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The cellular heterogeneity within a variety of solid tumors is influenced by several type of tumor-associated bacteria through yet poorly understood mechanisms. Unbiased genomic analysis revealed that right-sided colorectal tumors with poorer prognosis are preferentially populated with species related toEscherichia coli, including strains that are producing colibactin. By applying metabolomic profilingin situ, the presence of colibactin-producingEscherichia coli(CoPEC) was identified to establish a high-glycerophospholipid microenvironment within right-sided colorectal tumors that are bearing mutations in either APC or KRAS. Using spatial approaches, we revealed that bacterial microniches are poorly infiltrated with CD8+T cells. The aforementioned alterations in lipid metabolism negatively correlated with immunomodulatory and neurotrophic factors among which the human regenerating family member 3 alpha gene (REG3A). Accordingly, the loss of its ortholog enhances growth at the proximal colon of tumors with a microenvironment that impedes immune surveillance and shares similarities to human right-sided colorectal tumors. This work clarifies how the presence of colibactin-producing bacteria may locally establish tumor heterogeneity for lipid metabolism to promote cancer progression and provide unique insights both for therapeutic intervention and enabling basic research into the mechanisms of microbiota-host interaction.STATEMENT OF SIGNIFICANCEApplyingin situmetabolic imaging to patients’ tumors revealed that the accumulation of colibactin-producing bacteria that locally creates a potential vulnerability to cancer treatments through lipid metabolic reprogramming of tumor cells.

SummaryRecently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here we characterized and investigated one of these oral pathobionts, the Gram-positive anaerobic coccusParvimonas micra.We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing anin vitrohypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we showed thatP. micraphylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carryingP. micraphylotype A, we found similar DNA methylations alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence ofP. micrapossible role in the carcinogenic process.

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.

ObjectiveType 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic β-cells producing insulin. Both T1D patients and animal models exhibit gut microbiota and mucosa alterations, although the exact cause for these remains poorly understood. We investigated the production of key cytokines controlling gut integrity, the abundance of segmented filamentous bacteria (SFB) involved in the production of these cytokines, and the respective role of autoimmune inflammation and hyperglycaemia.DesignWe used several mouse models of autoimmune T1D as well as mice rendered hyperglycaemic without inflammation to study gut mucosa and microbiota dysbiosis. We analysed cytokine expression in immune cells, epithelial cell function, SFB abundance and microbiota composition by 16S sequencing. We assessed the role of anti-tumour necrosis factor α on gut mucosa inflammation and T1D onset.ResultsWe show in models of autoimmune T1D a conserved loss of interleukin (IL)-17A, IL-22 and IL-23A in gut mucosa. Intestinal epithelial cell function was altered and gut integrity was impaired. These defects were associated with dysbiosis including progressive loss of SFB. Transfer of diabetogenic T-cells recapitulated these gut alterations, whereas induction of hyperglycaemia with no inflammation failed to do so. Moreover, anti-inflammatory treatment restored gut mucosa and immune cell function and dampened diabetes incidence.ConclusionOur results demonstrate that gut mucosa alterations and dysbiosis in T1D are primarily linked to inflammation rather than hyperglycaemia. Anti-inflammatory treatment preserves gut homeostasis and protective commensal flora reducing T1D incidence.

International audience; The ecological dynamics underlying the coexistence between antagonistic populations of bacteria and their viruses, bacteriophages (phages), in the mammalian gut microbiota remain poorly understood. We challenged a murine synthetic bacterial community with phages to study the factors allowing phages-bacteria coexistence. Coexistence was not dependent on the development of phage-resistant clones nor on the ability of phages to extend their host range. Instead, our data suggest that phage-inaccessible sites in the mucosa serve as a spatial refuge for bacteria. From there, bacteria disseminate in the gut lumen where they are predated by luminal phages fostering the presence of intestinal phage populations. The heterogeneous biogeography of microbes contributes to the long-term coexistence of phages with phage-susceptible bacteria. This observation could explain the persistence of intestinal phages in humans as well as the low efficiency of oral phage therapy against enteric pathogens in animal models and clinical trials.

International audience; Editorial

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.

International audience; Several diseases and vulnerabilities associated with genetic or microbial factors are more frequent among populations of Oceanian, Non-European, Non-Asian descent (ONENA). ONENA are specific and have long been isolated geographically. To our knowledge, there are no published official, quantitative, aggregated data on the populations impacted by these excess vulnerabilities in Oceania. We searched official census reports for updated estimates of the total population for each of the Pacific Island Countries and Territories (including Australia) and the US State of Hawaii, privileging local official statistical or censual sources. We multiplied the most recent total population estimate by the cumulative percentage of the ONENA population as determined in official reports. Including Australia and the US State of Hawaii, Oceania counts 27 countries and territories, populated in 2016 by approximately 41 M inhabitants (17 M not counting Australia) among which approximately 12.5 M (11.6 M not counting Australia) consider themselves of entire or partial ONENA ancestry. Specific genetic and microbiome traits of ONENA may be unique and need further investigation to adjust risk estimates, risk prevention, diagnostic and therapeutic strategies, to the benefit of populations in the Pacific and beyond.

