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The intestinal epithelium is continuously renewed by intestinal epithelial stem cells (IESCs) positioned at the base of each crypt. Mesenchymal-derived factors are essential to maintain IESCs; however, the cellular composition and development of such mesenchymal niche remains unclear. Here, we identify pericryptal CD34⁺ Gp38⁺ αSMA⁻ mesenchymal cells closely associated with Lgr5⁺ IESCs. We demonstrate that CD34⁺ Gp38⁺ cells are the major intestinal producers of the niche factors Wnt2b, Gremlin1, and R-spondin1, and are sufficient to promote maintenance of Lgr5⁺ IESCs in intestinal organoids, an effect mainly mediated by Gremlin1. CD34⁺ Gp38⁺ cells develop after birth in the intestinal submucosa and expand around the crypts during the third week of life in mice, independently of the microbiota. We further show that pericryptal CD34⁺gp38⁺ cells are rapidly activated by intestinal injury, up-regulating niche factors Gremlin1 and R-spondin1 as well as chemokines, proinflammatory cytokines, and growth factors with key roles in gut immunity and tissue repair, including IL-7, Ccl2, Ptgs2, and Amphiregulin. Our results indicate that CD34⁺ Gp38⁺ mesenchymal cells are programmed to develop in the intestine after birth to constitute a specialized microenvironment that maintains IESCs at homeostasis and contribute to intestinal inflammation and repair after injury.

Sporadic colorectal cancer (CRC) is a result of complex interactions between the host and its environment. Environmental stressors act by causing host cell DNA alterations implicated in the onset of cancer. Here we investigate the stressor ability of CRC-associated gut dysbiosis as causal agent of host DNA alterations. The epigenetic nature of these alterations was investigated in humans and in mice. Germ-free mice receiving fecal samples from subjects with normal colonoscopy or from CRC patients were monitored for 7 or 14 wk. Aberrant crypt foci, luminal microbiota, and DNA alterations (colonic exome sequencing and methylation patterns) were monitored following human feces transfer. CRC-associated microbiota induced higher numbers of hypermethylated genes in murine colonic mucosa (vs. healthy controls' microbiota recipients). Several gene promoters including SFRP1,2,3, PENK, NPY, ALX4, SEPT9, and WIF1 promoters were found hypermethylated in CRC but not in normal tissues or effluents from fecal donors. In a pilot study (n = 266), the blood methylation levels of 3 genes (Wif1, PENK, and NPY) were shown closely associated with CRC dysbiosis. In a validation study (n = 1,000), the cumulative methylation index (CMI) of these genes was significantly higher in CRCs than in controls. Further, CMI appeared as an independent risk factor for CRC diagnosis as shown by multivariate analysis that included fecal immunochemical blood test. Consequently, fecal bacterial species in individuals with higher CMI in blood were identified by whole metagenomic analysis. Thus, CRC-related dysbiosis induces methylation of host genes, and corresponding CMIs together with associated bacteria are potential biomarkers for CRC.

Graphical Abstract What is COVID‐19? What are the causes, parameters, and effects of this disease? What are the short‐ and long‐term prospects? Philippe Sansonetti, Infectious disease specialist and Chief Editor of EMBO Molecular Medicine , explains why the fate of the epidemic is in our hands.

Due to the huge number of bacteria constituting the human colon microbiota, alteration in the balance of its constitutive taxa (i.e., dysbiosis) is highly suspected of being involved in colorectal oncogenesis. Indeed, bacterial signatures in association with CRC have been described. These signatures may vary if bacteria are identified in feces or in association with tumor tissues. Here, we show that bacteria colonize human colonic crypts in tissues obtained from patients with CRC and with normal colonoscopy results. Aerobic nonfermentative Proteobacteria previously identified as constitutive of the crypt-specific core microbiota in murine colonic samples are similarly prevalent in human colonic crypts in combination with other anaerobic taxa. We also show that bacterial signatures characterizing the crypts of colonic tumors vary depending whether right-side or left-side tumors are analyzed. We have previously identified a crypt-specific core microbiota (CSCM) in the colons of healthy laboratory mice and related wild rodents. Here, we confirm that a CSCM also exists in the human colon and appears to be altered during colon cancer. The colonic microbiota is suggested to be involved in the development of colorectal cancer (CRC). Because the microbiota identified in fecal samples from CRC patients does not directly reflect the microbiota associated with tumor tissues themselves, we sought to characterize the bacterial communities from the crypts and associated adjacent mucosal surfaces of 58 patients (tumor and normal homologous tissue) and 9 controls with normal colonoscopy results. Here, we confirm that bacteria colonize human colonic crypts in both control and CRC tissues, and using laser-microdissected tissues and 16S rRNA gene sequencing, we further show that right and left crypt- and mucosa-associated bacterial communities are significantly different. In addition to Bacteroidetes and Firmicutes , and as with murine proximal colon crypts, environmental nonfermentative Proteobacteria are found in human colonic crypts. Fusobacterium and Bacteroides fragilis are more abundant in right-side tumors, whereas Parvimonas micra is more prevalent in left-side tumors. More precisely, Fusobacterium periodonticum is more abundant in crypts from cancerous samples in the right colon than in associated nontumoral samples from adjacent areas but not in left-side colonic samples. Future analysis of the interaction between these bacteria and the crypt epithelium, particularly intestinal stem cells, will allow deciphering of their possible oncogenic potential. IMPORTANCE Due to the huge number of bacteria constituting the human colon microbiota, alteration in the balance of its constitutive taxa (i.e., dysbiosis) is highly suspected of being involved in colorectal oncogenesis. Indeed, bacterial signatures in association with CRC have been described. These signatures may vary if bacteria are identified in feces or in association with tumor tissues. Here, we show that bacteria colonize human colonic crypts in tissues obtained from patients with CRC and with normal colonoscopy results. Aerobic nonfermentative Proteobacteria previously identified as constitutive of the crypt-specific core microbiota in murine colonic samples are similarly prevalent in human colonic crypts in combination with other anaerobic taxa. We also show that bacterial signatures characterizing the crypts of colonic tumors vary depending whether right-side or left-side tumors are analyzed.

