Intestinal mucosal adherence and translocation of commensal bacteria at the early onset of type 2 diabetes: molecular mechanisms and probiotic treatmentстатья из журнала
Аннотация: Research Article3 August 2011Open Access Intestinal mucosal adherence and translocation of commensal bacteria at the early onset of type 2 diabetes: molecular mechanisms and probiotic treatment Jacques Amar Corresponding Author Jacques Amar [email protected] Rangueil Hospital, Department of Therapeutics, Toulouse, France Institut National de la Santé et de la Recherche Médicale (INSERM), U558, Toulouse, France These authors contributed equally to this work. Search for more papers by this author Chantal Chabo Chantal Chabo Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France These authors contributed equally to this work. Search for more papers by this author Aurélie Waget Aurélie Waget Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Pascale Klopp Pascale Klopp Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Christelle Vachoux Christelle Vachoux Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Luis G. Bermúdez-Humarán Luis G. Bermúdez-Humarán Unité d'Ecologie et de Physiologie du Système Digestif, INRA, Domaine de Vilvert, Jouy en Josas Cedex, France Search for more papers by this author Natalia Smirnova Natalia Smirnova Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Mathieu Bergé Mathieu Bergé Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France Centre National de la Recherche Scientifique, LMGM-UMR5100, Toulouse, France Search for more papers by this author Thierry Sulpice Thierry Sulpice Physiogenex SAS, Prologue Biotech, Labège Innopole, France Search for more papers by this author Sampo Lahtinen Sampo Lahtinen Danisco Health & Nutrition, Kantvik, Finland Search for more papers by this author Arthur Ouwehand Arthur Ouwehand Danisco Health & Nutrition, Kantvik, Finland Search for more papers by this author Philippe Langella Philippe Langella Unité d'Ecologie et de Physiologie du Système Digestif, INRA, Domaine de Vilvert, Jouy en Josas Cedex, France Search for more papers by this author Nina Rautonen Nina Rautonen Danisco Health & Nutrition, Kantvik, Finland Search for more papers by this author Philippe J. Sansonetti Philippe J. Sansonetti Unité de Pathogénie Microbienne Moléculaire and Unité INSERM 786, Institut Pasteur, Paris Cedex 15, France Search for more papers by this author Rémy Burcelin Corresponding Author Rémy Burcelin [email protected] Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Jacques Amar Corresponding Author Jacques Amar [email protected] Rangueil Hospital, Department of Therapeutics, Toulouse, France Institut National de la Santé et de la Recherche Médicale (INSERM), U558, Toulouse, France These authors contributed equally to this work. Search for more papers by this author Chantal Chabo Chantal Chabo Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France These authors contributed equally to this work. Search for more papers by this author Aurélie Waget Aurélie Waget Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Pascale Klopp Pascale Klopp Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Christelle Vachoux Christelle Vachoux Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Luis G. Bermúdez-Humarán Luis G. Bermúdez-Humarán Unité d'Ecologie et de Physiologie du Système Digestif, INRA, Domaine de Vilvert, Jouy en Josas Cedex, France Search for more papers by this author Natalia Smirnova Natalia Smirnova Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Mathieu Bergé Mathieu Bergé Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France Centre National de la Recherche Scientifique, LMGM-UMR5100, Toulouse, France Search for more papers by this author Thierry Sulpice Thierry Sulpice Physiogenex SAS, Prologue Biotech, Labège Innopole, France Search for more papers by this author Sampo Lahtinen Sampo Lahtinen Danisco Health & Nutrition, Kantvik, Finland Search for more papers by this author Arthur Ouwehand