The specific contribution of each bacterial species within a complex microbiota to the regulation of host lipid metabolism remains largely unknown. Using two model commensal microorganisms, L. paracasei and E. coli, we demonstrated that both bacterial species impacted host lipid metabolism in a diet-dependent manner and, notably, that L. paracasei-colonized mice but not E. coli-colonized mice resisted high-fat-diet-induced body weight gain. In addition, we set up cellular models of fatty acid absorption and secretion by enterocytes cocultured with bacteria and showed that, in vitro, both L. paracasei and E. coli inhibited lipid secretion, through increased intracellular fat storage and enhanced lipid catabolism, respectively., The gut microbiota contributes to nutrients absorption and metabolism by enterocytes, but the molecular mechanisms involved remain poorly understood, and most conclusions are inferred from studies comparing germfree and conventional animals colonized with diverse bacterial species. We selected two model commensal microorganisms, Escherichia coli and Lactobacillus paracasei, to assess the role of the small-intestinal microbiota in modulating lipid absorption and metabolism by the epithelium. Using an integrated approach encompassing cellular and murine models and combining metabolic parameters measurement, lipid droplet imaging, and gene expression analysis, we demonstrated that under homeostatic conditions, L. paracasei promotes fat storage in enterocytes, whereas E. coli enhances lipid catabolism and reduces chylomicron circulating levels. The Akt/mammalian target of sirolimus (mTOR) pathway is inhibited by both bacterial species in vitro, indicating that several regulatory pathways are involved in the distinct intracellular lipid outcomes associated with each bacterial species. Moreover, soluble bacterial factors partially reproduce the effects observed with live microorganisms. However, reduction of chylomicron circulating levels in E. coli-colonized animals is lost under high-fat-diet conditions, whereas it is potentiated by L. paracasei colonization accompanied by resistance to hypercholesterolemia and excess body weight gain.

We report here the complete genome sequence of Delftia tsuruhatensis CM13, isolated from murine proximal colonic tissue. The genome assembly using PacBio single-molecule real-time sequencing resulted in a single scaffold of 7.19 Mb.

Metagenomic analysis of the human intestinal microbiome has provided a wealth of information that allowed an exceptionally detailed description of its microbial content and physiological potential. It also set the basis for studies allowing correlation of alterations in the balance of this microbiota and the occurrence of a certain number of emerging diseases, such as inflammatory bowel diseases, obesity and diabetes, and possibly colorectal cancer. The time has come to give the intestinal microbiota in symbiosis with its host an experimental dimension. This brief review summarizes our attempt at developing a cellular microbiology of the mutualistic symbiosis established between the gut microbiota and the host intestinal surface. Particular attention is paid to the intestinal crypt, due to its role in epithelial regeneration. This article is part of the themed issue ‘The new bacteriology’.

International audience; Neisseria meningitidis is a life-threatening human bacterial pathogen responsible for pneumonia, sepsis, and meningitis. Meningococcal strains with reduced susceptibility to penicillin G (Pen(I)) carry a mutated penicillin-binding protein (PBP2) resulting in a modified peptidoglycan structure. Despite their antibiotic resistance, Pen(I) strains have failed to expand clonally. We analyzed the biological consequences of PBP2 alteration among clinical meningococcal strains and found that peptidoglycan modifications of the Pen(I) strain resulted in diminished in vitro Nod1-dependent proinflammatory activity. In an influenza virus-meningococcal sequential mouse model mimicking human disease, wild-type meningococci induced a Nod1-dependent inflammatory response, colonizing the lungs and surviving in the blood. In contrast, isogenic Pen(I) strains were attenuated for such response and were out-competed by meningococci sensitive to penicillin G. Our results suggest that antibiotic resistance imposes a cost to the success of the pathogen and may potentially explain the lack of clonal expansion of Pen(I) strains.

International audience; Diet is among the most important factors contributing to intestinal homeostasis, and basic functions performed by the small intestine need to be tightly preserved to maintain health. Little is known about the direct impact of high-fat (HF) diet on small-intestinal mucosal defenses and spatial distribution of the microbiota during the early phase of its administration. We observed that only 30 d after HF diet initiation, the intervillous zone of the ileum-which is usually described as free of bacteria-became occupied by a dense microbiota. In addition to affecting its spatial distribution, HF diet also drastically affected microbiota composition with a profile characterized by the expansion of Firmicutes (appearance of Erysipelotrichi), Proteobacteria (Desulfovibrionales) and Verrucomicrobia, and decrease of Bacteroidetes (family S24-7) and Candidatus arthromitus A decrease in antimicrobial peptide expression was predominantly observed in the ileum where bacterial density appeared highest. In addition, HF diet increased intestinal permeability and decreased cystic fibrosis transmembrane conductance regulator (Cftr) and the Na-K-2Cl cotransporter 1 (Nkcc1) gene and protein expressions, leading to a decrease in ileal secretion of chloride, likely responsible for massive alteration in mucus phenotype. This complex phenotype triggered by HF diet at the interface between the microbiota and the mucosal surface was reversed when the diet was switched back to standard composition or when mice were treated for 1 wk with rosiglitazone, a specific agonist of peroxisome proliferator-activated receptor-γ (PPAR-γ). Moreover, weaker expression of antimicrobial peptide-encoding genes and intervillous bacterial colonization were observed in Ppar-γ-deficient mice, highlighting the major role of lipids in modulation of mucosal immune defenses.

