SummaryExcessive shedding of enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. However, the mechanisms underlying this phenomenon remain unclear. Intraepithelial lymphocytes (IEL) expressing the γδ T-cell receptor (TCR) provide surveillance of the intestinal mucosa at steady-state, which is regulated, in part, by CD103. Intravital microscopy of lipopolysaccharide (LPS)-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, as CD103 blockade significantly reduces LPS-induced shedding. Furthermore, we find that granzymes A and B, but not perforin, are required for cell shedding, and that these granzymes are released by γδ IELs both constitutively and following CD103/E-cadherin ligation. These findings indicate that extracellular granzyme facilitates shedding, likely through cleavage of extracellular matrix proteins. Our results uncover a previously unrecognized role for γδ IELs in facilitating pathological cell shedding in a CD103- and granzyme-dependent manner.

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.

We describe a precision medicine workflow, the integrated single nucleotide polymorphism network platform (iSNP), designed to determine the mechanisms by which SNPs affect cellular regulatory networks, and how SNP co-occurrences contribute to disease pathogenesis in ulcerative colitis (UC). Using SNP profiles of 378 UC patients we map the regulatory effects of the SNPs to a human signalling network containing protein-protein, miRNA-mRNA and transcription factor binding interactions. With unsupervised clustering algorithms we group these patient-specific networks into four distinct clusters driven by PRKCB, HLA, SNAI1/CEBPB/PTPN1 and VEGFA/XPO5/POLH hubs. The pathway analysis identifies calcium homeostasis, wound healing and cell motility as key processes in UC pathogenesis. Using transcriptomic data from an independent patient cohort, with three complementary validation approaches focusing on the SNP-affected genes, the patient specific modules and affected functions, we confirm the regulatory impact of non-coding SNPs. iSNP identified regulatory effects for disease-associated non-coding SNPs, and by predicting the patient-specific pathogenic processes, we propose a systems-level way to stratify patients.

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.

OBJECTIVE:To study the role of α4β7 integrin for gut homing of monocytes and to explore the biological consequences of therapeutic α4β7 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 α4β7 integrin.RESULTS:Classical and non-classical monocytes were clearly dichotomous regarding homing marker expression including relevant expression of α4β7 integrin on human and mouse non-classical monocytes but not on classical monocytes. Monocyte-expressed α4β7 integrin was functionally important for dynamic adhesion to mucosal vascular addressin cell adhesion molecule 1 and in vivo gut homing. Impaired α4β7-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-α4β7 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.

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.

demonstrating that mir-181a-5p increases intestinal enterocyte differentiation via inhibition of Akt.Furthermore, overexpression of mir-181a-5p increased expression of MUC2, a goblet cell marker, and LYZ, a Paneth cell marker.This induction was associated with decreased expression of the Notch target gene, Hes5, but not Hes1, in HT29 cells, suggesting that mir-181a-5p increases secretory cell differentiation via inhibition of Hes5.Conclusions:Through a global miRNA analysis, we have identifiedthe essential role of miR-181a-5p in intestinal cell differentiation.Moreover, we demonstrate that mir-181a-5p increases intestinal cell differentiation through inhibition of Akt and Hes5.

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.

Abstract Background Microbiome dysbiosis predisposes to colorectal cancer (CRC), but a population-based study of oral antibiotic exposure and CRC risk is lacking. Methods A matched case–control study (incident CRC cases and up to 5 matched controls) was conducted in the Clinical Practice Research Datalink (CPRD; 1989–2012). The CRPD is validated as 92% and 99% sensitive and specific for CRC detection (98% PPV). Antibiotic exposure [categorical and continuous terms (spline)] was investigated for risk pattern, stratified by tumor location, using conditional logistic regression and adjusting for known confounders. Results In total, 28,980 CRC cases and 137,077 controls were identified. Oral antibiotic use increased risk of colon cancer in a dose-dependent fashion (Ptrend < 0.001), but effects differed by anatomic location. Colon cancer risk was greatest in the proximal colon and with antibiotics with anti-anaerobic activity (Figure 1). 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 odds ratio [AOR], 0.85, 95% CI 0.79–0.93) when compared with no antibiotic exposure. Nonlinearity models showed significantly increased colon cancer risk after minimal antibiotic use, but decreased rectum cancer risk with cumulative use of over 30 days (Figure 2). Penicillins, particularly ampicillin/amoxicillin, increased risk of colon cancer (AOR,1.09, [1.05–1.13]) whereas tetracyclines reduced risk for rectal cancer (AOR, 0.90, [0.84–0.97]). Significant interactions were detected between antibiotic use and tumor location (colon vs. rectum, Pinteraction < 0.001). The antibiotic-cancer association was found for antibiotic exposure occurring >10 years before diagnosis (AOR, 1.17, [1.06–1.31]). Conclusion We conclude that oral antibiotic use associates with increased colon cancer risk, particularly in the right colon, but a reduced risk for rectal cancer. This effect heterogeneity suggests unabsorbed antibiotics impact gut microbiota in the right colon to enhance carcinogenesis whereas antibiotic anti-inflammatory or anti-proliferative actions may yield an inverse effect on carcinogenesis in the rectum. Disclosures Sara E. Cosgrove, MD, MS, Basilea: Consultant; Theravance: Consultant.