Arthur Ouwehand Danisco Health & Nutrition, Kantvik, Finland Search for more papers by this author Philippe Langella Philippe Langella Unité d'Ecologie et de Physiologie du Système Digestif, INRA, Domaine de Vilvert, Jouy en Josas Cedex, France Search for more papers by this author Nina Rautonen Nina Rautonen Danisco Health & Nutrition, Kantvik, Finland Search for more papers by this author Philippe J. Sansonetti Philippe J. Sansonetti Unité de Pathogénie Microbienne Moléculaire and Unité INSERM 786, Institut Pasteur, Paris Cedex 15, France Search for more papers by this author Rémy Burcelin Corresponding Author Rémy Burcelin [email protected] Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France Search for more papers by this author Author Information Jacques Amar *,1,2, Chantal Chabo3, Aurélie Waget3, Pascale Klopp3, Christelle Vachoux3, Luis G. Bermúdez-Humarán4, Natalia Smirnova3, Mathieu Bergé5,6, Thierry Sulpice7, Sampo Lahtinen8, Arthur Ouwehand8, Philippe Langella4, Nina Rautonen8, Philippe J. Sansonetti9 and Rémy Burcelin *,3 1Rangueil Hospital, Department of Therapeutics, Toulouse, France 2Institut National de la Santé et de la Recherche Médicale (INSERM), U558, Toulouse, France 3Institut National de la Santé et de la Recherche Médicale (INSERM), U1048, Institut de Recherche sur les Maladies Métaboliques et Cardiovasculaires de Rangueil (I2MC), Toulouse, France 4Unité d'Ecologie et de Physiologie du Système Digestif, INRA, Domaine de Vilvert, Jouy en Josas Cedex, France 5Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France 6Centre National de la Recherche Scientifique, LMGM-UMR5100, Toulouse, France 7Physiogenex SAS, Prologue Biotech, Labège Innopole, France 8Danisco Health & Nutrition, Kantvik, Finland 9Unité de Pathogénie Microbienne Moléculaire and Unité INSERM 786, Institut Pasteur, Paris Cedex 15, France *Jacques Amar, Tel: +33 5 61 32 30 72, Fax: +33 5 61 32 27 10Rémy Burcelin, Tel: +33 5 61 32 56 14, Fax: +33 561 325 621 EMBO Mol Med (2011)3:559-572https://doi.org/10.1002/emmm.201100159 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract A fat-enriched diet modifies intestinal microbiota and initiates a low-grade inflammation, insulin resistance and type-2 diabetes. Here, we demonstrate that before the onset of diabetes, after only one week of a high-fat diet (HFD), live commensal intestinal bacteria are present in large numbers in the adipose tissue and the blood where they can induce inflammation. This translocation is prevented in mice lacking the microbial pattern recognition receptors Nod1 or CD14, but overtly increased in Myd88 knockout and ob/ob mouse. This 'metabolic bacteremia' is characterized by an increased co-localization with dendritic cells from the intestinal lamina propria and by an augmented intestinal mucosal adherence of non-pathogenic Escherichia coli. The bacterial translocation process from intestine towards tissue can be reversed by six weeks of treatment with the probiotic strain Bifidobacterium animalis subsp. lactis 420, which improves the animals' overall inflammatory and metabolic status. Altogether, these data demonstrate that the early onset of HFD-induced hyperglycemia is characterized by an increased bacterial translocation from intestine towards tissues, fuelling a continuous metabolic bacteremia, which could represent new therapeutic targets. The paper explained PROBLEM: We are facing an epidemic of metabolic diseases, which the classical pharmacological strategies have failed to overcome. Hence, new paradigms are required. We have previously shown that bacterial fragments such as lipopolysaccharides (LPS) are present in increased amounts in the blood of diabetic mice, and induce adipose tissue inflammation, which is the first step leading to insulin resistance and adipose tissue expansion, resulting in obesity. Our previous data suggested that targeting mechanisms involved in the recognition of bacterial fragments could be an original therapeutic strategy to overcome the