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

We compared gene expression in blood neutrophils (polymorphonuclear leukocytes, or PMNs) collected from healthy subjects with those of cystic fibrosis (CF) patients devoid of bacterial colonization. Macroarray analysis of 1050 genes revealed upregulation of 62 genes and downregulation expression of 27 genes in CF blood PMNs. Among upregulated genes were those coding for vitronectin, some chemokines (particularly CCL17 and CCL18), some interleukin (IL) receptors (IL-3, IL-8, IL-10, IL-12), all three colony-stimulating factors (G-, M-, GM-CSF), numerous genes coding for molecules involved in signal transduction, and a few genes under the control of gamma-interferon. In contrast, none of the genes coding for adhesion molecules were modulated. The upregulation of six genes in CF PMNs (coding for thrombospondin-1, G-CSF, CXCL10, CCL17, IKKvarepsilon, IL-10Ra) was further confirmed by qPCR. In addition, the increased presence of G-CSF, CCL17, and CXCL10 was confirmed by ELISA in supernatants of neutrophils from CF patients. When comparison was performed between blood and airway PMNs of CF patients, there was a limited difference in terms of gene expression. Only the mRNA expression of amphiregulin and tumor necrosis factor (TNF) receptor p55 were significantly higher in airway PMNs. The presence of amphiregulin was confirmed by ELISA in the sputum of CF patients, suggesting for the first time a role of amphiregulin in cystic fibrosis. Altogether, this study clearly demonstrates that blood PMNs from CF patients display a profound modification of gene expression profile associated with the disease, suggesting a state of activation of these cells.

Here, we report three genome sequences of bacteria isolated from murine proximal colonic tissue and identified as Acinetobacter parvus CM11, Acinetobacter radioresistens CM38, and Stenotrophomonas maltophilia BR12.

International audience; Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable identification of transposon insertion sites in a L. casei mutant library of 9250 mutants. DNA extraction procedure based on silica membranes in 96-column format was optimized to obtain genomic DNA from a large number of mutants. Then reliable direct genomic sequencing was improved to fit the obtained genomic DNA extracts. Using this procedure, readable and identifiable sequences were obtained for 87% of the L. casei mutants. This method extends the applications of a library of this type, reduces the number of insertions needed to be screened, and allows selection of specific mutants from an arrayed and stored mutant library. This method is applicable to any already existing mutant library (obtained by transposon or insertional mutagenesis) and could be useful for other bacterial species, especially for highly lysis-resistant bacteria species such as lactic acid bacteria.

International audience; Intestinal epithelial cells (IEC) play a central role in innate and acquired mucosal immunity. They ensure early signaling to trigger an inflammatory response against pathogens. Moreover, IEC mediate transcytosis of dimeric IgA (dIgA), through the polymeric-immunoglobulin receptor (pIgR), to provide secretory IgA, the major protective Ig in mucosal secretions. Using an in vitro model of polarized IEC, we describe an additional anti-inflammatory mechanism of dIgA-mediated protection against intracellular bacterial components involved in the proinflammatory activation of IEC. Specific dIgA colocalizes to lipopolysaccharide (LPS) in the apical recycling endosome compartment, preventing LPS-induced NF-kappaB translocation and subsequent proinflammatory response. Thus, intracellular neutralization by dIgA limits the acute local inflammation induced by proinflammatory pathogen-associated molecular patterns such as LPS.

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment., Author Summary Shigellosis is a major global health concern. Recently, a shift in the dominance of types of Shigella that cause disease has been observed with S. sonnei increasing in prevalence under improved socio-economic conditions leading to a replacement of S. flexneri. Most of the knowledge of Shigella disease mechanisms has been obtained from studies of S. flexneri. We found that S. sonnei possesses a high molecular weight sugar capsule that is absent in S. flexneri 2a. Removal of the capsule made S. sonnei bacteria highly invasive in vitro and strongly inflammatory in vivo, but in contrast, there was reduced spreading of these mutant bacteria from the gut to peripheral organs in rabbits and higher sensitivity to complement-mediated lysis. Thus, the capsule plays a role in both, invasion and protection of the bacteria against the innate immune defense of the host during the infection. These findings indicate that the capsule is an important virulence factor for S. sonnei. Based on the substantial changes in pathogenesis observed upon removal and overexpression of the capsule, we hypothesize that its level of expression may be under significant evolutionary pressure.

International audience; Although the composition of the gut microbiota and its symbiotic contribution to key host physiological functions are well established, little is known as yet about the bacterial factors that account for this symbiosis. We selected Lactobacillus casei as a model microorganism to proceed to genomewide identification of the functions required for a symbiont to establish colonization in the gut. As a result of our recent development of a transposon-mutagenesis tool that overcomes the barrier that had prevented L. casei random mutagenesis, we developed a signature-tagged mutagenesis approach combining whole-genome reverse genetics using a set of tagged transposons and in vivo screening using the rabbit ligated ileal loop model. After sequencing transposon insertion sites in 9,250 random mutants, we assembled a library of 1,110 independent mutants, all disrupted in a different gene, that provides a representative view of the L. casei genome. By determining the relative quantity of each of the 1,110 mutants before and after the in vivo challenge, we identified a core of 47 L. casei genes necessary for its establishment in the gut. They are involved in housekeeping functions, metabolism (sugar, amino acids), cell wall biogenesis, and adaptation to environment. Hence we provide what is, to our knowledge, the first global functional genomics analysis of L. casei symbiosis.