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 (p trend <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 (p trend =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, p interaction <0.001; proximal colon versus distal colon, p interaction =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.

ABSTRACT Background Environmental enteropathy (EE) refers to villus blunting, reduced absorption, and microbial translocation in children and adults in tropical or deprived residential areas. In previous work we observed an effect of micronutrients on villus height (VH). Objective We aimed to determine, in a randomized controlled trial, if amino acid (AA) or multiple micronutrient (MM) supplementation can improve intestinal structure or barrier dysfunction in Zambian adults with EE. Methods AA (tryptophan, leucine, and glutamine) and/or MM supplements were given for 16 wk in a 2 × 2 factorial comparison against placebo. Primary outcomes were changes in VH, in vivo small intestinal barrier dysfunction assessed by confocal laser endomicroscopy (CLE), and mechanistic (or mammalian) target of rapamycin complex 1 (MTORC1) nutrient responsiveness in lamina propria CD4+ lymphocytes. Results Over 16 wk AA, but not MM, supplementation increased VH by 16% (34.5 μm) compared with placebo (P = 0.04). Fluorescein leak, measured by CLE, improved only in those allocated to both AA and MM supplementation. No effect was seen on MTORC1 activation, but posttreatment MTORC1 and VH were correlated (ρ = 0.51; P = 0.001), and change in MTORC1 was correlated with change in VH in the placebo group (ρ = 0.63; P = 0.03). In secondary analyses no effect was observed on biomarkers of microbial translocation. Metabolomic analyses suggest alterations in a number of microbial- and host-derived metabolites including the leucine metabolite β-hydroxy-β-methylbutyrate, which was increased by AA supplementation and correlated with VH. Conclusions In this phase 2 trial, AA supplementation protected against a decline in VH over the supplementation period, and improved barrier function when combined with micronutrients. Leucine and MTORC1 metabolism may be involved in the mechanism of effect. This trial was registered at www.pactr.org as PACTR201505001104412.

IntroductionAcute severe ulcerative colitis (ASUC) is a severe manifestation of ulcerative colitis (UC) that warrants hospitalisation. Despite significant advances in therapeutic options for UC and in the medical management of steroid-refractory ASUC, the initial treatment paradigm has not changed since 1955 and is based on the use of intravenous corticosteroids. This treatment is successful in approximately 50% of patients but failure of this and subsequent medical therapy still occurs, with colectomy rates of up to 40% reported. The Interleukin 1 (IL-1) blockade in Acute Severe Colitis (IASO) trial aims to investigate whether antagonism of IL-1 signalling using anakinra in addition to intravenous corticosteroid treatment can improve outcomes in patients with ASUC.Methods and analysisIASO is a phase II, multicentre, two-arm (parallel group), randomised (1:1), placebo-controlled, double-blinded trial of short-duration anakinra in ASUC. Its primary outcome will be the incidence of medical (eg, infliximab/ciclosporin) or surgical rescue therapy (colectomy) within 10 days following the commencement of intravenous corticosteroid therapy. Secondary outcomes will include disease activity, time to clinical response, time to rescue therapy, colectomy incidence by day 98 post intravenous corticosteroids and safety. The trial aims to recruit 214 patients across 20 sites in the UK.Ethics and disseminationThe trial has received approval from the Cambridge Central Research Ethics Committee (Ref: 17/EE/0347), the Health Research Authority (Ref: 201505) and Clinical Trials Authorisation from the Medicines and Healthcare products Regulatory Agency. We plan to present trial findings at scientific conferences and publish in high-impact peer-reviewed journals.Trial registration numberISRCTN43717130; EudraCT 2017-001389-10.

