRACT FROM AUTHOR]

Epithelial cells that line lung airways produce and secrete proteins with important roles in barrier function and host defense. Secretion of airway goblet cells is controlled by autophagy proteins during inflammatory conditions, resulting in accumulation of mucin proteins. We hypothesized that autophagy proteins would also be important in the function of club cells, dominant secretory airway epithelial cells that are dysregulated in chronic lung disease. We found that in the absence of an inflammatory stimulus, mice with club cells deficient for the autophagy protein Atg5 had a markedly diminished expression of secreted host defense proteins secretoglobulin family 1A, member 1 (Scgb1a1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology. Adult mice with targeted loss of Atg5 also showed diminished levels of host defense proteins in regenerating cells following ablation with naphthalene. A mouse strain with global deficiency of Atg16-like 1 (Atg16l1), an Atg5 binding partner, had a similar loss of host defense proteins and abnormal club cell morphology. Cigarette smoke exposure reduced levels of Scgb1a1 in wild-type mice as expected. Smoke exposure was not required to trigger club cell abnormalities in mice bearing the human ATG16 variant Atg16l1T300A/T300A, which had low Scgb1a1 levels independent of this environmental stress. Evaluation of lung tissues from former smokers with severe chronic obstructive pulmonary disease showed evidence of reduced autophagy and SCGB1A1 expression in club cells. Thus, autophagy proteins are required for the function of club cells, independent of the cellular stress of cigarette smoke, with roles that appear to be distinct from those of other secretory cell types. [ABSTRACT FROM AUTHOR]

Transplantable kidneys are in very limited supply. Accurate viability assessment prior to transplantation could minimize organ discard. Rapid and accurate evaluation of intra-operative donor kidney biopsies is essential for determining which kidneys are eligible for transplantation. The criterion for accepting or rejecting donor kidneys relies heavily on pathologist determination of the percent of glomeruli (determined from a frozen section) that are normal and sclerotic. This percentage is a critical measurement that correlates with transplant outcome. Inter- and intra-observer variability in donor biopsy evaluation is, however, significant. An automated method for determination of percent global glomerulosclerosis could prove useful in decreasing evaluation variability, increasing throughput, and easing the burden on pathologists. Here, we describe the development of a deep learning model that identifies and classifies non-sclerosed and sclerosed glomeruli in whole-slide images of donor kidney frozen section biopsies. This model extends a convolutional neural network (CNN) pre-trained on a large database of digital images. The extended model, when trained on just 48 whole slide images, exhibits slide-level evaluation performance on par with expert renal pathologists. Encouragingly, the model’s performance is robust to slide preparation artifacts associated with frozen section preparation. The model substantially outperforms a model trained on image patches of isolated glomeruli, in terms of both accuracy and speed. The methodology overcomes the technical challenge of applying a pretrained CNN bottleneck model to whole-slide image classification. The traditional patch-based approach, while exhibiting deceptively good performance classifying isolated patches, does not translate successfully to whole-slide image segmentation in this setting. As the first model reported that identifies and classifies normal and sclerotic glomeruli in frozen kidney sections, and thus the first model reported in the literature relevant to kidney transplantation, it may become an essential part of donor kidney biopsy evaluation in the clinical setting. [ABSTRACT FROM AUTHOR]

It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding preclinical models with the same G+E combinations is useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full-thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including proapoptosis, metabolic dysregulation, and selective downregulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease-relevant phenotype and provides rational strategies for identifying actionable targets. [ABSTRACT FROM AUTHOR]

Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract remains the major cause of morbidity and nonrelapse mortality after BM transplantation (BMT). The Paneth cell protein regenerating islet-derived 3α (REG3α) is a biomarker specific for GI GVHD. REG3α serum levels rose in the systematic circulation as GVHD progressively destroyed Paneth cells and reduced GI epithelial barrier function. Paradoxically, GVHD suppressed intestinal REG3γ (the mouse homolog of human REG3α), and the absence of REG3γ in BMT recipients intensified GVHD but did not change the composition of the microbiome. IL-22 administration restored REG3γ production and prevented apoptosis of both intestinal stem cells (ISCs) and Paneth cells, but this protection was completely abrogated in Reg3g-/- mice. In vitro, addition of REG3α reduced the apoptosis of colonic cell lines. Strategies that increase intestinal REG3α/γ to promote crypt regeneration may offer a novel, nonimmunosuppressive approach for GVHD and perhaps for other diseases involving the ISC niche, such as inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