We established in previous studies that a constitutive lipopolysaccharide (LPS) receptor of low affinity is present on mouse bone marrow granulocytes (BMG). This yet-unidentified receptor is involved in the LPS-induced expression of a second LPS receptor, CD14. Because it has been claimed that L-selectin (CD62L) is a low-affinity LPS receptor in mature granulocytes (polymorphonuclear leukocytes), it may be asked whether this molecule could be the constitutive LPS receptor in BMG. We show in this study thatl-selectin is constitutively present on BMG and is down-regulated after exposure of the cells to LPS. A phorbol ester induced a down-regulation of CD62L and blocked the LPS-induced expression of CD14. However, a metalloproteinase inhibitor (BB-3103) blocked the former but not the latter effect of PMA. We also observed an absence of cross-reactivity between LPS and a CD62L ligand (fucoidan) in binding studies with radiolabeled derivatives of the two agents. Furthermore, BMG froml-selectin-deficient mice expressed normal levels of CD14 in response to LPS. Taken together, these results demonstrate that in BMG,l-selectin is not the constitutive LPS receptor required for the LPS-induced expression of CD14.

We have previously shown that stimulation of mouse bone marrow granulocytes (BMC) by lipopolysaccharide (LPS) induces the expression of CD14. We found here that phorbol 12-myristate 13-acetate (PMA) blocks this LPS effect. The aim of this study was to investigate the mechanism by which PMA can block the LPS signaling pathway in BMC. The unmodified binding of a radiolabeled LPS in PMA-treated cells indicated that the PMA effect was not the consequence of a shedding or an internalization of the LPS receptor, but was rather due to a biochemical event that follows the interaction of LPS with its receptor. The observations that a selective activator of protein kinase C (PKC)-alpha (sapintoxin D) mimics the PMA effect, whereas a selective PKC-alpha inhibitor (Ro-320432) antagonizes this effect, suggest a regulatory role of PKC-alpha in the LPS signaling pathway in mouse BMC.

We established in previous studies that the binding of Salmonella lipopolysaccharide (LPS) to constitutive receptors of low affinity triggers the expression of the inducible LPS-binding molecule CD14 in bone marrow cells (BMC) of C3H/HeOU mice, but not in BMC from C3H/HeJ mice. We show in this study that BMC from C3H/HeJ and C57BL/10ScCr mice do not express CD14 after exposure to LPSs from Salmonella enterica and Bordetella pertussis, but do express this marker when treated with several LPSs from Rhizobiaceae, or their lipid A fragments. This shows that the constitutive LPS receptor in BMC from C3H/HeJ and C57BL/10ScCr mice is fully able to trigger a complete signalling cascade. Results of cross-inhibition of the binding of radiolabelled LPS indicated that active LPSs (from R. species Sin-1 and R. galegae) and inactive LPSs (from S. enterica and B. pertussis) bind to the same site of the constitutive LPS receptor of C3H/HeJ cells. Furthermore, binding of R. species Sin-1 LPS, and signalling induced by this LPS, were both inhibited by pre-exposure of C3H/HeJ cells to B. pertussis lipid A. This correlation between binding and signalling suggests that in C3H/HeJ cells, the constitutive receptor, which recognizes a large panel of LPSs from different origins, appears selectively unable to be activated by some particular LPSs, such as those of Enterobacteria and Bordetella.

Little is known about the mechanisms of lipopolysaccharide (LPS) signaling in immature cells that do not express the LPS receptor CD14 yet. Bone marrow granulocytes do not constitutively express CD14 but can be stimulated by low doses of LPS in the absence of serum and then express an inducible form of LPS receptor (iLpsR). We show that in addition to LPS, cholera toxin (CT) and various cyclic AMP (cAMP) analogs can also induce the expression of iLpsR, which was identified as CD14. Induction was independent of intracellular cAMP. The hypothesis that cAMP analogs act via a cell surface receptor was suggested by the unresponsiveness of trypsin-treated cells to these inducers and by the specific binding of [ 3 H]cAMP to the cells. This binding was not inhibited by LPS or CT but was inhibited by various purine derivatives. However, the receptor involved is not a conventional purinoreceptor since both an agonist and an antagonist of such receptors were able to induce iLpsR expression. The results suggest that cAMP analogs and other purine derivatives induce iLpsR after interaction with an unconventional, trypsin-sensitive, purinoreceptor distinct from LPS and CT receptors.

A new method to obtain the gate coupling ratio (@a"g) and oxide trapped charge (Q"o"x) as a result of cycling in flash memory cells is described here. Three cells with an equivalent physical structure but different erase characteristics are measured. The threshold changes versus erase times are fitted to the charge removal rate calculated based on Fowler-Nordheim (FN) tunneling and the capacitive relations among all nodes. The extracted @a"g is independent of technologies and this method is particular useful when the profile of the floating gate is not traditionally rectangular owing to advanced processes such as trapezoidal poly etch or a poly-spacer addition on the floating gate sidewall. The Q"o"x can also be determined once @a"g is extracted.

