Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation.

OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1b iΔIEC and Rac1 iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.

γδ Intraepithelial Lymphocytes Facilitate Pathological Epithelial Cell Shedding Via CD103-Mediated Granzyme Release.

BACKGROUND & AIMS: Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) are actively involved in the shedding of enterocytes into the lumen. METHODS: Intravital microscopy was performed on GFP γδ T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated γδ IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for γδ T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. RESULTS: Intravital microscopy of lipopolysaccharide-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by γδ IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of γδ IEL localization to CC3-positive enterocytes is increased in Crohn's disease biopsies compared with healthy controls. CONCLUSIONS: Our results uncover a previously unrecognized role for γδ IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular granzyme release.

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

The early life microbiota protects neonatal mice from pathological small intestinal epithelial cell shedding.

The early life gut microbiota plays a crucial role in regulating and maintaining the intestinal barrier, with disturbances in these communities linked to dysregulated renewal and replenishment of intestinal epithelial cells. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period, and which host and microbial factors mediate these responses. Surprisingly, neonatal mice (Day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of liposaccharide (LPS), with Day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses. When we profiled microbiota and metabolites, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia, and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes, and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Antibiotic treatment of neonates restored LPS-induced small intestinal cell shedding, whereas adult fecal microbiota transplant alone had no effect. Our findings further support the importance of the early life window for microbiota-epithelial interactions in the presence of inflammatory stimuli and highlights areas for further investigation.

Non-classical monocyte homing to the gut via α4ß7 integrin mediates macrophage-dependent intestinal wound healing.

OBJECTIVE: To study the role of α4ß7 integrin for gut homing of monocytes and to explore the biological consequences of therapeutic α4ß7 inhibition with regard to intestinal wound healing. DESIGN: We studied the expression of homing markers on monocyte subsets in the peripheral blood and on macrophage subsets in the gut of patients with IBD and controls with flow cytometry and immunohistochemistry. Integrin function was addressed with dynamic adhesion assays and in vivo gut homing assays. In vivo wound healing was studied in mice deficient for or depleted of α4ß7 integrin. RESULTS: Classical and non-classical monocytes were clearly dichotomous regarding homing marker expression including relevant expression of α4ß7 integrin on human and mouse non-classical monocytes but not on classical monocytes. Monocyte-expressed α4ß7 integrin was functionally important for dynamic adhesion to mucosal vascular addressin cell adhesion molecule 1 and in vivo gut homing. Impaired α4ß7-dependent gut homing was associated with reduced (effect size about 20%) and delayed wound healing and suppressed perilesional presence of wound healing macrophages. Non-classical monocytes in the peripheral blood were increased in patients with IBD under clinical treatment with vedolizumab. CONCLUSION: In addition to reported effects on lymphocytes, anti-α4ß7 therapy in IBD also targets non-classical monocytes. Impaired gut homing of such monocytes might lead to a reduction of wound healing macrophages and could potentially explain increased rates of postoperative complications in vedolizumab-treated patients, which have been observed in some studies.

Oral antibiotic use and risk of colorectal cancer in the United Kingdom, 1989-2012: a matched case-control study.

BACKGROUND: Microbiome dysbiosis predisposes to colorectal cancer (CRC), but a population-based study of oral antibiotic exposure and risk patterns is lacking. OBJECTIVE: To assess the association between oral antibiotic use and CRC risk. DESIGN: A matched case-control study (incident CRC cases and up to five matched controls) was performed using the Clinical Practice Research Datalink from 1989 to 2012. RESULTS: 28 980 CRC cases and 137 077 controls were identified. Oral antibiotic use was associated with CRC risk, but effects differed by anatomical location. Antibiotic use increased the risk of colon cancer in a dose-dependent fashion (ptrend <0.001). The risk was observed after minimal use, and was greatest in the proximal colon and with antibiotics with anti-anaerobic activity. In contrast, an inverse association was detected between antibiotic use and rectal cancers (ptrend=0.003), particularly with length of antibiotic exposure >60 days (adjusted OR (aOR), 0.85, 95% CI 0.79 to 0.93) as compared with no antibiotic exposure. Penicillins, particularly ampicillin/amoxicillin increased the risk of colon cancer (aOR=1.09 (1.05 to 1.13)), whereas tetracyclines reduced the risk of rectal cancer (aOR=0.90 (0.84 to 0.97)). Significant interactions were detected between antibiotic use and tumour location (colon vs rectum, pinteraction<0.001; proximal colon versus distal colon, pinteraction=0.019). The antibiotic-cancer association was found for antibiotic exposure occurring >10 years before diagnosis (aOR=1.17 (1.06 to 1.31)). CONCLUSION: Oral antibiotic use is associated with an increased risk of colon cancer but a reduced risk of rectal cancer. This effect heterogeneity may suggest differences in gut microbiota and carcinogenesis mechanisms along the lower intestinal tract.

Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy.

Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.

Elevated apoptosis impairs epithelial cell turnover and shortens villi in TNF-driven intestinal inflammation.

The intestinal epithelial monolayer, at the boundary between microbes and the host immune system, plays an important role in the development of inflammatory bowel disease (IBD), particularly as a target and producer of pro-inflammatory TNF. Chronic overexpression of TNF leads to IBD-like pathology over time, but the mechanisms driving early pathogenesis events are not clear. We studied the epithelial response to inflammation by combining mathematical models with in vivo experimental models resembling acute and chronic TNF-mediated injury. We found significant villus atrophy with increased epithelial cell death along the crypt-villus axis, most dramatically at the villus tips, in both acute and chronic inflammation. In the acute model, we observed overexpression of TNF receptor I in the villus tip rapidly after TNF injection and concurrent with elevated levels of intracellular TNF and rapid shedding at the tip. In the chronic model, sustained villus atrophy was accompanied by a reduction in absolute epithelial cell turnover. Mathematical modelling demonstrated that increased cell apoptosis on the villus body explains the reduction in epithelial cell turnover along the crypt-villus axis observed in chronic inflammation. Cell destruction in the villus was not accompanied by changes in proliferative cell number or division rate within the crypt. Epithelial morphology and immunological changes in the chronic setting suggest a repair response to cell damage although the villus length is not recovered. A better understanding of how this state is further destabilised and results in clinical pathology resembling IBD will help identify suitable pathways for therapeutic intervention.

Chronic TNFα-driven injury delays cell migration to villi in the intestinal epithelium.

The intestinal epithelium is a single layer of cells which provides the first line of defence of the intestinal mucosa to bacterial infection. Cohesion of this physical barrier is supported by renewal of epithelial stem cells, residing in invaginations called crypts, and by crypt cell migration onto protrusions called villi; dysregulation of such mechanisms may render the gut susceptible to chronic inflammation. The impact that excessive or misplaced epithelial cell death may have on villus cell migration is currently unknown. We integrated cell-tracking methods with computational models to determine how epithelial homeostasis is affected by acute and chronic TNFα-driven epithelial cell death. Parameter inference reveals that acute inflammatory cell death has a transient effect on epithelial cell dynamics, whereas cell death caused by chronic elevated TNFα causes a delay in the accumulation of labelled cells onto the villus compared to the control. Such a delay may be reproduced by using a cell-based model to simulate the dynamics of each cell in a crypt-villus geometry, showing that a prolonged increase in cell death slows the migration of cells from the crypt to the villus. This investigation highlights which injuries (acute or chronic) may be regenerated and which cause disruption of healthy epithelial homeostasis.

Deciphering the Complex Signaling Systems That Regulate Intestinal Epithelial Cell Death Processes and Shedding.

Intestinal epithelial cells play a fundamental role in maintaining homeostasis. Shedding of intestinal cells in a controlled manner is critical to maintenance of barrier function. Barrier function is maintained during this shedding process by a redistribution of tight junctional proteins to facilitate closure of the gap left by the shedding cell. However, despite the obvious importance of epithelial cell shedding to gut health, a central question is how the extrusion of epithelial cells is achieved, enabling barrier integrity to be maintained in the healthy gut and restored during inflammation remains largely unanswered. Recent studies have provided evidence that excessive epithelial cell shedding and loss of epithelial barrier integrity is triggered by exposure to lipopolysaccharide or tumor necrosis factor alpha. Subsequent studies have provided evidence of the involvement of specific cellular components and signaling mechanisms as well as the functionality of microbiota that can be either detrimental or beneficial for intestinal barrier integrity. This review will focus on the evidence and decipher how the signaling systems through which the mucosal immune system and microbiota can regulate epithelial cell shedding and how these mechanisms interact to preserve the viability of the epithelium.