low-grade inflammation and the development of metabolic diseases. Here, we aimed at identifying such mechanisms. RESULTS: We show that at the early onset of HFD-induced diabetes and obesity, the mucosal adherence of commensal bacteria is increased dramatically in the mouse intestine. This is accompanied by an augmentation of the translocation of these bacteria into the adipose tissue. These living bacteria then co-localized with dendritic cells of the intestinal lamina propria and the mesenteric lymph nodes when diabetes is established. Importantly, we demonstrate that this mechanism requires CD14 and Nod1, which are receptors that recognize different fragments of gram-negative bacteria. Furthermore, leptin reduces intestinal mucosal adherence and translocation of bacteria, since in the absence of the hormone, the mice are dramatically obese and inflamed. Finally, to overcome the bacterial translocation, we used a probiotic treatment and showed that glucose metabolism was indeed improved, demonstrating the importance of the control of intestinal adherence and translocation of commensal bacteria in the regulation of metabolic diseases. IMPACT: We thus propose a new paradigm for the treatment of metabolic disease. We suggest that targeting intestinal bacterial adherence, bacterial translocation, or receptors of bacterial fragments would be an original and novel strategy to prevent or reverse the occurrence of metabolic diseases. Therefore, new probiotics, which aim to target this function, can be defined. INTRODUCTION Over the last decades, the incidence of diabetes and obesity has risen dramatically and this unabated increase can be justly described as a world-wide epidemic. A dramatic change in dietary habits, characterized by an increased intake of lipids, has been suggested to be mainly responsible for the dramatic rise in metabolic diseases through mechanisms, which induce low grade inflammation (Hotamisligil, 2006; Shi et al, 2006; Shoelson et al, 2006). TNF-α continuously released by the adipose tissue during obesity has been shown to activate protein kinase C (PKC) and to increase the phosphorylation of insulin receptor substrates on serine residues such as Ser-307, leading to the inactivation of this insulin signalling molecule and hence to insulin resistance (Tanti et al, 2004). However, the origin of the antigens responsible for the inflammatory process has not been described. Recently, the causal role of the intestinal microbiota in weight gain has been demonstrated in experiments in which germ-free mice colonized with the intestinal microbiota from genetically obese ob/ob mice gained more weight than their counterparts colonized with microbiota from lean animals. Hence, obesity could be transferred by the intestinal microbiota (Backhed et al, 2004, 2005; Ley et al, 2005, 2006; Turnbaugh et al, 2006, 2007). Altogether, these data demonstrated that the intestinal microbiota and the interactions between the host and the microbiota are involved in the control of energy metabolism. A key question is to understand the mechanism through which the intestinal microflora could specifically affect tissue inflammation. An important observation in this respect is that intestinal phagocytes such as dendritic cells and macrophages capture bacterial intestinal antigens and transfer them into lysosomes for degradation (Sansonetti & Di Santo, 2007), thereby providing a direct cellular link between the intestinal microbiota and the host, named bacterial translocation. This process requires bacterial antigen receptors such as toll-like receptors (TLR) (Keita et al, 2008; Neal et al, 2006) and nod-like receptors (NLR) (Kufer et al, 2008), through which viable and dead bacteria and their components initiate the activation of innate immune cells. Furthermore, cells from the innate immune system infiltrate the adipose depots during metabolic diseases (Weisberg et al, 2003, 2006). Therefore, we suggest that during high-fat diet-induced diabetes, commensal intestinal