Although lipopolysaccharide (LPS)-induced overproduction of cytokines, involved in the pathogenesis of septic shock, occupies the spotlight of endotoxin research, another LPS effect, the differentiation of various cell types including haematopoietic bone marrow cells (BMC), which is probably related to its radioprotective activity, deserves equal attention. We have previously established that nanomolar concentrations of LPS trigger in human BMC the expression of CD14 by an induction mechanism independent of CD14 or any other molecule anchored to the cell membrane by a glycosyl phosphatidylinositol glycolipid. We now show that this LPS-induced stimulation is triggered by the binding of a small number of LPS molecules (13,000 molecules/cell) to constitutive LPS receptors of low affinity (Kd = 480 nM). This interaction, which was inhibited by a synthetic LPS antagonist, appeared specific, reversible, saturable, time- and temperature-dependent, but was independent of divalent cations, and was inhibited by serum. Exposure of BMC to LPS did not induce a down-modulation of these receptors, but enhanced their sensitivity to trypsin degradation. Inhibition of LPS binding following different treatments correlated with inhibition of BMC stimulation, thus suggesting that the sparse constitutive receptors of low affinity are efficient signalling receptors for LPS.

International audience; Rhinoscleroma is a human specific chronic disease characterized by the formation of granuloma in the airways, caused by the bacterium Klebsiella pneumoniae subspecies rhinoscleromatis, a species very closely related to K. pneumoniae subspecies pneumoniae. It is characterized by the appearance of specific foamy macrophages called Mikulicz cells. However, very little is known about the pathophysiological processes underlying rhinoscleroma. Herein, we characterized a murine model recapitulating the formation of Mikulicz cells in lungs and identified them as atypical inflammatory monocytes specifically recruited from the bone marrow upon K. rhinoscleromatis infection in a CCR2-independent manner. While K. pneumoniae and K. rhinoscleromatis infections induced a classical inflammatory reaction, K. rhinoscleromatis infection was characterized by a strong production of IL-10 concomitant to the appearance of Mikulicz cells. Strikingly, in the absence of IL-10, very few Mikulicz cells were observed, confirming a crucial role of IL-10 in the establishment of a proper environment leading to the maturation of these atypical monocytes. This is the first characterization of the environment leading to Mikulicz cells maturation and their identification as inflammatory monocytes.

International audience

In an attempt to explore the microbial content of functionally critical niches of the mouse gastrointestinal tract, we targeted molecular microbial diagnostics of the crypts that contain the intestinal stem cells, which account for epithelial regeneration. As current evidence indicates, the gut microbiota affects epithelial regeneration; bacteria that are likely to primarily participate in this essential step of the gut, microbiota cross talk, have been identified. We show in this article that only the cecal and colonic crypts harbor resident microbiota in the mouse and that regardless of the line and breeding origin of these mice, this bacterial population is unexpectedly dominated by aerobic genera. Interestingly, this microbiota resembles the restricted microbiota found in the midgut of invertebrates; thus, the presence of our so-called “crypt-specific core microbiota” (CSCM) in the mouse colon potentially reflects a coevolutionary process under selective conditions that can now be addressed. We suggest that CSCM could play both a protective and a homeostatic role within the colon. This article is setting the bases for such studies, particularly by providing a bona fide—and essentially cultivable—crypt microbiota of reference., IMPORTANCE Metagenomic typing of the whole-gut luminal microbiome was recently provided, revealing great opportunities for physiological and physiopathological analysis of the host-microbiota interface. On this basis, it appears increasingly important to analyze which niches of the gut exposed to a particular microbiota are of major functional importance, specifically focusing on the crypt, which accounts for permanent epithelial renewal, and to analyze how this microbiota compares to its luminal counterpart in composition and quantity. Crypt-specific core microbiotas may show themselves as important elements regarding crypt protection and homeostasis of its functions.

We have previously established that lipopolysaccharide (LPS) induces the expression of new specific LPS-binding sites (LpsR) in mouse bone marrow cells (BMC). We now show that exposure of human BMC to LPS elicits the production of both CD14 molecules (detectable with monoclonal antibody My4) and LpsR (detectable with fluorescein isothiocyanate-LPS). Pretreatment of stimulated human BMC with My4 inhibited the binding of fluorescein isothiocyanate-LPS. The stimulation of human BMC, but not mouse BMC, required the presence of serum. Other characteristics of mouse and human BMC examined were very similar. Their inducible LpsR interacted with the lipid moieties of LPS and Leishmania donovani lipophosphoglycan and with a soluble preparation of peptidoglycan. Moreover, mouse and human LpsR were susceptible to treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), thus suggesting that both are PI-anchored CD14 molecules. Neither LpsR appeared able to interact with a synthetic LPS antagonist (compound PPDm2) structurally related to the lipid region of LPS. However, PPDm2 blocked LPS-induced expression of LpsR in both BMC. Furthermore, in both species, pretreatment of BMC with PI-PLC did not prevent the cells from expressing LpsR in response to LPS. The results support the hypothesis that the elicited LpsR of mouse and human BMC is an inducible form of CD14, whereas the putative "signaling LPS receptor" of these cells is not CD14 or any other PI-anchored molecule.