We recently demonstrated that cellular responses to butyrate depend on the differentiation status of the colonic epithelium. Here, we apply the implications of these findings to cancer biology and discuss discrepancies in the effects of butyrate on cancer progression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The microbiota is known to modulate the host response to influenza infection through as-yet-unclear mechanisms. We hypothesized that components of the microbiota exert effects through type I interferon (IFN), a hypothesis supported by analysis of influenza in a gain-of-function genetic mouse model. Here we show that a microbially associated metabolite, desaminotyrosine (DAT), protects from influenza through augmentation of type I IFN signaling and diminution of lung immunopathology. A specific humanassociated gut microbe, Clostridium orbiscindens, produced DATand rescued antibiotictreated influenza-infected mice. DAT protected the host by priming the amplification loop of type I IFN signaling. These findings show that specific components of the enteric microbiota have distal effects on responses to lethal infections through modulation of type I IFN. [ABSTRACT FROM AUTHOR]

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound-associated epithelial ( WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 ( PGE2) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β-catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE2-Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury. [ABSTRACT FROM AUTHOR]

The organization of the lining of the gastrointestinal tube is similar for the stomach, small intestine and colon. It is composed of a continuous simple epithelium and underlying mesenchyme, including blood vessels, fibroblasts/myofibroblasts and immune cells. Individual organs contain modifications of this mucosa that are critical for their functions. We hypothesize that changes in the composition of the epithelial basement membrane are a critical determinant for structure and function of the local mucosa. Laminins are excellent candidates for such a role, as they comprise a large family of αβγ heterotrimeric glycoproteins that demonstrate developmental, organ and cell type specificity. We have found that adult mice lacking the normal villus laminin, α5, demonstrate a compensatory increase in α1 and α4, which are normally present in the colon. This laminin switch results in colonic metaplasia of the distal small intestine, which showed deepened crypts, villus atrophy and subsequent replacement by flat surface epithelial structures, and expression of colonic markers. This change was associated with aberrant intestinal epithelial cell proliferation, differentiation and migration. Here, unlike pathologic processes in humans that result in colonic metaplasia, there was no infiltration of inflammatory cells. These observations highlight the important and distinct roles of different laminin isoforms on the patterning of intestinal crypt-villus architecture and the regulation of intestinal epithelial cell behavior. Our data also suggest that differences in laminin composition along the gut's rostrocaudal axis contribute to its regionalization into small and large intestine. [ABSTRACT FROM AUTHOR]

Summary In the mammalian intestine, crypts of Leiberkühn house intestinal epithelial stem/progenitor cells at their base. The mammalian intestine also harbors a diverse array of microbial metabolite compounds that potentially modulate stem/progenitor cell activity. Unbiased screening identified butyrate, a prominent bacterial metabolite, as a potent inhibitor of intestinal stem/progenitor proliferation at physiologic concentrations. During homeostasis, differentiated colonocytes metabolized butyrate likely preventing it from reaching proliferating epithelial stem/progenitor cells within the crypt. Exposure of stem/progenitor cells in vivo to butyrate through either mucosal injury or application to a naturally crypt-less host organism led to inhibition of proliferation and delayed wound repair. The mechanism of butyrate action depended on the transcription factor Foxo3. Our findings indicate that mammalian crypt architecture protects stem/progenitor cell proliferation in part through a metabolic barrier formed by differentiated colonocytes that consume butyrate and stimulate future studies on the interplay of host anatomy and microbiome metabolism. [ABSTRACT FROM AUTHOR]

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to highdose etoposide. Nearly all SI stemcells were lost in fedmice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations,marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy. [ABSTRACT FROM AUTHOR]

