Purpose: To study the therapeutic effects of probiotic Escherichia coli Nissle 1917 (EcN) in irritable bowel syndrome (IBS) and identify subgroups benefiting most. Background: Some trials investigating therapeutic effects in irritable bowel syndrome have shown benefits in IBS subgroups only. Probiotic treatment seems to be promising. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E Escherichia coli Nissle 1917 (EcN) treatment promotes recovery of DSS-induced intestinal injury and inflammation in mice. (A) Histological images of colonic tissue stained with hematoxylin and eosin showing the effect of EcN on DSS-induced colitis. OBJECTIVES Different probiotic strains used in clinical trials have shown prophylactic properties in different inflammatory diseases of the gastrointestinal tract. This study was aimed to investigate the influence of Escherichia coli strain Nissle 1917 (EcN) components on the integrity of the Caco-2 cell monolayer (human adenocarcinoma cell line). The delivery of probiotics to the microbiota is a promising method to prevent and treat diseases. However, oral probiotics will suffer from gastrointestinal insults, especially the pathological microenvironment of inflammatory diseases such as reactive oxygen species (ROS) and the exhausted mucus layer, which can limit their survival and colonization in the intestinal tract. Inspired by the Vay Tiền Trả Góp Theo Tháng Chỉ Cần Cmnd Hỗ Trợ Nợ Xấu. Use of Escherichia coli Nissle 1917 producing recombinant colicins for treatment of IBD patients Roman Kotłowski. Med Hypotheses. 2016 Aug. Abstract Patients with Crohn's Disease and Ulcerative Colitis infected with Adherent-Invasive Escherichia coli strains constitute the largest group among Inflammatory Bowel Disease subjects, when taking into account all known etiological agents of the disease. A possible link between these pathogenic bacteria and inflammation process has gained the confidence in recently published papers. Observed enteric neuroglial cells apoptosis and epithelial gaps of ileum are probably the key manifestations of inflammation, which has been shown in IBD patients in contrary to the samples taken from healthy individuals. The cascade of consecutive events from bacterial infection via inflammation to excessive apoptosis in IBD patients leads up to the aim of our hypothesis about designing of new therapeutic strategy directed to Adherent-Invasive E. coli strains. The main advantage of biological method, which will rely on application of E. coli Nissle 1917 strain as a carrier for specific recombinant colicins against AIEC strains, could probably cause a long-lasting remission of inflammation in CD and UC patients. Copyright © 2016 Elsevier Ltd. All rights reserved. Similar articles Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response. Dreux N, Denizot J, Martinez-Medina M, Mellmann A, Billig M, Kisiela D, Chattopadhyay S, Sokurenko E, Neut C, Gower-Rousseau C, Colombel JF, Bonnet R, Darfeuille-Michaud A, Barnich N. Dreux N, et al. PLoS Pathog. 2013 Jan;9(1):e1003141. doi: Epub 2013 Jan 24. PLoS Pathog. 2013. PMID: 23358328 Free PMC article. Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation. Dogan B, Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL, Stewart K, Scherl EJ, Araz Y, Bitar PP, Lefébure T, Chandler B, Schukken YH, Stanhope MJ, Simpson KW. Dogan B, et al. Inflamm Bowel Dis. 2014 Nov;20(11):1919-32. doi: Inflamm Bowel Dis. 2014. PMID: 25230163 Invasive Escherichia coli are a feature of Crohn's disease. Sasaki M, Sitaraman SV, Babbin BA, Gerner-Smidt P, Ribot EM, Garrett N, Alpern JA, Akyildiz A, Theiss AL, Nusrat A, Klapproth JM. Sasaki M, et al. Lab Invest. 2007 Oct;87(10):1042-54. doi: Epub 2007 Jul 30. Lab Invest. 2007. PMID: 17660846 Activity of Species-specific Antibiotics Against Crohn's Disease-Associated Adherent-invasive Escherichia coli. Brown CL, Smith K, Wall DM, Walker D. Brown CL, et al. Inflamm Bowel Dis. 2015 Oct;21(10):2372-82. doi: Inflamm Bowel Dis. 2015. PMID: 26177305 [Crohn disease, ulcerative colitis. When bacteria attack the intestinal wall....]. Duchmann R, Lochs H, Kruis W. Duchmann R, et al. MMW Fortschr Med. 1999 Dec 16;141(51-52):48-51. MMW Fortschr Med. 1999. PMID: 10949626 Review. German. Cited by Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation. Seco EM, Fernández LÁ. Seco EM, et al. Microb Biotechnol. 2022 May;15(5):1374-1391. doi: Epub 2021 Nov 9. Microb Biotechnol. 2022. PMID: 34755474 Free PMC article. Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease. Chervy M, Barnich N, Denizot J. Chervy M, et al. Int J Mol Sci. 2020 May 25;21(10):3734. doi: Int J Mol Sci. 2020. PMID: 32466328 Free PMC article. Review. New Approaches for Escherichia coli Genotyping. Kotłowski R, Grecka K, Kot B, Szweda P. Kotłowski R, et al. Pathogens. 2020 Jan 21;9(2):73. doi: Pathogens. 2020. PMID: 31973175 Free PMC article. K5 Capsule and Lipopolysaccharide Are Important in Resistance to T4 Phage Attack in Probiotic E. coli Strain Nissle 1917. Soundararajan M, von Bünau R, Oelschlaeger TA. Soundararajan M, et al. Front Microbiol. 2019 Nov 29;10:2783. doi: eCollection 2019. Front Microbiol. 2019. PMID: 31849915 Free PMC article. Integrating omics for a better understanding of Inflammatory Bowel Disease: a step towards personalized medicine. Kumar M, Garand M, Al Khodor S. Kumar M, et al. J Transl Med. 2019 Dec 13;17(1):419. doi: J Transl Med. 2019. PMID: 31836022 Free PMC article. Review. MeSH terms Substances LinkOut - more resources Full Text Sources ClinicalKey Elsevier Science Other Literature Sources scite Smart Citations Medical MedlinePlus Health Information AbstractBackgroundGenetically modified probiotics have potential for use as a novel approach to express bioactive molecules for the treatment of obesity. The objective of the present study was to investigate the beneficial effect of genetically modified Escherichia coli Nissle 1917 (EcN-GM) in obese C57BL/6J an obesity model in C57BL/6J mice was successfully established. Then, the obese mice were randomly assigned into three groups: obese mice (OB), obese mice + EcN-GM (OB + EcN-GM), and obese mice + orlistat (OB + orlistat) (n = 10 in each group). The three groups were gavaged with ml of 1010 CFU/ml control EcN, EcN-GM (genetically engineered EcN) and 10 ml/kg orlistat. Body weight, food consumption, fat pad and organ weight, hepatic biochemistry and hepatic histopathological alterations were measured. The effects of EcN-GM on the levels of endocrine peptides and the intestinal microbiota were also supplementation for 8 weeks, EcN-GM was associated with decreases in body weight gain, food intake, fat pad and liver weight, and alleviation hepatocyte steatosis in obese mice. EcN-GM also increased the level of GLP-1 in serum and alleviated leptin and insulin resistance. Moreover, supplementation with EcN-GM increased the α-diversity of the intestinal microbiota but did not significantly influence the relative abundance of Firmicutes and results indicated that EcN-GM, a genetically modified E. coli strain, may be a potential therapeutic approach to treat obesity. The beneficial effect of EcN-GM may be independent of the alteration of the diversity and composition of the intestinal microbiota in obese mice. This is a preview of subscription content Access options Subscribe to JournalGet full journal access for 1 year111,22 €only 9,27 € per issueAll prices are NET prices. VAT will be added later in the calculation will be finalised during articleGet time limited or full article access on ReadCube.$ prices are NET prices. Additional access options: Log in Learn about institutional subscriptions ReferencesKyle TK, Dhurandhar EJ, Allison DB. 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Am J Physiol Regul Integr Comp Physiol. 2016;310:R885– PubMed Central Google Scholar Download referencesAuthor informationAuthors and AffiliationsDepartment of Research and Development, Weichuang Tianyi Biotechnology Co., Ltd, Chengdu, Sichuan, PR ChinaJie MaDepartment of Research and Development, LiTong Bio-Medical Science, Chengdu, Sichuan, PR ChinaJie Ma & Lu XuSavaid Medical School, University of Chinese Academy of Sciences, Beijing, PR ChinaJunrui WangDepartment of Orthopaedics, Chengdu Second People’s Hospital, Chengdu, Sichuan, PR ChinaJunrui WangCollege of Comprehensive Health Management, Xihua University, Chengdu, Sichuan, PR ChinaYuanqi LiuDepartment of Neurosurgery, PLA Strategic Support Force Characteristic Medical Center, Beijing, PR ChinaJianwen GuAuthorsJie MaYou can also search for this author in PubMed Google ScholarJunrui WangYou can also search for this author in PubMed Google ScholarLu XuYou can also search for this author in PubMed Google ScholarYuanqi LiuYou can also search for this author in PubMed Google ScholarJianwen GuYou can also search for this author in PubMed Google ScholarContributionsAll authors contributed to this work. JM, JW, and JG designed the experiment. JM and JW performed the experiment. LX and YL analyzed the data. JM and JW drafted the manuscript. JM, LX, and YL prepared the figures. JM, JW, LX, and JG critically revised the manuscript. All the listed authors reviewed and approved the submitted authorsCorrespondence to Jie Ma or Jianwen declarations Competing interests The authors declare no competing interests. Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional and permissionsAbout this articleCite this articleMa, J., Wang, J., Xu, L. et al. The beneficial effects of genetically engineered Escherichia coli Nissle 1917 in obese C57BL/6J mice. Int J Obes 46, 1002–1008 (2022). citationReceived: 17 June 2021Revised: 07 January 2022Accepted: 12 January 2022Published: 25 January 2022Issue Date: May 2022DOI: Loading metrics Open Access Peer-reviewed Research Article Anurag Singh, Ulrike Dringenberg, Regina Engelhardt, Ursula Seidler, Wiebke Hansen, André Bleich, Dunja Bruder, Anke Franzke, Gerhard Rogler, Sebastian Suerbaum, Jan Buer, Florian Gunzer, Astrid M. Westendorf Probiotic Escherichia coli Nissle 1917 Inhibits Leaky Gut by Enhancing Mucosal Integrity Sya N. Ukena, Anurag Singh, Ulrike Dringenberg, Regina Engelhardt, Ursula Seidler, Wiebke Hansen, André Bleich, Dunja Bruder, Anke Franzke, Gerhard Rogler x Published: December 12, 2007 Figures Abstract Background Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs). Methodology and Principal Findings To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1. Conclusion and Significance This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances. Citation: Ukena SN, Singh A, Dringenberg U, Engelhardt R, Seidler U, Hansen W, et al. (2007) Probiotic Escherichia coli Nissle 1917 Inhibits Leaky Gut by Enhancing Mucosal Integrity. PLoS ONE 2(12): e1308. Editor: Debbie Fox, The Research Institute for Children, United States of AmericaReceived: September 17, 2007; Accepted: November 21, 2007; Published: December 12, 2007Copyright: © 2007 Ukena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 621).Competing interests: The authors have declared that no competing interests exist. IntroductionThe epithelial layer of the gastrointestinal tract serves as one of the primary interfaces with the outside world. The mucosal surface of the intestinal epithelium is in constant contact with abundant populations of microbes and their metabolites. The intestinal barrier formed by the epithelial cells and the junctional complex, consisting of tight junctions (TJ), adherens junctions, gap junctions and desmosomes, excludes the majority of these microbes and their metabolites from access to the subepithelial cells. Its effectiveness and stability are ensured by the junctional complex [1]. Compromising the integrity of this barrier can promote manifestation of enteric infections and is a key feature of IBDs like Crohn's disease (CD) and ulcerative colitis (UC). Intestinal permeability was also found to be increased in HIV infection [2], and diarrhea is one of the most predominant symptoms of HIV-infected patients. The diarrhea of these patients is mainly due to infections with enteropathogens. However, in a number of HIV patients with gastrointestinal complaints no enteropathogen can be identified [3], [4]. The role of a ‘leaky gut’ in the pathogenesis of gastrointestinal diseases is increasingly recognized. Consequently, reduction of the increased permeability is an interesting target for improvement of the clinical status of gastrointestinal diseases. Tight junctions are intricate macromolecular protein structures located at the most apical regions of the junctional complex, sealing the spaces between the IECs. The first junction-associated protein identified was zonula occludens 1 (ZO-1), with a molecular mass between ∼210–225 kDa [5]. This molecule constitutes the structural link between the cytoskeleton and the tight junction by binding to both actin filaments and the TJ protein occludin [6]. Reorganization of TJ proteins like ZO-1, triggered by cytokines produced secondary to the inflammatory processes in IBDs, results in increased intestinal permeability [7], [8]. ZO-2 is another TJ associated protein that forms a complex together with ZO-1 [9] and has recently been shown to be up-regulated by EcN in vitro [10]. Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer health benefits to the host [11]. The use of such microorganisms as novel therapeutic agents and as an alternative to standard medication in gastrointestinal diseases is promising, although their mechanism of action is still under investigation. EcN has evolved into one of the best characterized probiotics, and its therapeutic efficacy and safety have convincingly been proven [12]–[16]. A potential mechanism by which probiotics may exhibit their beneficial activities is modulation of the epithelial barrier function [17], [18]. This hypothesis is also supported by a recent study demonstrating that probiotic Streptococcus thermophilus and Lactobacillus acidophilus can prevent invasion of enteroinvasive E. coli and enhance intestinal epithelial barrier function by amplifying phosphorylation of occludin and ZO-1 in vitro [19]. Driven by mounting evidence affirming the beneficial effects of probiotics on the intestinal epithelial barrier and the already well-documented therapeutic efficacy of EcN, we set out to investigate the impact of EcN on the intestinal epithelial barrier function in vivo. Materials and Methods Mice All mice used in this study were 6–8 weeks old females. Conventional BALB/c mice were obtained from Harlan (Borchen, Germany). Gnotobiotic BALB/c mice were obtained from colonies maintained germfree at the Central Animal Facility of Hannover Medical School, as described previously [20]. The animal experiments reported here were conducted in accordance with the German Animal Welfare Law and with the European Communities Council Directive 86/609/EEC for the protection of animals used for experimental purposes. All experiments were approved by the local institutional animal care and research advisory committee and authorized by the district authority of Braunschweig and Hannover. Bacterial colonization of gnotobiotic BALB/c mice EcN or E. coli MG1655 was freshly grown to an OD600 = 1 (∼ CFU/ml) from an overnight culture diluted 1:500 in LB media [21]. Bacteria were collected by centrifugation (1 ml, 3 min at 1000×g). The resulting pellet was redissolved in 200 µl sterile PBS and administered by oral gavage. Application was repeated two days later. After 6 days of colonization, fecal CFU were determined by plate count from pooled stool samples and were calculated per gram feces. The animals grew comparable numbers of both bacterial strains in all experiments averaging CFU/g feces with EcN and CFU/g feces with E. coli MG1655. Feces of mice that had received PBS remained sterile. Induction of colitis Acute colitis was induced in BALB/c mice by addition of 4–6% dextran sodium sulfate (DSS) (MP Biomedicals, Eschwege, Germany) to drinking water for a period of 8 days, according to a protocol recently described by Grabig et al. [22]. Animals were separated into the following groups: Group I was treated orally with PBS two times a day. Group II received drinking water with 4–6% DSS and was treated orally with PBS twice daily. Group III also received drinking water with 4–6% DSS and was given CFU (Mutaflor mite, Ardeypharm, Germany) EcN twice daily by oral application (DSS+EcN) (Figure 1). Colitis induction indicated by weight loss of the mice was monitored by comparing the body weight upon DSS treatment to the initial body weight of the respective animals. Figure 1. Experimental design of the DSS colitis colitis was induced by administration of 4–6% DSS to drinking water (DSS). (A) Group I was orally treated with PBS twice daily. Group II was given 4–6% DSS in drinking water and orally treated with PBS twice daily. Group III received CFU EcN two times a day by oral application in combination with 4–6% DSS in drinking water (DSS+EcN). Isolation of IECs IECs were isolated as described elsewhere [23]. Briefly, the small and/or large intestine were isolated, rinsed with PBS and opened longitudinally. Mucus was removed by treatment with DTT for 15 min at 37°C on a shaker. Having washed the mucosa in PBS, it was placed in HBSS/ mM EDTA and tumbled for 10 min at 37°C. The supernatant was collected and the remaining mucosa was vortexed in PBS. This supernatant was also collected. Pooled IECs were centrifuged with HBSS/PBS; the pellet was resuspended in FACS buffer (PBS+2% FCS+2 mM EDTA) and stained with anti-CD45 APC antibody (BD Biosciences, Heidelberg, Germany) to deplete hematopoietic cells [24]. IECs were sorted with a MoFlow cell sorter (Cytomation, Fort Collins, CO, USA). RNA isolation and expression analysis To analyze ZO-1 and ZO-2 mRNA expression in murine IECs, total RNA was isolated using the RNeasy