A monoclonal antibody (E1), which reacts with the 3-deoxy-D-manno-2-octulosonic acid region of lipopolysaccharides belonging to the rough Re chemotype (LPS-Re) was used to analyze in vivo and in vitro effects of endotoxin. E1 inhibited LPS-Re-induced activation of ZOZ/3 pre-B cells (expression of surface immunoglobulins) and mature splenic B cells (DNA synthesis) and blocked the binding of biotin-labeled LPS-Re to mouse peritoneal macrophages. However, other effects elicited by LPS-Re, such as the production of interleukin 6 and tumor necrosis factor α by macrophages, and the acute lethality in galactosamine-sensitized mice, were not inhibited by E1. The results suggest that the specificity of the LPS receptor which triggers the production of cytokines in macrophages is different from that of the LPS receptor which induces activation of pre-B and B lymphocytes, and is also different from that of the major LPS-binding sites of macrophages. This is consistent with the hypothesis that the large majority or LPS-binding sites of macrophages do not trigger cytokine production, and that the small number of "signaling LPS receptors" of macrophages have fine specificities which are different from those of B cells.

BACKGROUND: Shigella, an enteroinvasive bacteria induces a major inflammatory response responsible for acute rectocolitis in humans. However, early effect of Shigella flexneri (S. flexneri) infection upon the human mucosa and its microenvironement, in particular the enteric nervous system, remains currently unknown. Therefore, in this study, we sought to characterize ex vivo the early events of shigellosis in a model of human colonic explants. In particular, we aimed at identifying factors produced by S. flexneri and responsible for the lesions of the barrier. We also aimed at determining the putative lesions of the enteric nervous system induced by S. flexneri. METHODOLOGY/PRINCIPAL FINDINGS: We first showed that, following 3 h of infection, the invasive but not the non-invasive strain of S. flexneri induced significant desquamation of the intestinal epithelial barrier and a reduction of epithelial height. These changes were significantly reduced following infection with SepA deficient S. flexneri strains. Secondly, S. flexneri induced rapid neuronal morphological alterations suggestive of cell death in enteric submucosal neurones. These alterations were associated with a significant increase in the proportion of vasoactive intestinal peptide (VIP) immunoreactive (IR) neurons but not in total VIP levels. The NMDA receptor antagonist MK-801 blocked neuronal morphological changes induced by S. flexneri, but not the increase in the proportion of VIP-IR. CONCLUSIONS/SIGNIFICANCE: This human explant model can be used to gain better insight into the early pathogenic events following S. flexneri infection and the mechanisms involved.

According to a classical view of bacterial-host interactions at intestinal surfaces, the commensal microbiota establishes tolerance, and invasive pathogens cause stereotypic inflammation. The reality is more complex, marked by a “ménage à trois” situation encompassing three emerging concepts: (1) pathogens take advantage of inflammation to cross the epithelial barrier, (2) pathogens reduce the commensal flora to invade their niche, and (3) pathogens express dedicated effectors that modulate inflammation.

The intestinal tract of adult mice is naturally resistant to infection by Shigella, the causative agent of bacillary dysentery in humans. Conversely, newborn mice are highly susceptible to intragastric Shigella infection and develop inflammatory lesions of the jejunal mucosa, very similar to those observed in the colon of dysenteric patients. However, the susceptibility period is short and one week after birth, animals have acquired a status of resistance characteristic of adult animals. To identify the developmental changes controlling the switch from disease susceptibility to resistance, we performed global gene expression analysis on noninfected and infected intestinal tissues taken from 4-day- and 7-day-old animals. Transcriptomic analysis of 4-day-old mice infected with the invasive Shigella strain showed a profile reflecting a strong inflammatory response with no evidence for retro-control, suggesting that the invasive process had occurred, whereas inflammation had been controlled after infection with the noninvasive strain. Differences in gene expression profiles between noninfected 4-day- and 7-day-old mice corresponded mainly to genes encoding anti-microbial peptides and proteases, suggesting that these molecules could be candidates for host antimicrobial resistance in the course of shigellosis. Indeed, expression of genes specific of Paneth cells was higher in 7-day- than in 4-day-old mice, and histological analysis indicated that Paneth cells were present only at day 7. Finally, using Sox9flox/flox-vil-cre mice, we showed that depletion of Paneth cells restored the susceptibility to Shigella of 7-day-old mice, clearly indicating that Paneth cells development is crucial for the clearance of intestinal infection.

The relative paucity of data about the development of the stem cell pool present in the spleen prompted this study. During in vitro cultures of B-enriched lymphocytes from mouse spleens and in the presence of a culture supernatant of WEHI-3 cells (WEHI-SUP), a population of cells expressing the BP-1 antigen appears progressively, reaches an optimal size 8 days after initiation of the culture, and disappears on day 28. In 8-day-old cultures, a minor population of cells bearing both BP-1 and B220 can be detected. The growth of this cell population, with characteristics of the B lymphoid lineage (pro-B), is strictly dependent on the presence of WEHI-SUP in the medium. After 2 weeks of culture, the BP-1 antigen is expressed on a cell population, which is essentially constituted of B220-, polynuclear cells. The BP-1 antigen, which is considered as characteristic of early cells of the B lymphoid lineage, can therefore also be expressed on cells of the myeloid lineage. The injection of BP-1+ or B220+ cells in irradiated mice can hardly reconstitute their B cell pool, whereas BP-1- and B220- cells are much more efficient in vivo progenitors of this cell lineage.

Phosphorylation of histone H3 at Ser10 increases chromatin accessibility to transcription factor NF-kappaB on a subset of genes involved in immune responses. Here we report that a bacterial pathogen abrogated phosphorylation of histone H3 to 'shape' the transcriptional responses of infected host cells. We identify the Shigella flexneri protein effector OspF as a dually specific phosphatase that dephosphorylated mitogen-activated protein kinases in the nucleus, thus preventing histone H3 phosphorylation at Ser10 in a gene-specific way. That activity of OspF enabled shigella to block the activation of a subset of NF-kappaB-responsive genes, leading to compromised recruitment of polymorphonuclear leukocytes to infected tissues. S. flexneri has thus evolved the capacity to precisely modulate host cell epigenetic 'information' as a strategy for repressing innate immunity.