Subsets of innate lymphoid cells (ILCs) reside in the mucosa and regulate immune responses to external pathogens. While ILCs can be phenotypically classified into ILC1, ILC2 and ILC3 subsets, the transcriptional control of commitment to each ILC lineage is incompletely understood. Here we report that the transcription factor Runx3 was essential for the normal development of ILC1 and ILC3 cells but not of ILC2 cells. Runx3 controlled the survival of ILC1 cells but not of ILC3 cells. Runx3 was required for expression of the transcription factor RORγt and its downstream target, the transcription factor AHR, in ILC3 cells. The absence of Runx3 in ILCs exacerbated infection with Citrobacter rodentium. Therefore, our data establish Runx3 as a key transcription factor in the lineage-specific differentiation of ILC1 and ILC3 cells. [ABSTRACT FROM AUTHOR]

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 I2 (PGI2) 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. [ABSTRACT FROM AUTHOR]

The DNA methyltransferase Dnmt1 maintains DNA methylation patterns and genomic stability in several in vitro cell systems. Ablation of Dnmt1 in mouse embryos causes death at the postgastrulation stage; however, the functions of Dnmt1 and DNA methylation in organogenesis remain unclear. Here, we report that Dnmt1 is crucial during perinatal intestinal development. Loss of Dnmt1 in intervillus progenitor cells causes global hypomethylation, DNA damage, premature differentiation, apoptosis and, consequently, loss of nascent villi.We further confirmthe crucial role of Dnmt1 during crypt development using the in vitro organoid culture system, and illustrate a clear differential requirement for Dnmt1 in immature versus mature organoids. These results demonstrate an essential role for Dnmt1 in maintaining genomic stability during intestinal development and the establishment of intestinal crypts. [ABSTRACT FROM AUTHOR]

Objective: The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host-microbial interactions). Design: We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results: We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3-1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusions: This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host-microbial interactions [ABSTRACT FROM AUTHOR]

The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control. 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. In conventionally raised mice, the microbiome is transmitted from the dam. Here we show that microbially driven dichotomous faecal immunoglobulin-A (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. [ABSTRACT FROM AUTHOR]

Summary Decreases in the diversity of enteric bacterial populations are observed in patients with Crohn’s disease (CD) and ulcerative colitis (UC). Less is known about the virome in these diseases. We show that the enteric virome is abnormal in CD and UC patients. In-depth analysis of preparations enriched for free virions in the intestine revealed that CD and UC were associated with a significant expansion of Caudovirales bacteriophages. The viromes of CD and UC patients were disease and cohort specific. Importantly, it did not appear that expansion and diversification of the enteric virome was secondary to changes in bacterial populations. These data support a model in which changes in the virome may contribute to intestinal inflammation and bacterial dysbiosis. We conclude that the virome is a candidate for contributing to, or being a biomarker for, human inflammatory bowel disease and speculate that the enteric virome may play a role in other diseases. [ABSTRACT FROM AUTHOR]

IL-6 is a pleiotropic cytokine often associated with inflammation. Inhibition of this pathway has led to successful treatment of rheumatoid arthritis, but one unforeseen potential complication of anti-IL-6 therapy is bowel perforation. Within the intestine, IL-6 has been shown to prevent epithelial apoptosis during prolonged inflammation. The role of IL-6 in the intestine during an initial inflammatory insult is unknown. Here, we evaluate the role of IL-6 at the onset of an inflammatory injury. Using two murine models of bowel injury – wound by biopsy and bacterial triggered colitis – we demonstrated that IL-6 is induced soon after injury by multiple cell types including intraepithelial lymphocytes. Inhibition of IL-6 resulted in impaired wound healing due to decreased epithelial proliferation. Using intestinal tissue obtained from patients who underwent surgical resection of the colon due to traumatic perforation, we observed cells with detectable IL-6 within the area of perforation and not at distant sites. Our data demonstrate the important role of IL-6 produced in part by intraepithelial lymphocytes at the onset of an inflammatory injury for epithelial proliferation and wound repair. [ABSTRACT FROM AUTHOR]

Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi-vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3-positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3-positive vacuole-associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion. [ABSTRACT FROM AUTHOR]

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4+ and NKp46+ innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b+ cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103+ cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b+ cDCs in the response to pathogens in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]