bacteria translocate in a pathological manner from the intestine towards the tissues where they trigger a local inflammation. The present data show for the first time that during the early onset of high-fat diet-induced diabetes, bacteria from the intestine are actively translocated into the mesenteric adipose tissue (MAT) and the blood. This translocation initiates a 'low grade bacteremia' which depends on CD14, Nod1, but not Nod2. It is further regulated by leptin, which identifies a new function for this hormone. This metabolic bacteremia was reversed by a treatment using the probiotic strain Bifidobacterium animalis subsp. lactis 420, which reduced the mucosal adherence and bacterial translocation of gram-negative bacteria from the Enterobacteriaceae group. Consequently, adipose tissue inflammation and several features characteristic of diabetes were normalized. Therefore, the control of intestinal bacterial translocation and mucosal dysbiosis could be considered as a new therapeutic strategy for the control of high-fat diet-induced diabetes and metabolic syndrome. RESULTS High-fat feeding increases the translocation of live gram-negative bacteria through intestinal mucosa to blood and mesenteric adipose tissue To determine which tissues are targeted by the bacterial translocation, we first quantified the bacterial 16S rRNA DNA concentration in different tissues. The data show that bacterial DNA is detectable in the blood of normal chow fed mice (Fig 1A and D, Supporting information Fig S1A and C). However, there was roughly 1000 and 2000–10,000 times less bacterial DNA in MAT and blood, respectively, when compared to cæcal content (Supporting information Fig S1A). The overall microbial population was also evaluated by denaturing gradient gel electrophoresis (DGGE) analysis, and the number of bands showed that microbiota is less diverse in tissues than in the gut (Supporting information Fig S1B and C). Figure 1. High-fat diet increases the concentration of bacterial DNA in blood, mesenteric adipose tissue (MAT), and corresponding lymph nodes (MLN) before the onset of diabetes. The pathogen recognition receptors Nod1 and CD14, but not Nod2, control high-fat diet-induced bacterial DNA accumulation in tissues. The regulatory role of leptin. 16S rRNA DNA concentration (pg/µg total DNA; qPCR): A,B,C.. From total bacteria in blood (A), MAT (B) and MLN (C) of normal chow (NC)-fed, prediabetic (HFD 1 week), and diabetic mice (HFD 4 weeks) (mean ± SEM; n = 8). D,E.. From total bacteria in blood (D), and MAT (E) of Nod1-deficient mice (Nod1−/−), and Nod2-deficient mice (Nod2−/−), after 4 weeks of a normal chow (NC) or a high-fat diet (HFD 4 weeks) (mean ± SEM; n = 11). F,G.. From total bacteria in blood (F), and MAT (G), of ob/ob, ob/obxCD14−/−, and CD14−/− mice (all NC-fed) (mean ± SEM; n = 8). Data with identical superscript letters (A–G) do not differ from each other with p > 0.05. Download figure Download PowerPoint To follow the bacterial translocation from intestine, a possible mechanism for the existence of bacterial DNA of intestinal origin found in tissues, we generated a fluorescently labelled, ampicillin-resistant Escherichia coli (GFP-E. coli) from a mouse E. coli. After only 1 week of a high-fat diet (HFD), GFP-E. coli was gavaged to mice. As a result, the colony forming units (cfu) equivalent of fluorescent E. coli (as quantified by RT-qPCR) was increased in blood, 2 h after gavage, when compared with normal chow (NC)-fed mice (Supporting information Fig S2C). This was similar to what was observed for wild-type E. coli DNA quantified by specific E. coli 16S qPCR already present in blood (Supporting information Fig S2B), total gram-negative bacteria DNA (Supporting information Fig S2A), and for total bacteria (Fig 1A). After 4 weeks of HFD, when the diabetic state is established (Cani et al, 2008, 2009), the amounts of total bacterial DNA (Fig 1A), gram-negative bacterial DNA (Supporting information Fig S2A), and E. coli DNA (Supporting