Interleukin-10 (IL-10) is a well known anti-inflammatory cytokine. However, we previously showed that it could present pro-inflammatory properties on human monocytes in the absence of adherence. In the present study, using macroarray technology, we analyzed the effects of IL-10 and adherence on the expression of 1050 genes in human monocytes cultured for 3 hours on plastic or Teflon(R) (to avoid adherence). Adherence alone induced specifically the expression of 12 genes and repressed that of 25 genes. In adherent monocytes, IL-10 induced the expression of 21 genes and repressed that of 50 genes. In non-adherent monocytes, IL-10 induced the expression of 45 genes and repressed that of 67. Only 3 common genes were induced while 35 common genes were repressed by IL-10 in the two culture conditions. Interestingly, we showed that IL-10 modulated conversely on Teflon(R) and plastic the expression of 16 genes, of which SOCS molecules, coproporphyrinogen oxidase, matrix metalloproteinases and complement receptor-1 (CD35). This study demonstrates that adherence has profound modulatory effects on the properties and the signaling induced by IL-10. The discovery that IL-10 can inhibit the production of coproporphyrinogen oxidase (an enzyme involved in the synthesis of heme) may shed some lights on the mechanisms of anaemia induced by IL-10. Furthermore, the inhibition of the expression of SOCS1 by IL-10 in the absence of adherence, may explain its priming effects on a subsequent LPS stimulation that we previously described.

International audience; Shigella infection is characterized by the induction of acute inflammation, which is responsible for the massive tissue destruction of the intestinal mucosa. A murine model would be a valuable tool for gaining a better understanding of the physiopathology of shigellosis and the host immune response to Shigella infection, but adult mice do not develop disease upon oral inoculation. We therefore attempted to develop a model of infection in newborn mice. Four-day-old mice inoculated with 50 microl of 5 x 10(9) invasive wild-type Shigella flexneri 5a were susceptible to bacterial infection, but mice inoculated with the non-invasive strain BS176 were not. Histologically, 4-day-old mice infected with the invasive strain presented intestinal lesions and inflammation similar to those described in patients with shigellosis. Moreover, cytokine and chemokine responses consistent with inflammation were observed. Lower bacterial inocula induced less severe intestinal damage. In contrast, 5-day-old mice inoculated with either the invasive or the non-invasive strain were not infected. We have thus established a mouse model that is suitable for the study of the pathogenesis of intestinal Shigella infection.

Invasion of the human colonic epithelium by Shigella flexneri causes inflammation that disrupts the intestinal barrier. Invaded intestinal epithelial cells are the major source of mediators recruiting the inflammatory infiltrate. To better characterize the global response of intestinal epithelial cells to Shigella invasion, Caco-2 cells were infected by an invasive isolate of S. flexneri 5a, and their transcriptome was analyzed by Affymetrix (Santa Clara, CA) microarrays (12,000 genes) and compared with these elicited by a non-invasive Shigella mutant and tumor necrosis factor (TNF)-alpha. The invasive and non-invasive strains enhanced transcription of a common pattern of 240 genes, among which genes encoding isoforms of cytochrome P-450 were induced. These genes were not induced by TNF-alpha. Conversely, both the invasive strain and TNF-alpha induced a common set of 18 genes, mainly encoding proinflammatory molecules. They also induced specific sets of genes. The transcriptome induced by the invasive strain was characterized by the induction of early genes (i.e. expressed within the first 45 min of invasion) and late genes (i.e. after 60 min of invasion) whose pattern was strongly biased toward stimulation of granulopoiesis, chemoattraction, activation, and adherence of polymorphonuclear leukocytes. When compared with a non-invasive Shigella and TNF-alpha, invasive Shigella induced a narrow transcriptome that seems to program infected epithelial cells to recruit a mucosal polymorphonuclear leukocyte to infiltrate. Dramatic increase in IL-8 gene transcription points to this chemokine as the major molecule orchestrating mucosal inflammation in shigellosis.

We reported previously that bone marrow granulocytes respond to small amounts of enterobacterial lipopolysaccharide (LPS) via a CD14-independent and TLR4-mediated mechanism by de novo expression of an inducible receptor (CD14) and by down-modulation of a constitutive receptor (L-selectin). In this report we address another effect of LPS: the down-regulation of receptors for tumor necrosis factor-alpha. In mouse bone marrow cells (BMC), this down-regulation is detectable soon (20 min) after exposure of the cells to low levels (0.5 ng/ml) of LPS. This temperature-dependent effect is rather selective for LPS and requires the presence of a conventional lipid A structure in the LPS molecule and a functional TLR4 molecule in the cells. The down-modulation, due to a shedding of the receptors, is blocked by p38 MAPK inhibitors, by a furin inhibitor, and by three metalloproteinase inhibitors (BB-3103, TIMP-2, and TIMP-3). In contrast, inhibitors of MEK, protein kinase C, cAMP-dependent protein kinase, and kinases of the Src family do not block the shedding. Analysis of BMC from mice lacking tumor necrosis factor receptor-1 (CD120a-/-) or tumor necrosis factor receptor-2 (CD120b-/-) indicates that the LPS-induced shedding is specific for CD120b. Thus, exposure of BMC to LPS triggers a rapid shedding of CD120b via a protein kinase C- and Src-independent pathway mediated by p38 MAPK, furin, and metalloproteinase. The additive effects of furin and metalloproteinase inhibitors suggest that these enzymes are involved in parallel shedding pathways.