Minikit (Qiagen, Hilden, Germany) with on-column DNase digestion using the RNase-Free DNase set (Qiagen). Isolated mRNA was reverse transcribed with 200 U Superscript II® (Invitrogen, Karlsruhe, Germany), oligo dT- and random hexamer primers (Invitrogen). PCR was performed using the following primers: ribosomal protein 9 (RPS9) mouse (mm) sense primer CTG GAC GAG GGC AAG ATG AAG C, RPS9 mm anti-sense primer TGA CGT TGG CGG ATG AGC ACA; ZO-1 mm sense primer TTT TTG ACA GGG GGA GTG G, ZO-1 mm anti-sense primer TGC TGC AGA GGT CAA AGT TCA AG; ZO-2 mm sense primer CTA GAC CCC CAG AGC CCC AGA AA, ZO-2 mm anti-sense primer TCG CAG GAG TCC ACG CAT ACA AG. Quantitative real-time RT-PCR was done with the GeneAmp 5700 Sequence Detection System (Perkin Elmer, Rodgau-Jügesheim, Germany) using Brilliant SYBR Green QPCR Core Reagent Kit (Stratagene, Heidelberg, Germany). RPS9 served as control. Western blot analysis To examine the ZO-1 protein expression in IECs, lysates of sorted cells from colonized gnotobiotic mice were homogenized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), followed by blotting the proteins on a PVDF membrane. After the blocking of unsaturated protein binding sites, the membrane was incubated with the primary antibody rabbit anti-ZO-1 (Zymed, South San Francisco, CA, USA) or rabbit anti-β actin (Sigma-Aldrich, Taufkirchen, Germany) and the secondary antibody goat anti-rabbit IgG (Dianova, Hamburg, Germany), respectively. Electricphysiologic measurements The colonic mucosa was mounted between two chambers with an exposed area of cm2 and placed in an Ussing chamber. Parafilm “O” rings were used to minimize edge damage to the tissue where it was secured between the chamber halves. Tissues were bathed with HCO3− containing solutions on both sides which were gassed with 95% O2/5% CO2. The composition (in mM) was 108 NaCl, 22 NaHCO3, 3 KCl, MgSO4, 2 CaCl2, KH2PO4, at pH The serosal bath contained (in mM) glucose, 10 sodium pyruvate, 10−3 indomethacin, and 10−3 tetrodotoxin; the luminal bath contained mannitol and 10−2 amiloride (to block potential amiloride-sensitive Na+ channels). Short-circuit current (Isc), potential difference (PD) and tissue resistance (R) were recorded using the Mussler 6-channel voltage clamp system (Mussler, Aachen, Germany). 22Na+ studies were performed during voltage clamp to zero PD. 74 kBq/ml 22Na+ was added to either the serosal or the mucosal solution after reaching stable electrical parameters. After stabilization (approximately 20–30 minutes after mounting), a 45-minute period of equilibration followed, then aliquots were taken in 15-minute intervals (two intervals for basal flux, two after forskolin, and two after luminal glucose). For the presented results, we used the values from the second basal flux period, the first period after forskolin, and the first period after glucose. There were no statistically significant differences between the values obtained in the first and second flux period after forskolin and after glucose. Radioactivity was determined in a liquid scintillation counter, and bidirectional flux rates for the respective substance were calculated. The values for Isc represent the average value of the 15-minute period. Measurement of colonic epithelial permeability Animals were maintained with free access to food and water. Induction of anesthesia was achieved by the administration of 10 µl/g intraperitoneal haloperidol/midazolam/fentanyl cocktail (haloperidol mg/kg, fentanyl mg/kg and midazolam 5 mg/kg body weight). The lower abdomen was opened by one small central incision, and a small polyethylene tube (PE100) with a distal flange was advanced to the proximal colon (immediately after the cecum), and secured by a ligature that served as inlet tube. A PE200 flanged tubing was inserted through the rectum and secured by ligature to allow for drainage through the rectum. The isolated colon segment with an intact blood supply was gently flushed and then perfused (Perfusor compact, BRAUN, Melsungen, Germany) at a rate of 30 ml/h with 150 mmol/l NaCl for 5 min, followed by perfusion with 1% Evans Blue in NaCl for 10 min. To wash-out the sticking dye in the mucus, the lumen was perfused with 6 mM acetylcysteine for 5 min followed by NaCl for 10 min. The animals were then sacrificed by cervical dislocation and the ligated colon was removed. The colon was rinsed once more with saline, its length was recorded and it was then placed in 5 ml N,N- dimethyl-formamide overnight to extract the Evans Blue. The dye concentration was measured spectrophotometrically at 620 nm (Hitachi U-2000 UV/VIS, Hitachi, Japan). Immunofluorescence Tissue sections were fixed with 4% paraformaldehyde, washed extensively and blocked with porcine serum. Subsequently, the sections were incubated with the primary antibody rabbit anti-ZO-1 (Zymed) followed by incubation with a Cy3 labeled secondary goat anti-rabbit IgG antibody (Jackson Immunoresearch, Cambridgeshire, UK). The sections were then dried, covered with gelatine and visualized by fluorescence microscopy. Statistical analysis Statistical analysis was performed with Origin software (OriginLab, Northampton, MA, USA). For analysis of numeric values, the one-tailed analysis of variance and the Student's t-test were used. A p-value of < was considered significant. Error bars represent the standard error of the mean. Results ZO-1 mRNA and protein expression are elevated in gnotobiotic mice colonized with EcN In order to investigate the impact of a single bacterial species on host IEC gene expression, we established a model for colonization of gnotobiotic mice with EcN and E. coli MG1655. To further investigate the in vivo impact of EcN on the epithelial barrier, primary IECs from the intestine of gnotobiotic mice were isolated and sorted by FACS resulting in an IEC population with greater than 94% purity (Figure 2A). ZO-1 is a TJ protein which has been described to play an important role in the prevention of intestinal barrier disruption by probiotics. Therefore, we investigated whether EcN differentially regulates ZO-1 mRNA expression in vivo. As depicted in figure 2B colonization of gnotobiotic mice with EcN resulted in a specific up-regulation of ZO-1 mRNA in IECs (p< To confirm these data at protein level, sections of ileum and colon from gnotobiotic control mice and animals colonized with EcN were stained with anti-ZO-1 antibody followed by a fluorescence-labeled secondary antibody (Figure 3A). Comparison of ZO-1 staining along the surface of the crypts revealed only a slight increase of ZO-1 protein in IECs of mice colonized with EcN. To further analyze the up-regulated ZO-1 expression at protein level, isolated IECs were investigated by Western blotting. In comparison to IECs isolated from control mice and those colonized with E. coli MG1655, IECs from EcN treated animals showed a markedly elevated expression of ZO-1 protein (Figure 3B). Thus, we could clearly demonstrate that colonization of gnotobiotic mice with EcN specifically up-regulates ZO-1 expression at the mRNA as well as at the protein level. Due to the important functional role of ZO-1 in the junctional complex, these findings suggest that enhancement of the intestinal epithelial barrier function by EcN could at least in part be attributed to up-regulation of ZO-1. It has also been previously demonstrated that treatment of T84 cells with EcN leads to an up-regulation of ZO-2 in vitro [10]. In contrast to the data generated with the T84 cell line, analysis of ZO-2 mRNA expression in IECs isolated from gnotobiotic mice colonized with EcN or E. coli MG1655 did not result in an increase of ZO-2 mRNA levels (fold change <2) in vivo (Figure 2C). Figure 2. Isolation of IECs from gnotobiotic mice colonized with EcN or E. coli MG1655 and ZO-1 expression BALB/c mice were colonized with E. coli MG1655 (K12) or EcN for 6 days, respectively. Application of PBS was used as control. (A) Whole intestinal cell populations were isolated from gnotobiotic mice treated either with PBS, K12 or EcN. For sorting of a pure intestinal epithelial cell population, cells were labeled with anti-CD45 antibody to exclude hematopoietic cells and further distinguished by cell granularity and size (SSC) (Pre sorting). IECs were FACS-sorted by negative selection. Re-analysis was performed to determine the purity of sorted IECs (Post sorting). (B) Quantitative ZO-1 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). (C) Quantitative ZO-2 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). *p< EcN vs. Ctrl (relative expression values). 3. ZO-1 protein expression in ileum and colon of gnotobiotic mice.