A hierarchical Bayesian model for understanding the spatiotemporal dynamics of the intestinal epithelium.

Our work addresses two key challenges, one biological and one methodological. First, we aim to understand how proliferation and cell migration rates in the intestinal epithelium are related under healthy, damaged (Ara-C treated) and recovering conditions, and how these relations can be used to identify mechanisms of repair and regeneration. We analyse new data, presented in more detail in a companion paper, in which BrdU/IdU cell-labelling experiments were performed under these respective conditions. Second, in considering how to more rigorously process these data and interpret them using mathematical models, we use a probabilistic, hierarchical approach. This provides a best-practice approach for systematically modelling and understanding the uncertainties that can otherwise undermine the generation of reliable conclusions-uncertainties in experimental measurement and treatment, difficult-to-compare mathematical models of underlying mechanisms, and unknown or unobserved parameters. Both spatially discrete and continuous mechanistic models are considered and related via hierarchical conditional probability assumptions. We perform model checks on both in-sample and out-of-sample datasets and use them to show how to test possible model improvements and assess the robustness of our conclusions. We conclude, for the present set of experiments, that a primarily proliferation-driven model suffices to predict labelled cell dynamics over most time-scales.

Epidermal growth factor suppresses intestinal epithelial cell shedding through a MAPK-dependent pathway.

Cell shedding from the intestinal villus is a key element of tissue turnover that is essential to maintain health and homeostasis. However, the signals regulating this process are not well understood. We asked whether shedding is controlled by epidermal growth factor receptor (EGFR), an important driver of intestinal growth and differentiation. In 3D ileal enteroid culture and cell culture models (MDCK, IEC-6 and IPEC-J2 cells), extrusion events were suppressed by EGF, as determined by direct counting of released cells or rhodamine-phalloidin labeling of condensed actin rings. Blockade of the MEK-ERK pathway, but not other downstream pathways such as phosphoinositide 3-kinase (PI3K) or protein kinase C (PKC), reversed EGF inhibition of shedding. These effects were not due to a change in cell viability. Furthermore, EGF-driven MAPK signaling inhibited both caspase-independent and -dependent shedding pathways. Similar results were found in vivo, in a novel zebrafish model for intestinal epithelial shedding. Taken together, the data show that EGF suppresses cell shedding in the intestinal epithelium through a selective MAPK-dependent pathway affecting multiple extrusion mechanisms. EGFR signaling might be a therapeutic target for disorders featuring excessive cell turnover, such as inflammatory bowel diseases.

Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi.

The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models.-Parker, A., Maclaren, O. J., Fletcher, A. G., Muraro, D., Kreuzaler, P. A., Byrne, H. M., Maini, P. K., Watson, A. J. M., Pin, C. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi.

Clostridium difficile: A healthcare-associated infection of unknown significance in adults in sub-Saharan Africa.

BACKGROUND: Clostridium difficile infection (CDI) causes a high burden of disease in high-resource healthcare systems, with significant morbidity, mortality, and financial implications. CDI is a healthcare-associated infection for which the primary risk factor is antibiotic usage, and it is the leading cause of bacterial diarrhoea in HIV-infected patients in the United States. Little is known about the disease burden of CDI in sub-Saharan Africa, where HIV and healthcare-associated infections are more prevalent and antibiotic usage is less restricted. This article reviews published literature on CDI in sub-Saharan Africa, highlighting areas for future research. METHODS: English language publications since 1995 were identified from online databases (PubMed, Medline, Google Scholar, and SCOPUS), using combinations of keywords "C. difficile", "Africa", and "HIV". RESULTS: Ten relevant studies were identified. There was considerable variation in the methodologies used to assess for carriage of toxigenic C. difficile and its associations. Eight studies reported carriage of toxigenic C. difficile. Three (of three) studies found an association with antibiotic usage. One (of four) studies showed an association with HIV infection. One study showed no association with degree of immunosuppression in HIV. Two (of three) studies showed an association between carriage of toxigenic C. difficile and diarrhoeal illness. CONCLUSIONS: While the carriage of toxigenic C. difficile is well described in sub-Saharan Africa, the impact of CDI in the region remains poorly understood and warrants further research.