was notably increased after Aicar and GW treatments in ileal organoids from either NTKO or NTR1KO mice compared with organoids from respective WT mice.CONCLUSIONS: Ileal mucosal AMPK activity is upregulated by NT deficiency in mice fed LFD but downregulated by NT deficiency in mice fed HFD.Our results demonstrate that AMPK activation increases FXR protein expression and transcriptional activity suggesting that AMPK plays a central role in NT/NTR1 signaling.NT contributes to the disruption of BA homeostasis induced by HFD through AMPK activation and its positive crosstalk with FXR.

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.

Cremonesi E, Governa V, Garzon JFG, et al. Gut microbiota modulate T cell trafficking into human colorectal cancer Gut 2018;67:1984–1994. Immune cells have an important effect of the prognosis of human colorectal cancer (CRC). Infiltration on CRC tissue by cytotoxic CD8 T cells, T helper 1 (Th1) cells, CXCR5+ follicular T helper cells (Tfh), and Foxp3+ T regulatory cells is associated with improved patient survival and a favorable clinical outcome (Nat Rev Cancer 2012;12;298–306). In this study, Cremonesi et al aimed to identify the nature of chemotactic factors promoting CRC infiltration by these T-cell populations and the stimuli responsible for inducing their expression in the CRC microenvironment. The investigators analyzed gene expression of a variety of T-cell markers in 62 CRC and corresponding tumor-free colonic tissues, to study the composition of T-cell populations within these tissues. They found that CRC tissues were only minimally infiltrated by Th2 cells, as indicated by undetectable levels of IL4 and minimal expression of IL5 and IL13. However, Th17 and T regulatory cell markers were highly expressed in CRC cells in comparison to control tissues, whereas CD4 T-helper cell and TfH cell markers were slightly reduced. Unsupervised hierarchical analysis of the data resulted in CRC samples being clustered into 3 main groups based on expression of T cell markers—cluster high (overexpression of most T-cell markers), cluster het (heterogeneous expression of T-cell markers), and cluster low (down-regulation of all T-cell markers). In the cluster high group, a specific panel of chemokine genes was found to be significantly up-regulated. Furthermore, specific highly significant correlations were noted between T-cell markers and chemokine genes in CRC samples, indicating particular chemokines could drive T-cell recruitment into CRC tissues. The investigators also studied chemokine receptor profiles of T cells in CRC samples in comparison with control tissues or peripheral blood mononuclear cells. In the group with an overexpression of T-cell markers, chemokine genes were found to be significantly up-regulated, indicating certain chemokines could drive T-cell recruitment into CRC tissues. Specifically, they found that CCR5, CXCR3, and CXCR4 were highly expressed on CD8, CD4, and FoxP3 T cells in both cancer and control tissues. However, significantly higher proportion of CCR5+ and CXCR4+ T cells were detected in cancer tissues in comparison with peripheral blood mononuclear cells. A further interrogation of the data, enabled the investigators to identify particular chemokine signatures for each CRC T-cell subset, that is, mainly CCR5-binding ligands (CCL3, CCL4, CCL5, CCL8), CXCR3-binding ligands (CXCL9 and CXCL10), and a CXCR4-binding ligand (CXCL12) for cytotoxic T lymphocytes; CCR4 ligands (CCL17, CCL22), CCR5-binding ligands (CCL3, CCL5), CXRC3-binding ligands (CXCL9, CXCL10), and CXCL12 for Th1 cells; CCR4 ligands (CCL17, CCL22), CXCL12, CCL5, and CXCL9 for T regulatory cells; CXCL13 for Tfh cells; and CCL20 and CCL17 for Th17 cells. The authors concluded from these data that the overexpression of the chemokines they identified associates with the infiltration of beneficial T-cell populations and an improved prognosis. Gene expression analysis of chemokines in CRC samples identified that tumor cells were the main source of T-cell–recruiting chemokines including CCL3, CCL4, CCL5, and CXCL10 (which bind to receptors on cytotoxic T cells and Th1 cells) but not CCL7, CCL8, CCL11, CCL13, CCL27, or the Th17 recruiting chemokines CCL17 and CCL22. Crucially, authors also noticed that in vitro cultured CRC cell lines expressed far fewer chemokine genes in comparison with primary tumor cells, suggesting that chemokine expression may require other environmental factors present in the in vivo setting. The authors hypothesized that this environmental signal may potentially arise from the gut microbiota, given that it has been previously demonstrated that commensal bacteria and/or their products can translocate across CRC epithelial tissues and thus come into direct contact with CRC cells (Science 2012;338:120–123). The authors exposed CRC cells from cell lines and CRC organoids to toll-like receptor agonists. This resulted in the up-regulation of constitutively expressed chemokine genes including CCL20, CXCL9, and CXCL10 and de novo expression of additional chemokine genes, including CCL3, CCL4, CCL5, and CCL22. Several chemokine genes were also induced on exposure of CRC cells to particular bacterial species known to be enriched in CRC tissues; Fusobacterium nucleatum, Bacteroides fragilis, and Escherichia coli. To determine