(A) Immunofluorescence staining of tissue sections from gnotobiotic control mice and mice colonized with EcN for 6 days with a fluorescent anti-ZO-1 antibody (orange). Original magnification×20. (B) Protein expression of ZO-1 in IECs. Western blot analysis of FACS sorted IECs from colonized mice was performed using anti-ZO-1 antibody. Anti-β actin antibody was used as internal control, binding to the corresponding protein with a molecular weight of 42 kDa. The ZO-1 antibody detects endogenous ZO-1 protein, displayed as a band at ∼210 kDa. Elevated ZO-1 expression after EcN treatment in experimental colitis It has been shown that the impaired barrier function in IBD is associated with an altered TJ structure [7], [8], [25]–[28]. Recently it was demonstrated that EcN, used as a therapeutic for the treatment of ulcerative colitis, ameliorates acute colitis in mice [29]. These observations raised the question whether an alleviated acute colitis-as a consequence of EcN treatment-may be due to its effect on the epithelial barrier. To investigate this aspect acute colitis was induced in BALB/c mice by administration of 4–6% DSS in drinking water for a period of 8 days [22]. In addition, mice were given CFU EcN or PBS orally two times a day. In contrast to untreated control mice of group I, mice exposed to DSS (group II) developed symptoms of acute colitis with diarrhea, rectal bleeding and wasting, loosing 10% of their initial body weight within 8 days (Figure 4A). Concomitant oral administration of EcN (group III) significantly ameliorated the severity of DSS-induced colitis and the loss of body weight was reduced (6%) (p< Healthy control mice (group I) exhibited an average colon length of (± cm, whereas, as a consequence of severe intestinal inflammation, the colon length of DSS treated diseased mice (group II) was reduced to (± cm (p< In contrast, in DSS and EcN treated mice (group III), this colon shortening was significantly attenuated with (± cm (p< (Figure 4B). Colonic inflammation is correlated with strong infiltration of hematopoietic cells into the intestine. To further elaborate the beneficial effect of EcN in DSS treated mice, FACS analysis of hematopoietic cells in the colon was performed. Consistent with the reduction in loss of body weight and colon shortening, mice treated with DSS and EcN (group III) exhibited significantly lower leukocyte infiltrates in the colon in comparison to DSS treated mice (group II) (Figure 4C). To analyze whether the improved state of health after EcN treatment is accompanied with an increased ZO-1 expression, IECs were isolated from the colon of treated mice and analyzed for ZO-1 gene expression. IECs of mice treated with DSS and EcN showed elevated ZO-1 mRNA levels in comparison to DSS treated animals (p< (Figure 5). Although ZO-2 mRNA was not up-regulated by EcN under healthy conditions, a slight increase of ZO-2 mRNA could be detected in DSS mice treated with EcN (data not shown). These results further underline the beneficial effects of EcN on the intestinal barrier even under inflammatory conditions. Figure 4. Administration of EcN in DSS induced colitis.(A) Disease severity was measured daily and is expressed in terms of body weight loss. (group II: n = 23, group III: n = 25, group I: n = 17) *p< group III vs. group II. (B) Reduction of colon length. Measurement of colon length [cm] after preparation. (group II: n = 24, group III: n = 25, group I: n = 13) *p< group III vs. group II or group III vs. group I **p< group II vs. group I. (C) Infiltration of hematopoietic cells into the colon. Whole intestinal cell populations were labeled with anti-CD45 APC antibody and measured by FACS analysis (n = 6/group). 5. Increased ZO-1 mRNA expression in mice treated with DSS and were isolated from indicated mice and relative levels of ZO-1 mRNA were normalized with respect to the expression level of IECs from DSS treated mice (fold change = 1). Data are presented as mean of four independent experiments (n = 3/group). *p< group III vs. group II (relative expression values). Electrolyte transport capacity and tissue resistance after EcN treatment in experimental colitis In order to assess the electrolyte transport capacity and the tissue resistance (R) in acute DSS colitis, and the influence of EcN treatment on these parameters, the basal and forskolin-stimulated Isc, basal and forskolin-inhibited Na+ absorption, and the tissue-resistance R in isolated colonic mucosa of inflamed DSS treated mice (group II), DSS and EcN treated mice (group III), and healthy controls (group I) were studied. To ensure that any potential inflammation-related changes would be detected, the mucosa was neither stripped nor were the prostaglandin production or neural transmission inhibited. Interestingly, no difference was found in either the basal and forskolin-stimulated Isc (Figure 6A), or in the transmucosal electrical resistance (R) between the different groups (data not shown). This demonstrates that in acute DSS colitis, the anion secretory capacity, which originates from the cryptal region of the colonic epithelium, is not perturbed. On the other hand, net Na+ absorption was significantly decreased in colonic mucosa of DSS and EcN treated mice (group III), and reversed to Na+ leakage into the luminal fluid in the mucosa of DSS treated mice (group II) (Figure 6B). Since the transporters for Na+ absorption are expressed in the surface colonic enterocytes, this demonstrates severe alterations in surface cell electrolyte transport following treatment with EcN. Figure 6. Secretory and absorptive function of isolated colonic mucosa from DSS-treated mice, with and without EcN administration, and healthy controls.(A) Basal and forskolin-stimulated Isc in DSS treated (group II n = 7), DSS+EcN (group III n = 12) and control animals (group I n = 10). The different numbers resulted from the fact that considerably more colonic segments from the DSS treated mice were so friable that they ruptured before measurements could be taken. (B) Basal and post-forskolin net Na+ flux rates in the three different groups. Whereas Na+ absorption was completely abolished in group II, active Na+ absorption was only partially inhibited in group III. Only in the control group I could an inhibition of Na+ absorption by forskolin be observed, indicating normal regulation (n = 6/group). *p< Reduction of colonic epithelial permeability after EcN treatment in experimental colitis In order to address the question whether the pronounced ZO-1 expression in EcN treated mice (group III) impacts the permeability of the colonic epithelium for transport of luminal substances, the uptake of Evans Blue into the mucosa in anesthetized mice after a short-term luminal perfusion with the dye was measured. In comparison to untreated mice (group I), a strong increase of Evans Blue uptake into the colonic mucosa of DSS treated mice (group II), and a much lesser increase into the colonic mucosa of DSS and EcN treated mice (group III) was detected (Figure 7). This demonstrates that the concomitant application of EcN during colitis induction with DSS markedly ameliorates the leakiness of the colonic epithelium. Figure 7. Permeability to Evans Blue in the colon of DSS treated (group II), DSS+EcN (group III) treated mice and healthy controls (group I).The graph shows a significant increase in Evans Blue uptake into the colonic mucosa of group II mice and a strong reduction in Evans Blue uptake in group III mice to almost normal values (n = 6/group). ***p< DiscussionThe current study establishes that E. coli Nissle 1917 positively impacts the intestinal epithelial barrier in vivo in three different ways. First, EcN is capable of producing a specific up-regulation of ZO-1 expression in IECs of healthy gnotobiotic mice. When treated concomitantly with EcN, IECs of mice with DSS-induced colitis also exhibit a pronounced expression of ZO-1 mRNA. Finally, EcN provides protection against the DSS-mediated leakiness of the gut in our mouse model. Our data strongly suggest that one of the protective effects of EcN treatment on colitis prevention could be a modulation of tight junctional integrity which in turn leads to preserved intestinal barrier function against noxious or infectious agents. Critical for the development of IBDs are imbalances in mucosal immunity as well as a disturbed function of the epithelial barrier, which leads to a marked infiltration of luminal microflora [30]. Moreover, in the majority of cases, gastrointestinal diseases develop from the disruption of the intestinal epithelial barrier by enteropathogenic bacteria that alter the cellular cytoskeleton [31], [32] or affect specific tight junction proteins like ZO-1 [33]. In the past years, probiotics have been shown to be effective in the treatment of mild to moderately active IBD [18] and to reduce inflammation in animal models of colitis [29], [34]–[36]. Three large clinical trials have investigated the therapeutic efficacy of EcN in maintaining remission of UC. EcN was reported to be as efficacious as standard medication in preventing the relapse of UC [12]–[14]. Although the mechanisms leading to relapses in the pathogenesis of IBDs have not yet