Key Points Bacterial pathogens exploit a range of niches within their hosts. A small number of bacteria, including Listeria monocytogenes , Shigella flexneri, Burkholderia pseudomallei , Francisella tularensis and Rickettsia spp., are able to gain access to and proliferate in the cell cytosol and are termed cytosolic bacteria. Escape from the vacuole following invasion is crucial for cytosolic pathogens. Bacteria escape rapidly from the vacuole through mechanisms that rely on the production of secreted enzymes and form pores to disrupt the vacuolar membrane. Interestingly, with the exception of F. tularensis , all cytosolic bacteria use actin-based motility after entry into the cytosol and spread to neighbouring cells. Little is known about the biochemical composition of the mammalian cell cytosol and a key, but unresolved, question is whether the cytosol is permissive for bacterial growth. Cytosolic bacteria are adapted to replicate in the cell cytosol. Studies identifying the bacterial genes and growth requirements that are important for intracellular replication have been informative regarding the nutrient availability within the cytosol. The cytosol seems to be limiting for compounds such as aromatic amino acids, and bacteria can readily use carbon sources, including pyruvate (by Rickettsia spp.) and hexose phosphates (by S. flexneri and L. monocytogenes ), that must be available to microorganisms in the cytosol. Bacteria in the cytosol are recognized by the innate immune system and autophagy is a key component of the host defence against cytosolic bacteria. There is increasing evidence that cytosolic bacteria interact and modify the autophagic pathway to promote their survival. Cytosolic bacteria have evolved several mechanisms to adapt to their preferred niche. Future work on these pathogens will provide information on the cytosol as a site for replication and the bacterial strategies required to survive within it. Furthermore, it is becoming appreciated that a wider range of bacteria can exploit this host niche during steps in their pathogenesis. Many bacterial pathogens can invade non-phagocytic cells and survive within a membrane-bound vacuole. However, few pathogens are able to escape the vacuoles and proliferate in the host cell cytosol. In this Review, Tang and colleagues discuss the mechanisms by which these pathogens enter the cytosol, obtain nutrients and subvert host immune responses. Bacterial pathogens exploit a huge range of niches within their hosts. Many pathogens can invade non-phagocytic cells and survive within a membrane-bound compartment. However, only a small number of bacteria, including Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis and Rickettsia spp., can gain access to and proliferate within the host cell cytosol. Here, we discuss the mechanisms by which these cytosolic pathogens escape into the cytosol, obtain nutrients to replicate and subvert host immune responses.

Quantitative reverse transcription PCR analysis is an important tool to monitor changes in gene expression in animal models. The rabbit is a widely accepted and commonly used animal model in the study of human diseases and infections by viral, fungal, bacterial and protozoan pathogens. Only a limited number of rabbit genes have, however, been analyzed by this method as the rabbit genome sequence remains unfinished. Recently, increasing coverage of the genome has permitted the prediction of a growing number of genes that are relevant in the context of the immune response. We hereby report the design of twenty-four quantitative PCR primer pairs covering common cytokines, chemoattractants, antimicrobials and enzymes for a rapid, sensitive and quantitative analysis of the rabbit immune response. Importantly, all primer pairs were designed to be used under identical experimental conditions, thereby enabling the simultaneous analysis of all genes in a high-throughput format. This tool was used to analyze the rabbit innate immune response to infection with the human gastrointestinal pathogen Shigella flexneri. Beyond the known inflammatory mediators, we identified IL-22, IL-17A and IL-17F as highly upregulated cytokines and as first responders to infection during the innate phase of the host immune response. This set of qPCR primers also provides a convenient tool for monitoring the rabbit immune response during infection with other pathogens and other inflammatory conditions.