whether such effects were also present in the in vivo setting, chemokine expression was measured in CRC tumor xenografts generated by injecting human CRC cells into NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). These mice, also called NOD scid gamma mice, are a type of immunodeficient mouse that lack mature T cells, B cells, and natural killer cells. The injection of tumor cells was performed either intraperitoneally or intracecally. Interestingly, they noticed that intracecal tumors contained significantly higher levels of a range of chemokines compared with intraperitoneal controls, namely, CCL5 (a 70-fold increase), CCL20 (a 19-fold increase), CXCL10 (a 12-fold increase), and CXCL11 (a 3-fold increase). Furthermore, levels of these chemokines significantly decreased after treatment with antibiotics and correlated with bacterial load. By adoptively transferring carboxyfluorescein diacetate succinimidyl ester–labelled tumor-infiltrating CD4+ and CD8+ T lymphocytes, into these tumor xenograft–bearing mice, the authors observed that these tumor-infiltrative lymphocytes homed to intracecal xenografts to a much greater extent than intraperitoneal xenografts. Hence, taken together, these experiments suggest that the commensal gut microbiota is a major factor in inducing chemokine expression in CRC cells which subsequently determines T-cell infiltration into tumor tissues. To characterize the microbiota–chemokine–T-cell relationship in clinical CRC samples, the investigators sought to identify significant biological correlations. Although greater quantities of gut bacteria were observed in highly T-cell–infiltrated CRC samples in comparison with poorly infiltrated tumors, total bacterial load (as assessed by 16S analysis) only weakly correlated with individual chemokine expression, in contrast with the observations in tumor xenografts. Instead, an analysis of individual bacteria genera in highly infiltrated and poorly infiltrated CRC tumor samples revealed significant correlations between specific bacteria and T-cell markers and chemokine gene expression. Lachnospiraceae and Ruminococcaceae, both part of the Firmicutes phylum, correlated strongly with CCR5 and CXCR3 binding chemokines. Bacteroides and Proteobacteria, in particular Methylobacteriaceae, also significantly correlated with the expression of all prognostically favorable T cell markers and corresponding chemokines. The authors concluded that the expression of chemokines by human CRC cells is associated with the abundance of specific bacteria within the tumors. The importance of immune cells in cancer pathogenesis has become increasingly appreciated. CRC, in particular, has become a paradigmatic tumor for understanding the complex role of immune cells in cancer. It is now recognized that the developing CRC resides within a rich microenvironment composed of a complex array of immune cell populations including T lymphocytes, B lymphocytes, macrophages, dendritic cells, natural killer cells, and mast cells, which are found either within the tumor core, the invasive margin, or in tertiary lymphoid structures. This is collectively referred to as the “immune contexture” (Nat Rev Cancer 2012;12:298–306). The clinical significance of these immune cells in human CRC was first noted in the 2000s, when it was demonstrated that the type, density, and location of immune cells within tumor samples are a better predictor of prognosis than the established histopathological Dukes’ staging system (Science 2006;313:1960–1964). A strong immune cell reaction in both the tumor core and invasive margin, comprising CD8 cytotoxic T cells and CD45RO memory T cells, correlated with a favorable prognosis regardless of cancer stage, while a poor immune reaction in both regions correlated with poor prognosis, even in those with minimally invasive (stage 1) tumors (Science 2006;313:1960–1964; J Clin Oncol 2009;27:5944–5951). This finding led to the development of a novel immune scoring system for CRC, based on memory and cytotoxic T-cell markers, which was found to be superior to the Dukes’ staging in predicting recurrence as well as survival (J Clin Oncol 2009;27:5944–5951; J Clin Oncol 2011;29:610–618). More recent studies have revealed that other components of the immune contexture, including T regulatory cells, B cells, natural killer cells, macrophages, as well as endothelial cells and fibroblasts, also correlate with CRC prognosis. In parallel to these developments, a major transcriptomic analysis identified that the vast majority of CRCs fall into four distinct consensus molecular subtypes: CMS1 (tumors exhibiting high microsatellite instability owing to mutations in genes encoding DNA mismatch-repair proteins), CMS2 (tumors with high chromosomal instability, and activation of the Wnt and MYC pathways), CMS3 (tumors with KRAS mutations and disruption of metabolic pathways), and CMS4 (tumors with a mesenchymal phenotype and frequent CpG island methylator phenotype; Nat Med 2015;21:1350–1356). This finding has prognostic implications, because CMS4 is associated with the worst disease-free survival and overall survival, and both CMS1 and CMS4 with poor survival after recurrence (Ann Oncol 2018;29[Suppl 8]:viii18). Using a transcriptome-based computational method, these molecular and immune-based classifications