been clarified, there is growing evidence that increased intestinal permeability plays a key role. This report is the first to demonstrate a direct influence of the therapeutic EcN on expression of the TJ associated molecule ZO-1 in vivo under healthy and inflammatory conditions. We demonstrate that EcN specifically up-regulates ZO-1 expression both at the mRNA and at the protein level in IECs of gnotobiotic mice. Very recently, Zyrek et al. described the up-regulation of ZO-2 after EcN exposure to the T84 cell line in vitro [10]. However, in this study analysis of IECs from gnotobiotic mice colonized with EcN did not reveal a differential ZO-2 mRNA expression in vivo following EcN treatment. In order to study functional consequences of DSS-mediated colitis and EcN treatment and also the potential significance of ZO-1 up-regulation for an enhancement of intestinal barrier function, we performed experiments aimed to assess transport and barrier function of the colonic epithelium of DSS-treated mice with and without application of EcN. To evaluate secretory and absorptive function of the colonic epithelium, we measured the basal and forskolin-stimulated Isc (which is an assessment of the electrogenic anion secretion, and its stimulation by an increase in intracellular cAMP levels), as well as net Na+ absorption before and after an increase in cAMP levels. Interestingly, after one week of DSS treatment, the acute colitis did not compromise the secretory function of the epithelium at all, but abolished the Na+ absorptive function completely. Na+ absorption is mediated by electroneutral (apical Na+/H+ exchangers NHE3 and possibly NHE2) and electrogenic (apical Na+ channel ENaC) pathways in the colon [37]. Na+ absorptive transporters are compromised during acute colitis, and this is one major reason for diarrhea during colonic inflammation [38]. Active fluid absorption is also dependent on intact tight junctions, otherwise a phenomenon called “leak flux diarrhea” occurs [39]–[41]. A cytokine-induced intestinal barrier dysfunction via a leak flux mechanism has recently been proposed by Schmitz et al. as potential cause for non-infectious diarrhea in HIV-infected patients, in addition to mucosal transformation with a consecutive malabsorptive mechanism [42]. When cholera toxin deletion mutants of Vibrio cholerae were given to healthy volunteers they still developed mild diarrhea [43]. Searching for the causative agent led to the detection of the ZOT, a toxin which causes a disruption of the tight junctions in isolated intestine [44]. Later studies revealed yet another agent from Vibrio cholerae, the HA/protease, that specifically interferes with the tight junction proteins occludin and ZO-1 resulting in barrier disruption [45], [46]. Thus, it became clear that the integrity of the tight junctions was necessary for the maintenance of an absorptive state of the gut epithelium. In addition, it was found that a disruption of the tight junctional complex allowed easier permeation of substances from the lumen [47]. Our experiments demonstrated a strongly elevated influx of Evans Blue into the colonic mucosa in the live mouse with DSS colitis which is indicative of increased gut permeability. Much less dye was bound to IECs of mice treated concomitantly with EcN. Combined with the absorptive Na+ flux in these animals, this is likely to explain the nearly normal appearance of the feces in the DSS plus EcN treated mice compared to the liquid stools of the mice with DSS colitis. It is feasible that the reduction of DSS-mediated downregulation of ZO-1 expression by EcN treatment is one reason for the enhanced barrier stability. Several lines of evidence suggest that increased intestinal permeability has a central role in the pathogenesis of IBDs. For example, between 10–20% of presymptomatic CD patients have been shown to exhibit increased gut permeability [48], [49]. Alteration of TJ structure in UC for instance results in impaired barrier function [40]. Localization studies in mucosal biopsies of IBD patients have revealed disappearance of key TJ proteins from intercellular junctions [26], [50]. Probiotics have been shown to reduce the increased intestinal permeability in vitro [51] as well as in clinical trials [52]. Here we demonstrate for the first time that the substantially increased intestinal permeability of mice treated with DSS is significantly alleviated by simultaneous oral application of EcN. In addition, we observed an altered ZO-1 expression profile in IECs of mice with DSS-induced colitis. Recently, a study described the translocation of ZO-1 from the apical to the basolateral side in CD patients [53] indicating an alteration of ZO-1 under pathological conditions. Moreover, using a mouse colitis model, Resta-Lenert et al. have shown that the increase in intestinal permeability is associated with a decrease of occludin and ZO-1 phosphorylation [54]. However, administration of EcN in our mouse models not only diminished the clinical signs of colitis like colon shortening and weight loss, but also prevented an increase in intestinal permeability while concurrently minimizing the down-regulation of IEC ZO-1 expression. This indicates a considerable association between the severity of colitis, as evidenced by increased gut permeability, and altered ZO-1 mRNA expression levels. It can be speculated that the rise of ZO-1 expression results in a reduced intestinal permeability by an enhanced junctional complex or a reinforced interaction of the junctional complex with actin. This hypothesis is consistent with recently published data regarding the antrum mucosal protein (AMP)-18 that ameliorates DSS colitis in mice and also enhances accumulation of occludin and ZO-1 in TJ domains in vitro [55]. Since AMP peptide also prevented a fall in transepithelial resistance during disruption of actin filaments and stabilized the perijunctional actin during oxidant injury, it has been suggested that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin [55]. Using another murine colitis model, administration of n-3 polyunsaturated fatty acids resulted not only in reduced pathological scores but also an increase of ZO-1 protein expression [56]. The present study clearly demonstrates that EcN specifically up-regulates ZO-1 mRNA expression in IECs in vivo. Together with the influence of EcN on intestinal permeability and an enhanced ZO-1 expression under pathological conditions, it can be speculated that EcN augments mucosal barrier function. These in vivo results corroborate the in vitro findings from other groups by demonstrating that probiotic EcN plays an important role in the maintenance of intestinal barrier function. An improved barrier integrity elicited by EcN is an appealing explanation for the success of this probiotic in the therapy of UC and could be an important aspect in treating further human intestinal disorders, including HIV-associated diarrhea. Acknowledgments We thank further Lothar Gröbe for FACS sorting, Marco Metzger for performing immunohistochemistry and Silvia Prettin for technical support as well as Anna Smoczek and Ina Köhn for animal care. The authors also gratefully acknowledge Michael J. Schubert for critically reading the manuscript. 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View Article Google Scholar AbstractAllergic asthma is characterized by a strong Th2 and Th17 response with inflammatory cell recruitment, airways hyperreactivity and structural changes in the lung. The protease allergen papain disrupts the airway epithelium triggering a rapid eosinophilic inflammation by innate lymphoid cell type 2 (ILC2) activation, leading to a Th2 immune response. Here we asked whether the daily oral administrations of the probiotic Escherichia coli strain Nissle 1917 (ECN) might affect the outcome of the papain protease induced allergic lung inflammation in BL6 mice. We find that ECN gavage significantly prevented the severe allergic response induced by repeated papain challenges and reduced lung inflammatory cell recruitment, Th2 and Th17 response and respiratory epithelial barrier disruption with emphysema and airway hyperreactivity. In conclusion, ECN administration attenuated severe protease induced allergic inflammation, which may be beneficial to prevent allergic asthma. IntroductionAllergic asthma is one of the most common chronic respiratory diseases with a significant impact on public health1,2. In recent years, the incidence of allergic asthma in developed countries has dramatically increased and it is predicted that the number of affected people worldwide will increase by 100 million by 20253. Risk alleles have been identified for the development of asthma4 but the rapidity of its increased incidence does not support solely a genetic basis and suggest the involvement of environmental factors. Long-term observations support the notion that urban life is associated with