information Fig S2B) were still increasing in blood. We also quantified total bacterial DNA in MAT and in the corresponding mesenteric lymph nodes (MLN), since innate immune cells, which were suspected to have phagocytosed translocated intestinal bacteria, could accumulate in this tissue. After 1 week of HFD, the amount of bacterial DNA was increased in MAT but not in MLN (Fig 1B and C), and a similar observation was made in regard to the cfu quantification from ampicillin-resistant E. coli in these tissues 2 h after gavage (Supporting information Fig S2D). Once the diabetic state was established (4 weeks HFD), the amount of bacterial DNA was increased in both MAT and MLN (Fig 1B and C). Translocation of intestinal bacteria, glucose metabolism and body weight are regulated by Nod1, leptin, Myd88 and CD14 We analysed first whether Nod1 and Nod2 were involved in the accumulation of tissue bacteria during HFD. When the Nod1−/− mice were fed with HFD for 1 month, fasting glycemia and glucose tolerance (Fig 2A and C), fat mass (Fig 2D) and body weight (Supporting information Fig S3A), fasted plasma insulin concentration (Fig 2E), and whole body insulin-sensitive glucose turnover rate (Fig 2F) were strictly similar between NC- and HFD-fed Nod1−/− mice as well as wild-type (WT) mice fed a normal chow, while WT mice fed a HFD were clearly sensitive to the dietary treatment for all parameters (Fig 2A, C, D, E and F, Supporting information Fig S3A). Conversely, Nod2−/− mice were still sensitive to HFD for all parameters studied (Fig 2B–F, Supporting information Fig S3A). Figure 2. The pathogen recognition receptors Nod1 and CD14, but not Nod2, control high-fat diet-induced glucose intolerance, diabetes and fat mass gain. The regulatory role of leptin. A,B,C.. Oral glucose tolerance test (OGTT) (1.5 g/kg) in Nod1-deficient mice (Nod1−/−) (A), Nod2-deficient mice (Nod2−/−) (B) and their corresponding wild-type controls (WT) after 4 weeks of a normal chow (NC) or a high-fat diet (HFD) and corresponding index (µM/min) from 15 to 120 min after glucose administration (C) (mean ± SEM; n = 11; *p < 0.05; data with identical superscript letters do not differ from each other with p > 0.05). D,E,F.. Fat mass (% of body weight; EchoMRI) (D), fasted plasma insulin (µU/ml) (E) and insulin sensitivity (glucose turnover rate in mg/kg min) (F), in Nod1−/− or Nod2−/− mice and their corresponding WT controls after 4 weeks of a NC or a HFD (mean ± SEM; n = 11 (D); 8 (E); or 5 (F); data with identical superscript letters do not differ from each other with p > 0.05). G.. Intraperitoneal glucose tolerance test (IPGTT) (1 g/kg) in WT, ob/ob, ob/obxCD14−/− and CD14−/− mice (all NC-fed) (mean ± SEM; n = 8; *p < 0.05 versus ob/ob). H.. IPGTT corresponding index (µM/min) from 30 min before to 90 min after glucose administration (mean ± SEM; n = 8; data with identical superscript letters do not differ from each other with p > 0.05). I.. Fat mass (% of body weight; EchoMRI) in WT, ob/ob, ob/obxCD14−/− and CD14−/− mice (all NC-fed) (mean ± SEM; n = 8; data with identical superscript letters do not differ from each other with p > 0.05). Download figure Download PowerPoint We also previously showed that CD14−/− mice resist the diabetic and obese phenotypes in response to HFD (Cani et al, 2008). We therefore mated leptin-deficient mice with CD14−/− mice, as described (Cani et al, 2008). In the double mutant mice, fasted glycemia and glucose tolerance (Fig 2G and H) as well as the fat mass percentage and whole body weight (Fig 2I, Supporting information Fig S3B) were improved when compared to the single mutant ob/ob mice. Myd88 is one of the major signalling molecules involved in TLR activation (except activation of TLR3). Therefore, we compared the corresponding mutant mice with WT mice and showed that fasted glycemia and glucose intolerance (Fig 3A), body weight (Fig 3B), fat mass (Fig 3C), plasma insulin (Fig 3D) and insulin resistance (Fig 3E) were increased when compared to the corresponding