of CRC were recently integrated (Clin Cancer Res 2016;22:4057–4066). It was found that, unlike CMS2 and CMS3 CRC subtypes, CMS1 and CMS4 subtypes displayed a strong immune and inflammatory contexture. CMS1 contained higher abundances of cytotoxic T cells, whereas CMS4 had higher expression of B cells, myeloid cells, fibroblasts, and endothelial cells. In addition, CMS1 subtype cancers expressed high levels of T-cell–attracting chemokines (including CXCL9, CXCL10, and CXCL16), Th1 cytokines (interferon-γ and IL15), immune checkpoints (eg, CTLA4, PD1), and MHC class 1. CMS4 subtype, however, expressed high levels of myeloid chemokines (eg, CCL2), complement components, angiogenic factors (vascular endothelial growth factor B, vascular endothelial growth factor C, and platelet-derived growth factor C), and immunosuppressive molecules (transforming growth factor-β1, transforming growth factor-β3, LGALS1, and CXCL12). CMS2 and CMS3 subtypes were relatively devoid of immune cell populations. These findings demonstrate that CRC is a heterogeneous disease, composed of distinct molecular and immune signatures, requiring different therapeutic strategies. For instance, CMS1 subtype cancers are most likely to respond to checkpoint inhibitor therapy such as pembrolizumab (an anti-PD1 antibody), whereas CMS4 subtype cancers may require a combination of antiangiogenic and anti–transforming growth factor-β checkpoint inhibitor therapies (Curr Opin Immunol 2016;39:7–13). In this study, Cremonesi et al aimed to further characterize the chemokine signals that drive favorable T-cell populations into the CRC microenvironment and identify the cellular sources of these chemokines, as well as the potential underlying stimuli responsible for inducing their expression. They found chemokine genes expressed by CRC cells in response to gut microbiota-derived stimuli are mainly responsible for the infiltration of favorable immune cell populations into the CRC microenvironment. This discovery is exciting, because it could potentially lead to the addition of an entirely new strategy in our armamentarium against CRC, one that involves modulation of the CRC immune contexture by targeting the gut microbiota. In the past decade, evidence of the involvement of bacterial populations during tumor progression was identified using metagenomic tools (Nat Genet 2002;30:141–142; PLoS ONE 2011;6:e19838.doi:10.1371). This approach demonstrated that a number of bacteria are involved in the pathogenesis of CRC, including Fusobacterium nucleatum, Bacteroides fragilis, and Escherichia coli. F nucleatum is an anaerobe that is highly invasive (Infect Immun 2000;68:3140–3146; Gut 2011;60:34–40), with proinflammatory characteristics (Infect Immun 2000;68:2907–2915; Cytokine 2009;46:201–210) and known to be present in CRC specimens. F nucleatum has been proposed to promote the pathogenesis of CRC by a variety of mechanisms, including creating a proinflammatory environment with increased levels of tumor necrosis factor and nuclear factor-κB, the activation of β-catenin signaling and reducing T-cell activation (World J Gastrointest Oncol 2018;10:71–81). B fragilis has been found to be an independent predictor of 3-year survival from CRC (Oncotarget 2016;7:46158–46172). It secretes a toxin that is a metalloprotease that cleaves E-cadherin, thereby activating the Wnt pathway (J Clin Invest 2014;124:4166–4172). E coli have been found to frequently colonize CRC and have been reported to have mutagenic effects (World J Gastroenterology 2014;20:6560–6572; Cell Host Microbe 2014;15:317–328). Some gut bacteria metabolize bile salts into procarcinogenic secondary bile acids, whereas bacteria fermentation of complex carbohydrates into short fatty acids is anticarcinogenic (Nat Rev Microbiol 2014;12:661). Biofilms, which are polybacterial communities encased in a polymeric matrix, are found in only 15% of healthy patients but in 100% of right-sided CRCs. The colonic mucosa under biofilms have decreased E-cadherin, and increased IL-6, Ki-67, and phospho-stat3, suggesting biofilms play a procarcinogenic role (Proc Natl Acad Sci U S A 2014;111:18321). In the current study, a range of bacteria including in particular Lachnospiraceae and Ruminococcaceae were associated with the expression of T-cell–recruiting chemokines. Fusobacteria, which have previously been associated with a poor prognosis, were found to evoke T-cell–recruiting chemokines in this study, suggesting that they may in some circumstances be associated with a good prognosis. Clearly, more detailed studies of individual bacteria grouping are required to define the specific circumstances in which they encourage recruitment of favorable T-cell populations. The potential effects of gut bacteria on the other cell populations comprising the CRC immune contexture also need to be investigated. In conclusion, the intestinal microbiota is being found to play an increasingly important role in determining the prognosis of a range of cancers including CRC. The gut microbiota is already known to be a key determinant of checkpoint inhibitor therapy (Gastroenterology 2018;154:2068–2070). These new observations that a range of gut bacteria can promote T-cell infiltration into CRC, which could confer a good prognosis, opens another exciting avenue for future treatments.