increased prevalence of chronic immunological disorders including asthma incidence as compared to children living in farms5. Early in life microbial exposure might modulate allergic disorders6. In addition, such favorable socioeconomic factors, like enriched dietary habits or increased level of hygiene are presumably important factors for a considerable shift in the gut microbiota and increased asthma susceptibility. Epidemiological and clinical studies indicate an association between alteration of intestinal microbial communities and increased incidence of allergic asthma7. Several studies revealed changes in gut microbiota composition in adults suffering from allergic diseases at distant body sites (eczema, rhinitis, asthma)8,9, which precede the development of allergic diseases10,11. Gut bacteria outnumber the human body cells and the microbiome encode approximately 100 times more genes than the human genome12. This impressive genetic capacity contribute to essential functions for the host including nutrients supply like short-chain fatty acids (SCFAs)13,14, vitamins and hormones15, energy balance16,17,18, metabolic signaling19, resistance to pathogens colonization20,21,22 and has a key role in promoting the postnatal maturation of the intestinal mucosal barrier23,24, etiology is complex, but exposure to allergens or air pollution, are clearly important factors for the pathogenesis5. Sensitization to allergen is one of the first steps involved in asthma. Various allergens, including house dust mite (HDM), fungi, cockroach and pollen have proteolytic activities26. Protease properties of allergens cause injury of the airway epithelium with increased permeability, airway remodeling, type 2 cytokine and chemokine production and cell recruitment27. Papain, a cysteine protease, induces a type 2 response characterized by interleukin (IL)-5 and IL-13 production, mediated by an IL-2-dependent IL-9 production28 and specific IgE production29,30. There is evidence that the commensal microflora is critical in the maintenance of systemic immune tolerance, which is instrumental in protecting against allergic asthma. Escherichia coli strain Nissle 1917 (Mutaflor®, ECN) is successfully used for the treatment of intestinal inflammation, especially in patients suffering from ulcerative colitis31. In the present study, we investigated the impact of the colonization by ECN on the allergic lung inflammatory response induced by single or repeated challenges to the protease allergen papain. We show here that chronic ECN administration reduces severe allergic lung inflammation, improves the respiratory epithelial barrier function and modulates emphysema in response to repeated papain colonization has a dual effect in acute papain-induced lung inflammationTo study the impact of the administration of the ECN strain on the development of allergic inflammation, we compared the susceptibility ECN treated mice to acute papain-induced lung inflammation in comparison to non-treated controls according to the protocol shown in Fig. 1a. ECN was administered by gavage over 6 days (108 cfu of live ECN/day) then the mice were challenged twice by intranasal instillation ( of the protease allergen papain (25 µg on day 7 and 8 and the inflammatory response was analyzed 24 h later as described before32. Microscopic examinations of the lungs revealed focal inflammatory cell infiltration around bronchi, capillaries and in alveoli, as well as mucus hypersecretion (Fig. 1b). The lung inflammation as assessed by a semi-quantitative score of microscopic lesions was not reduced in ECN fed mice (Fig. 1b,c), except for the production of mucus (Fig. 1d).Figure 1ECN colonization as a dual effect in acute papain-induced lung inflammation. (a) Experimental settings of acute papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (e) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (F) CCL11, (g) CCL17 and (h) CXCL1 were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imagePapain-induced lung inflammation is associated with enhanced cell recruitment in the lung, involving especially eosinophils32. Cell recruitment into the broncho-alveolar lavage fluid (BALF) was modulated with increased total cells, especially neutrophils upon ECN treatment as compared to control mice (Fig. 1e) with increased myeloperoxidase (MPO) (Supplementary Figure 1) and neutrophil chemoattractant CXCL1 levels (Fig. 1h). By contrast, the recruitment of eosinophils in the BALF was significantly decreased in ECN-treated animals as compared to papain controls (Fig. 1e). This was correlated with a lowered production of CCL17 (Fig. 1g) while CCL11 levels was not modified (Fig. 1f).Interestingly, mice treated with a non-probiotic K12 E. coli strain MG1655 and tested in the acute papain model (Supplementary Figure 2A) develop a similar lung neutrophilia as compared to ECN-treated animals (Supplementary Figure 2B–D), suggesting that this effect is probably mediated an E. coli genus dependent molecular determinant. On the contrary, MG1655 treatment has no protective effect on eosinophilia as observed with cell count and chemokine production (Supplementary Figure 2B,E,F). Taken together, these results suggest that gut colonization by ECN may modulate lung inflammation by enhancing neutrophil, but importantly reducing eosinophil cell recruitment in BALF and tissue. This data motivated studies in a chronic model of lung allergic lung inflammation induced by repeated papain challenges is attenuated by ECN administrationTo determine whether ECN modulates chronic airway inflammation induced by a protease allergen papain, BL6 mice were immunized with papain (25 µg on days 6, 7 by intranasal route), followed by two intranasal challenges at day 20 and 25 (25 µg). Control mice received vehicle (NaCl). In addition, mice were orally administered with 108 cfu of live ECN (Fig. 2a). 24 h after the last papain challenge, the mice were sacrificed and the extent of the lung inflammation was assessed. Histological analysis revealed a prominent lung inflammation characterized by perivascular, peribronchial and alveolar infiltration of eosinophils, neutrophils and air space enlargement with epithelial damage and disruption of alveolar septa, a hallmark of emphysema upon papain challenge (Fig. 2b,c). ECN-treated mice largely prevented lung inflammation, epithelial injury and emphysema (Fig. 2b–d). Finally, the extensive goblet cell hyperplasia and mucus production observed in primed/challenged mice was lowered in ECN probiotic treated mice (Fig. 2b,e). Diminished mucus expression was confirmed at the mRNA level for Muc5ac in lung (Fig. 2f). Interestingly, mice treated with E. coli strain MG1655 and tested in the chronic papain model develop a similar lung inflammation as compared to untreated animals, as revealed by the histological analysis (Supplementary Figure 3A–E), suggesting that the protective effect observed with ECN is due to intrinsic probiotic properties rather than a non-specific effect due to daily gavage E. coli species on the gut microbiota. The absence of protection with MG1655 is unlikely related to the lack of gut colonization, as we quantified equivalent Enterobacteria and E. coli colony counts in both ECN- and MG1655-treated animals along the treatment (Supplementary Figure 4).Figure 2Repeated papain challenges causing severe lung inflammation is attenuated by ECN administration. (a) Experimental settings of chronic papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of airway remodeling was performed on paraffin embedded section after HE staining. (e) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (f) Muc5ac relative gene expression levels in lung tissues was measured by qPCR. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papain chronic challengesPapain-induced chronic inflammation is characterized by a type 2 inflammatory response28. To determine whether ECN inhibited inflammatory cell recruitment, BALF cell counts were assessed for cell phenotyping. Saline sensitized and challenged mice present negligible leukocyte numbers in BALF, whereas papain-treated mice presented a dramatic increase of total cells, eosinophils and fewer neutrophils and macrophages (Fig. 3a). By contrast, ECN-treated mice had ~ less total BALF cell counts with a 2-fold reduction in eosinophils, neutrophils and macrophages. This was consistent with significant lower levels of eosinophils attracting chemokines CCL24 and CCL11 (Fig. 3b,d), EPO levels (Supplementary Figure 5) and neutrophils/monocytes chemoattractant CXCL1 (Fig. 3e), while CCL17 was unchanged in the lungs of ECN-treated mice as compared to controls. Moreover, Th2 cytokines such as IL-5 and to a lesser extent IL4 were significantly reduced in the lung of ECN-treated mice as compared to papain controls (Fig. 3f,g). The production of IFNγ was reduced, while IL17A level was unchanged in ECN probiotic-treated mice (Fig. 3h,i).Figure 3ECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papaïn chronic challenges. (a) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (b) CCL24, (C) CCL17, (D) CCL11, (e) CXCL1, (f) IL-4, (g) IL-5, (h) IL-17 and (i) IFNγ were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageTaking together, these data indicate that ECN gut colonization reduces papain induced Th2 immune airways hyperreactivity and respiratory barrier injury is attenuatedA hallmark of allergic lung inflammation is airways hyperreactivity (AHR), which is due functional changes of the respiratory barrier. AHR was assessed by invasive plethysmography in untreated and ECN-treated mice upon chronic papain exposure. Airway resistance and compliance in response to methacholine as a measure of AHR and were increased upon papain challenge. ECN administration reduced airway resistance and compliance indicating a significant amelioration of the lung function (Fig. 4a,b).Figure 4Papaïn-induced pulmonary dysfunction is attenuated by ECN. (a) Airway hyper-responsiveness to increasing doses of methacholine (Mch; 0−200 mg/ml) was measured by recording changes in lung resistance and (b) airway compliance. The pulmonary epithelial integrity was assessed by the leak of (c) Evans blue and (d) total protein in BAL. (e) Immunofluorescent staining for E-cadherin (green) on lung cryosections. (f) Quantitative evaluation of E-cadherin expression on lung sections. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageThe protease papain induces inflammation and injury of the lung epithelium and capillaries with increased vascular permeability. The probiotic ECN has the ability to strengthen the epithelial barrier33. We used Evans Blue (EB), which binds to serum albumin, as a tracer of the capillary leak of macromolecules from the circulation into the BALF. Our data reveal that ECN treatment reduced the acute lung capillary/epithelial leak of intravenous administered EB upon papain exposure (Fig. 4c). Furthermore, total protein in BALF was also reduced (Fig. 4d). To get further insights into the role of ECN in the improvement of lung epithelial barrier function during allergic asthma, lung histological sections were analyzed for the expression of E-cadherin, a critical component of the epithelial barrier, which is crucial in the maintenance of the immunologic tolerance during airway allergic sensitization34. Immunofluorescence analysis revealed reduced E-cadherin expression concomitant with epithelial cell injury upon papain exposure, while ECN feeding attenuated the reduction of E-cadherin expression (Fig. 4e), which was confirmed by a semi-quantitative assessment of E-cadherin immunostaining (Fig. 4f).Therefore ECN colonization attenuated papain protease induced allergic lung inflammation with reduced Th2 response and airways hyperreactivity. Importantly the protease induced injury of the alveolar septae reflected by emphysema and of the respiratory barrier were significantly diminished by the probiotic strain mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challengesTh2 lymphocytes and ILC2 accumulate in lungs after papaïn exposure and produce IL-5 and IL-1335. We determine the relative contribution of ECN on Th2 and ILC2 activation 24 h after the last allergen challenge. Lung cells were restimulated by papain and the production of cytokines was analyzed. IL-5 (Fig. 5a) and to a lesser extent IL-13 (Fig. 5b) was significantly reduced upon ECN treatment while IL-33 levels remain unchanged (Fig. 5c). Total Th2 and ILC2 producing IL-5 and IL-13 were analyzed by flow cytometry (Supplementary Figures 6 and 7). The frequency of CD3+ CD4+ IL5+ or IL13+ cells were significantly reduced in ECN-treated mice as compared to untreated controls (Fig. 5d–f). This was associated with a similar decrease of ILC2+ and ILC2+ IL13+ (Fig. 5g–i). These data indicate that ECN was able to dampen Th2 and ILC2 activation and the production of the prototypal pro-allergenic IL-5 and 5ECN-treated mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challenges. IL-5 (a), IL-13 (b) and IL-33 (c) levels after lung mononuclear cell restimulation with papaïn for 72 h. Frequency of CD3+ CD4+ lymphocytes (d) producing IL-5 (e) or IL-13 (f) are shown. Frequency of ILC2 (g) producing IL-5 (h) or IL-13 (i) are shown. Data are expressed as mean + SEM from a single experiment with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. * and ** refer to P < and P < size imageDiscussionAllergic asthma is a major health issue with increasing incidence especially in developed countries with an epidemic feature36. Asthma etiology is complex including both genetic and environmental factors, such as exposure to allergens and/or air pollution, are important for the pathogenesis5. Data regarding the use of probiotics in the prevention of allergic diseases and asthma are conflicting37. Several different bacterial strains or combinations have been used in clinical trials to assess protective effects in the context of allergic asthma with significant reduction of both incidence and severity of allergic diseases38 which were not confirmed by others39. A meta-analysis concluded that probiotic are not efficient for the prevention of allergy40. This discrepancy may be related to the dose and duration of probiotic administration, immunomodulatory differences41 among strains, mostly Lactobacillus or Bifidobacterium probiotics42. Here we evaluated the probiotic potential of the Gram negative ECN to prevent allergic lung inflammatory allergic response induced by the protease papain. ECN drastically reduced the severity of chronic lung inflammation through the modulation of the Th2 inflammatory response, injury of the respiratory barrier and airways hyperreactivity. The beneficial effects of ECN has been demonstrated before in intestinal inflammatory disorders, especially in ulcerative colitis43. Two previous studies investigated ECN in experimental asthma. Bickert et al. using the inert protein allergen OVA observed a protection upon oral administration of ECN, but no inhibition of the Th2 immune response44. Adam et al. evaluated the prophylactic potential of ECN on recombinant house mite antigen Derp1 as mucosal antigen. ECN strongly reduced the antigen specific humoral response45. Here, using oral prophylactic administration of ECN we demonstrate for the first time a reduction of papain-induced lung inflammation and amelioration of AHR. In contrast, mice administered K12 E. coli strain MG1655 were as sensitive to lung inflammation as untreated papain challenged mice suggesting that the genetic background of the strain is of particular importance and determines its ability to act as a probiotic. Nevertheless, we observed that both E. coli strains has the ability to induce a potent lung neutrophilia. These results are in line with several papers demonstrating that ECN capsule antigen K5 was an important contributor the recruitment of neutrophil46,47. More generally, it has also been suggested that the presence of capsular antigen may induce an increased influx of pulmonary neutrophils48,49. The mechanisms by which capsular antigen modulate neutrophil response are not completely understood but may include direct effect such an upregulation of shed bacterial formylmethionyl-leucyl-phenylalanine50, a potent neutrophil chemotactic factor; or indirect by modulating the host’s generation of chemokines, including CXCL1 or IL-8 which was observed upon ECN or MG1655 of the best-characterized features contributing to the effectiveness of ECN is its ability to strengthen the epithelial barrier function51. This probiotic property of ECN has been extensively demonstrated in the context of intestinal inflammatory diseases. Asthma is often associated with mucosal barrier dysfunction52. We found that respiratory barrier dysfunction due to papain-induced inflammation and injury is alleviated by ECN with reduced protein leak and upregulation of E-cadherin. Recent studies suggests that this adhesion molecule contributes to the structural and immunological function of the airway epithelium, acting as a rheostat through the regulation of epithelial junctions and production of pro-inflammatory mediators34. Alterations of the airway epithelium enhance both allergic sensitization and airway remodeling including goblet cell hyperplasia, mucus hyperproduction and subepithelial fibrosis53 thus contributing to severe airways hyperreactivity. ECN conferred a significant reduction of inflammatory cell recruitment in BALF, lung tissue inflammation and disruption of alveolar septa with epithelial cells participate in the innate immune response of the lung and have barrier function. Barrier dysfunction favors the access of noxious or immunogenic protein or chemicals to the mucosa-associated lymphoid tissues. Thus, regulation of airway epithelial barrier function is an important checkpoint of the immune response during