WT mice. Figure 3. Metabolic disturbances and increased bacterial translocation to mesenteric adipose tissue (MAT) in Myd88 knockout mice. A.. Intraperitoneal glucose tolerance test (IPGTT) (1 g/kg) in Myd88-deficient mice (Myd88−/−) and their corresponding wild-type controls (WT) fed a normal chow (NC) diet (mean ± SEM; n = 7; *p < 0.05 versus controls). B,C,D,E.. Body weight (B), fat mass (% of body weight; EchoMRI) (C), fasted plasma insulin (µU/ml) (D) and insulin sensitivity (glucose turnover rate in mg/kg min) (E), in Myd88−/− mice and their corresponding WT controls fed a NC diet (mean ± SEM; n = 5; data with identical superscript letters do not differ from each other with p > 0.05). F.. Number of GFP-E. coli cfu per g of MAT in Myd88−/− mice and their corresponding WT controls fed a NC diet, 2 h post-gavage with 109 cfu of GFP-E. coli (mean ± SEM; n = 7; data with identical superscript letters do not differ from each other with p > 0.05). G.. 16S rRNA DNA concentration (pg/µg total DNA; qPCR) from total bacteria in MAT of Myd88−/− mice and their corresponding WT controls fed a NC diet (mean ± SEM; n = 7; data with identical superscript letters do not differ from each other with p > 0.05). Download figure Download PowerPoint Total bacterial DNA concentration, in blood and MAT, was unchanged or even reduced in Nod1−/− mice after HFD treatment, whereas it was conversely increased in Nod2−/− mice (Fig 1D and E, and compare with wild-type mice Fig 1A and B). The absence of leptin increased the total amount of bacterial DNA in blood (Fig 1F compared with Fig 1A) and MAT (Fig 1G compared with Fig 1B). A similar observation was made when GFP-E. coli was quantified in MAT 2 h following the gavage (Supporting information Fig S2E). In addition, the absence of CD14 in leptin-deficient mice decreased the bacterial DNA content in blood and MAT (Fig 1F and G), as well as the cfu count of GFP-E. coli in MAT 2 h after gavage (Supporting information Fig S2E) compared with ob/ob mice. In Myd88-deleted mice, the translocation of living E. coli (Fig 3F) and the total content of bacterial DNA (Fig 3G) were dramatically increased in MAT compared with WT mice. High-fat feeding increases the adherence of a gram-negative bacteria species to intestinal mucosa and its co-localization with dendritic cells in lamina propria and mesenteric lymph nodes Two hours after gavage with GFP-E. coli, adherence of the bacteria to mucosal surface of duodenum, jejunum, ileum and cæcum was increased in mice fed with a HFD for only 1 week, when compared with normal chow-fed mice (Fig 4A), while after 5 h the difference was more evident in cæcum (Supporting information Fig S4A). Importantly, 1 week of HFD is not sufficient to induce diabetes showing that bacterial translocation precedes the occurrence of diabetes. This increased adherence of GFP-E. coli to mucosa persisted significantly in ileum and cæcum until the 4th week of HFD (Fig 4A), by which time HFD-fed mice have developed diabetes. In addition, examination by fluorescence microscopy of ileal mucosa obtained by scraping 2 h after the oral gavage, confirmed the presence of fluorescent E. coli mainly in ileal mucosa of HFD-fed mice (Fig 4B and C). This was similar, although to a lower extent, after 5 h (Supporting information Fig S4B and C). Fluorescent bacteria could also be detected in sections of ileum, inside lamina propria, 2 h post-gavage (Fig 4D) and inside submucosa 5 h post-gavage (Supporting information Fig S4D). The number of transmucosal GFP-E. coli was estimated to be 5–10 times higher, 2 and 5 h post-gavage in HFD-fed mice compared to normal chow-fed mice (Fig 4E, Supporting information Fig S4E). Figure 4. Intestinal mucosal adherence and transmucosal passage of bacteria are increased during high-fat diet (HFD) treatment before the onset of diabetes, and intestinal bacteria co-localize with dendritic cells of lamina propria and mesenteric lymph nodes (MLN). A.. Bacterial adherence (GFP-E. coli cfu per cm of