Autophagy is a highly conserved catabolic pathway that eliminates damaged organelles, invading pathogens and specifically degrades proteins. Mutation in autophagy genes and deregulated autophagy are related to various human diseases including Crohn’s disease (CD) where autophagy impairment was shown to affect Paneth cells. Previously, we developed the Autophagy Regulatory Network resource (http://autophagyregulation.org) to better understand the mechanism and regulation of autophagy in disease pathomechanisms. To investigate autophagy-related processes in Paneth cells, we combined ARN with multi-omics data from intestinal organoids. In particular, we investigated how autophagy impairment, often observed in CD, could affect the key cell functions of Paneth cells. We generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells making these cells impaired in autophagy. Using a 3D intestinal organoid culture model that we enriched for Paneth cells, we compared the proteomic profiles of organoids derived from the wild-type (WT) and Atg16l1 KO mice. We developed 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 cellular processes. We detected 284 proteins with altered protein levels by comparing the proteomic profiles of organoids derived from normal mice or mice with impaired autophagy. Our integrated analysis—combination of proteomics and network biology approaches—revealed autophagy-mediated mechanisms which degrade essential proteins belonging to key Paneth cell functions such as exocytosis, apoptosis, and DNA damage repair. We performed validation experiments by generating full transcriptomics profiles of both organoid types, and by specifically focussing on Paneth cell-derived lysozyme to confirm our inferred observation of down-regulated exocytosis. We used both experimental and computational approaches to analyse and uncover the systems-level regulation of cellular processes dependent on autophagy in Paneth cells enriched organoids. Strikingly, the analysis revealed that when autophagy is impaired, nearly 300 proteins display increased or decreased abundance, encompassing at least 18 functional processes. Our observations could explain how protein-level alterations in CD as a result of autophagy-impairment could affect Paneth cell functions. The established workflow enables assessing the potential intestinal effect of autophagy-related mutations in CD patients, and prioritise the key affected processes.