The innate immune system of mammals has been forged by coevolution with microbes in response to the double constraint of preserving a symbiotic interaction with commensal flora and eliminating intrusion of those commensals or invasion by pathogens. Thus, a 'sensing' network, accompanied by or lacking inflammatory responses, is controlled by elaborate mechanisms of regulation that maintain balance in the basal state. A growing number of non–Toll-like innate immune receptors is recognized as part of this surveillance network.

The interleukin-2 receptor (IL-2R) is a cytokine receptor essential for immunity that transduces proliferative signals regulated by its uptake and degradation. IL-2R is a well-known marker of clathrin-independent endocytosis (CIE), a process devoid of any coat protein, raising the question of how the CIE vesicle is generated. Here, we investigated the impact of IL-2Rγ clustering in its endocytosis. Combining total internal reflection fluorescence (TIRF) live imaging of a CRISPR-edited T cell line endogenously expressing IL-2Rγ taggedwith green fluorescent protein (GFP), with multichannel imaging, single-molecule tracking, and quantitative analysis, we were able to decipher IL-2Rγ stoichiometry at the plasma membrane in real time. We identified three distinct IL-2Rγ cluster populations. IL-2Rγ is secreted to the cell surface as a preassembled small cluster of three molecules maximum, rapidly diffusing at the plasma membrane. A medium-sized cluster composed of four to six molecules is key for IL-2R internalization and is promoted by interleukin 2 (IL-2) binding, while larger clusters (more than six molecules) are static and inefficiently internalized. Moreover, we identified membrane cholesterol and the branched actin cytoskeleton as key regulators of IL-2Rγ clustering and IL-2–induced signaling. Both cholesterol depletion and Arp2/3 inhibition lead to the assembly of large IL-2Rγ clusters, arising from the stochastic interaction of receptor molecules in close correlation with their enhanced lateral diffusion at the membrane, thus resulting in a default in IL-2R endocytosis. Despite similar clustering outcomes, while cholesterol depletion leads to a sustained IL-2–dependent signaling, Arp2/3 inhibition prevents signal initiation. Taken together, our results reveal the importance of cytokine receptor clustering for CIE initiation and signal transduction.

The gut microbiota plays a crucial role in the maturation of the intestinal mucosal immune system of its host(1,2). Within the thousand bacterial species present in the intestine, the symbiont segmented filamentous bacterium(SFB) is unique in its ability to potently stimulate the post-natal maturation of the B-and T-cell compartments and induce a striking increase in the small-intestinal Th17 responses(3-5). Unlike other commensals, SFB intimately attaches to absorptive epithelial cells in the ileum and cells overlying Peyer's patches(6,7). This colonization does not result in pathology; rather, it protects the host from pathogens(4). Yet, little is known about the SFB-host interaction that underlies the important immunostimulatory properties of SFB, because SFB have resisted in vitro culturing for more than 50 years. Here we grow mouse SFB outside their host in an SFB-host cell co-culturing system. Single-celled SFB isolated from mono-colonized mice undergo filamentation, segmentation, and differentiation to release viable infectious particles, the intracellular offspring, which can colonize mice to induce signature immune responses. In vitro, intracellular offspring can attach to mouse and human host cells and recruit actin. In addition, SFB can potently stimulate the upregulation of host innate defence genes, inflammatory cytokines, and chemokines. In vitro culturing thereby mimics the in vivo niche, provides new insights into SFB growth requirements and their immunostimulatory potential, and makes possible the investigation of the complex developmental stages of SFB and the detailed dissection of the unique SFB-host interaction at the cellular and molecular levels.

We identified a crypt-specific core microbiota (CSCM) dominated by strictly aerobic, nonfermentative bacteria in murine cecal and proximal colonic (PC) crypts and hypothesized that, among its possible functions, it may affect epithelial regeneration. In the present work, we isolated representative CSCM strains using selective media based upon our initial 16S rRNA-based molecular identification (i.e., Acinetobacter , Delftia , and Stenotrophomonas ). Their tropism for the crypt was confirmed, and their influence on epithelial regeneration was demonstrated in vivo by monocolonization of germfree mice. We also showed that lipopolysaccharide (LPS), through its endotoxin activity, was the dominant bacterial agonist controlling proliferation. The relevant molecular mechanisms were analyzed using colonic crypt-derived organoids exposed to bacterial sonicates or highly purified LPS as agonists. We identified a Toll-like receptor 4 (TLR4)-dependent program affecting crypts at different stages of epithelial differentiation. LPS played a dual role: it repressed cell proliferation through RIPK3-mediated necroptosis of stem cells and cells of the transit-amplifying compartment and concurrently enhanced cell differentiation, particularly the goblet cell lineage. IMPORTANCE The LPS from crypt-specific core microbiota controls intestinal epithelium proliferation through necroptosis of stem cells and enhances cell differentiation, mainly the goblet cell lineage. The LPS from crypt-specific core microbiota controls intestinal epithelium proliferation through necroptosis of stem cells and enhances cell differentiation, mainly the goblet cell lineage.