mucosa/GFP-E. coli cfu per cm of lumen) in normal chow (NC)-fed (Control NC), prediabetic (HFD 1 week), and diabetic mice (HFD 4 weeks), 2 h after gavage with 109 cfu of GFP-E. coli (Duo, duodenum; Jej, jejunum; Ile, ileum; Cæ, cæcum) (mean ± SEM; n = 10–12; data with identical superscript letters do not differ from each other with p > 0.05). B.. Fluorescence microscopy of scrapped ileal mucosa from NC-fed (Control NC) and prediabetic mice (HFD 1 week), 2 h after gavage with 109 cfu of GFP-E. coli. Bars = 20 µm. C.. Corresponding number of GFP-E. coli/mm2 of scrapped ileal mucosa (fluorescence microscopy) (mean ± SEM; n = 6; data with identical superscript letters do not differ from each other with p > 0.05). D.. Fluorescence microscopy of DAPI-counterstained cryosections of ileum from NC-fed (Control NC) and prediabetic mice (HFD 1 week), 2 h after gavage with 109 cfu of GFP-E. coli. Right panel corresponds to magnification of surrounded region. Bars = 20 µm. E.. Corresponding number of transmucosal GFP-E. coli/mm2 of ileum section (fluorescence microscopy) (mean ± SEM; n = 6; data with identical superscript letters do not differ from each other with p > 0.05). F,H.. Immunofluorescent labelling of CD11c-positive cells [dendritic cells (DC) in red] and co-localization with fluorescent GFP-E. coli (green) on DAPI-counterstained (nuclei in blue) cryosections of ileum (F), and MLN (H), from NC-fed (Control NC), and diabetic mice (HFD 4 weeks), 2 h after gavage with 109 cfu of GFP-E. coli. Arrows point co-localizations (yellow). Right panels correspond to magnification of surrounded regions. Bars = 20 µm. G,I.. Corresponding quantification of CD11c-positive cells co-localized with GFP-E. coli (% of total DC) in ileum (G) and MLN (I) (mean ± SEM; n = 6; data with identical superscript letters do not differ from each other with p > 0.05). Download figure Download PowerPoint After 4 weeks of HFD, when the diabetic state is established (Burcelin et al, 2002; Cook et al, 2004), we were able to detect co-localization of fluorescent bacteria with dendritic cells (DC) in the lamina propria. In normal chow-fed mice, DC were restricted inside the lamina propria, whereas after 4 weeks of HFD, DC were also seen between enterocytes, extending large dendrites towards the intestinal lumen. Importantly, fluorescent E. coli could be co-localized with CD11c-positive cells, i.e. DC, on the luminal side of villi and in lamina propria. These co-localizations were detected in HFD-fed mice only (Fig 4F). The percentage of DC that co-localized with fluorescent E. coli was estimated to be between 5 and 10% of all DC in lamina propria 2 h post-gavage in HFD-fed mice, whereas no co-localization was detectable in normal chow-fed controls (Fig 4G). Interestingly, fluorescent E. coli also co-localized with DC in MLN, although to a lower extent than what was observed in the lamina propria (Fig 4H). The percentage of DC co-localizing with fluorescent E. coli in MLN was also estimated to be between 5 and 10% 2 h post-gavage in HFD-fed mice, while they were undetectable in normal chow-fed animals (Fig 4I). To analyse whether there was a differential distribution of intestinal microbiota in the intestine, we quantified the mucosal and luminal bacterial DNA content by qPCR and showed that 1 week of HFD increased the microbial population in the lumen and the mucosa (Fig 5A). This was further reinforced in the mucosa after 4 weeks of HFD. The expression of markers of inflammation wa
Год издания: 2011
Авторы: Jacques Amar, Chantal Chabo, Aurélie Waget, Pascale Klopp, Christelle Vachoux, Luis G. Bermúdez‐Humarán, Natalia Smirnova, Mathieu Bergé, Thierry Sulpice, Sampo J. Lahtinen, Arthur C. Ouwehand, Philippe Langella, Nina Rautonen, Philippe Sansonetti, Rémy Burcelin
Издательство: Springer Nature
Источник: EMBO Molecular Medicine
Ключевые слова: Gut microbiota and health, Probiotics and Fermented Foods, Gastrointestinal motility and disorders
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