In addition to their effects on alveolar surface tension, some components of lung surfactant also have immunological functions. We found recently that the hydrophobic lung surfactant protein SP-C specifically binds to the lipid A region of lipopolysaccharide (LPS). In this study, we show that SP-C also interacts with CD14. Four observations showed cross talk between the three molecules SP-C, LPS, and CD14. (i) Like LBP, SP-C allows the binding of a fluorescent LPS to cells expressing CD14 (the other surfactant components were ineffective). (ii) Recombinant radiolabeled CD14 and SP-C (or a synthetic analog of SP-C) interact in a dose-dependent manner. (iii) LPS blocks the binding of radiolabeled CD14 to SP-C-coated wells. (iv) SP-C enhances the binding of radiolabeled CD14 to LPS-coated wells. These results, obtained with native murine SP-C and with three synthetic analogs, suggest that LPS and CD14 interact with the same region of SP-C and that binding of SP-C modifies the conformation of CD14 or the accessibility of its LPS-binding site, allowing it to bind LPS. This ability of SP-C to interact with the pattern recognition molecule CD14 extends the possible immunological targets of SP-C to a large panel of microorganisms that can enter the airways.

Lipopolysaccharide (LPS) derived from enterobacteria elicit in several cell types cellular responses that are restricted in the use of Toll-like receptor 4 (TLR4) as the principal signal-transducing molecule. A tendency to consider enterobacterial LPS as a prototypic LPS led some authors to present this mechanism as a paradigm accounting for all LPSs in all cell types. However, the structural diversity of LPS does not allow such a general statement. By using LPSs from bacteria that do not belong to the Enterobacteriaceae, we show that in bone marrow cells (BMCs) the LPS of Rhizobium species Sin-1 and of three strains of Legionella pneumophila require TLR2 rather than TLR4 to elicit the expression of CD14. In addition, exposure of BMCs from TLR4-deficient (C3H/HeJ) mice to the lipid A fragment of the Bordetella pertussis LPS inhibits their activation by the Legionella lipid A. The data show selective action of different LPSs via different TLRs, and suggest that TLR2 can interact with many lipid A structures, leading to either agonistic or specific antagonistic effects.

Lipopolysaccharide (LPS) of Gram-negative bacteria interacts with a CD14-independent receptor of mouse bone marrow granulocytes (BMC), and triggers in these cells the expression of CD14, an inducible type of LPS receptor (iLpsR). This particular response of BMC to LPS required the activation of protein tyrosine kinase and p38 MAP kinase. The inhibition of the LPS effect by the MEK inhibitor PD-98059 suggested that the ERK pathway was also involved. Unexpectedly, protein kinase C, myosin light chain kinase, cAMP-, cGMP-, and Ca(2+)/calmodulin-dependent kinases, as well as ecto-protein kinases, were not required for iLpsR expression. However, other yet unidentified serine/threonine protein kinase(s) were implied since the BMC response to LPS was markedly reduced after exposure to three inhibitors of such kinases (K-252a, H-7, and KT-5823). The atypical kinase requirements observed in this study may be due either to a novel signaling LPS receptor complex present in BMC, or to the particular events involved in CD14 biosynthesis.

We established previously that lipopolysaccharide (LPS) can induce the expression of LPS-binding sites on bone marrow cells (BMC). We now report that staurosporine (STP), a glycosylated indolocarbazole alkaloid with potent inhibitory activity for various protein kinases, can induce the same effect. With both agents, the newly expressed LPS receptor was found to be CD14. The STP-induced effect was independent of its protein kinase inhibitory activity because several other protein kinase inhibitors, such as the indolocarbazole K-252a, the bisindolylmaleimide RO-31–8220, the perylenequinone calphostin C, and the isoquinolinesulfonamide H7, did not induce CD14 expression. The observation that the STP analog K-252a with an identical polyaromatic aglycon moiety was inactive yet the analog UCN-01 with an identical glycoside ring was active suggests that the induction of CD14 expression is triggered by the sugar moiety of STP. Three lines of evidence show that the mechanism of CD14 expression induced by STP differs from that induced by LPS: (i) unlike LPS, STP can stimulate BMC from LPS-unresponsive C3H/HeJ mice, (ii) LPS and STP effects are additive at a saturating dose of LPS, and (iii) the protein kinase inhibitor K-252a inhibits the LPS-induced but not STP-induced stimulation. Therefore, our findings show that both a protein kinase-dependent (LPS-induced) and a protein kinase-independent (STP-induced) mechanism can lead to the expression of the LPS receptor CD14 on BMC. We also found that the STP-induced stimulation of BMC is modulated by cyclosporin A, vinblastine, and verapamil. This observation may suggest that the inducible effect of STP could be initiated by its interaction with P-glycoprotein, a membrane pump with drug efflux function that plays a critical role in the multidrug resistance of cancer cells.

In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA) were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.