asthma54. In the present study, we show that ECN treatment affects a prevalent Th2 response known for papain induced lung inflammation28. We observed a significant reduction of eosinophils and eosinophil-related chemokines/cytokines associated with diminished recruitment of neutrophils and CXCL1 and IFN-γ levels. The data are consistent with previous studies showing that colonization by ECN lead to a modification of the cytokines repertoire55,56. In addition, we show for the first time that ECN treatment reduce Th2 CD4+ lymphocytes as well as ILC2 activation, resulting in decreased IL-5 and IL-13 production. The latter population is known to precede Th2 activation which is the cardinal feature of allergic asthma, culminating in airway hyperresponsiveness and Th2 cytokines and chemokines. In this setting, we investigated IL-33, which is known to be involved in ILC2 activation35 but we did not find any difference upon ECN treatment, which was also the case in another reduced allergic asthma molecular rationale behind the immunomodulatory properties of ECN has not yet been elucidated and is under investigation58. The beneficial effect of ECN could rely on the improvement of the intestinal barrier function and the resulting prevention of a continuous stimulation of the host innate immune system by the gut components. Indeed, we have recently demonstrated that ECN was able to prevent CNS inflammation through the improvement of the intestinal permeability59 showing that modulation of the gut microbiota with ECN exerts remote immunological imprinting. ECN genome encodes the production of specialized molecules that may modulate immune functions60,61,62. The intestinal mucosa represents an interface between bacterial-derived metabolites and mucosal immune processes that will influence immunological processes on the host conclusion, our findings indicate that ECN is able to prevent papain-induced lung inflammation after high dose per os administration supporting a gut-lung mucosal communication64. In addition, our results suggest that the prevention of the respiratory barrier dysfunction by probiotic treatment may be important to control allergic lung inflammation. Therefore, ECN might be considered as a valuable prophylactic or diet supplement to prevent allergic (B6) mice were bred in our specific pathogen free animal facility at TAAM-CNRS, Orleans, France (agreement D-45-234-6 delivered on March, 10 of 2014). Mice were maintained in a temperature-controlled (23 °C) facility with a strict 12 h light/dark cycle and were given free access to food and water. The experiments were performed with female mice aged 8–10 weeks using 5 mice per group, and the experiments were repeated at least twice. All animal experimental protocols were carried out in accordance with the French ethical and animal experiments regulations (see Charte Nationale, Code Rural R 214-122, 214-124 and European Union Directive 86/609/EEC) and were approved by the “Ethics Committee for Animal Experimentation of CNRS Campus Orleans” (CCO), registered (N°3) by the French National Committee of Ethical Reflexion for Animal Experimentation (CLE CCO 2013-1006).Bacterial preparation, growth conditions and administrationThe strains used in this study are the probiotic Escherichia coli Nissle 1917 (ECN) and the archetypal K12 E. coli strain MG1655. Both strains were engineered to exhibit a mutation in the rpsL gene, which is known to confer resistance to streptomycin62. Before oral administrations, ECN and MG1655 strains were grown for 6 h in LB broth supplemented with streptomycin (50 µg/mL) at 37 °C with shaking. This culture was diluted 1:100 in LB broth without antibiotics and cultured overnight at 37 °C with shaking. Bacterial pellets from this overnight culture were diluted in sterile PBS to the concentration of 109 colony forming units (cfu)/ml. Mice were treated by oral gavage with 108 cfu of ECN or MG1655 in 100 µl of PBS or 100 µl of PBS as negative lung inflammation model in miceMice were anesthetized by an iv injection of ketamine/xylazine followed by an intranasal administration of 25 µg of papain (Calbiochem, Darmstadt, Germany) in 40 µL of saline solution. Mice were euthanized by CO2 inhalation 24 h after papain administration and BALF was collected. After a hearth perfusion with ISOTON II (Acid free balanced electrolyte solution Beckman Coulter, Krefeld, Germany) lung were collected and sampled for alveolar lavage (BAL)BAL was performed by 4 lavages of lung with 500 µL of saline solution via a cannula introduced into mice trachea. BAL fluids were centrifuged at 400 g for 10 min at 4 °C, the supernatants were stored at −20 °C for ELISA analysis and pellets were recovered to prepare cytospin (Thermo scientific, Waltham, USA) glass slides followed by a Diff-Quik (Merz & Dade Dudingen, Switzerland) staining. Differential cell counts were performed with at least 400 eosinophil peroxidase (EPO) activityEPO activity was determined in order to estimate the recruitment of eosinophil counts in lung parenchyma as expressionTotal RNA was isolated from homogenized mouse lung using Tri Reagent (Sigma) and quantified by NanoDrop (Nd-1000). Reverse transcription was performed withSuperScript III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected to quantitative PCR using primers for Muc5ac (sense 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and anti-sense 5′-AGTCTCTCTCCGCTCCTCTCA-3′; Sigma). Relative transcript expression of a gene is given as 2−ΔCt(ΔCt = Cttarget−Ctreference), and relative changes compared with control are 2−ΔΔCtvalues (ΔΔCt = ΔCttreated−ΔCtcontrol) {John, 2014 #340}.Enzyme-linked Immunosorbent assay (ELISA)Homogenized lung or cell supernatant were tested for MPO, CXCL1, CCL24, CCL11, CCL17, IL-4, IL17A and IFNγ (R&D systems Abingdon, UK), IL-13, IL-5, IL-33 (eBiosciences, San-5, Diego, USA) using commercial ELISA kits according to the manufacturer’s left lobe of lung was fixed in 4% buffered formaldehyde and paraffin embedded under standard conditions. Tissue sections (3 µm) were stained with PAS. Histological changes such as inflammation and emphysema were assessed by a semi-quantitative score from 0 to 5 for cell infiltration (with increasing severity) as described before66. The slides were examined by two blinded investigators with a Leica microscope (Leica, Germany).Determination of bronchial hyperresponsiveness (AHR)For invasive measurement of dynamic resistance, mice were anesthetized with intra-peritoneal injection of solution containing ketamine (100 mg/kg, Merial) and xylasine (10 mg/kg, Bayer), paralyzed using D-tubocuranine ( Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per min with a tidal volume of ml and a positive end-expiratory pressure of 2 ml H2O. Increasing concentrations of aerosolized methacholine ( 75 and 150 mg/ml) were administered. Resistance was recorded using an invasive plethysmograph (Buxco, London, UK). Baseline resistance was restored before administering the subsequent doses of immunofluorescence stainingLungs were fixed for 3 days in 4% PFA and submerged in 20% sucrose for 1 week. Lungs were embedded in OCT (Tissue-Teck) and 10 µM sections were prepared with cryotome (Leica). Slides were incubated 30 min in citrate buffer at 80 °C, washed in TBS-Tween and then incubated overnight with mouse-anti-mouse-E-cadherin (1 µg/ml, ab76055, Abcam). After washing with slides were treated with 0,05% pontamin sky blue (Sigma) for 15 min and then incubated with secondary AF-546 goat anti-mouse antibody (Abcam) for 30 min at room temperature. After washing, slides were incubated with DAPI (Fisher Scientific) and mounted in fluoromount® (SouthernBiotech). Lung sections were observed on a fluorescence microscope Leica (Leica, CTR6000) at x200 magnification. The slides were analyzed and semi-quantitatively scored and the MFI was epithelial barrier functionTotal protein in BAL fluid and Evans blue EB leak in BAL fluid was determined as described mononuclear cell isolation and stimulationLung mononuclear cells were isolated from mice 24 h after the last challenge as described previously67. Briefly the aorta and the inferior vena cava were sectioned and the lungs were perfused with 10 mL of saline. The lobes of the lungs were sliced into small cubes and then incubated for 45 min in 1 ml of RPMI 1640 solution and digested in 1,25 mg/ml of Liberase TL (Roche Diagnostics) and 1 mg/ml DNAse 1 (Sigma) during 1 h at 37 °C. Red blood cells were lysed with lysing buffer (BD Pharm LyseTM – BD Pharmingen). Isolated lung mononuclear single live cells were plated in round bottom 96-well plates (1 × 106/ml) and restimulated 3 h at 37 °C with PMA (50 ng/mL) and ionomicyn (750 ng/mL) in the presence of Brefeldin A (1 μl/1 × 106 cells, BD Biosciences) for intracellular flow cytometry analysis. Lung mononuclear cell (1 × 106 cells) were restimulated with 25 µg of papain in RPMI and 10% SVF at 37 °C in a 96 well plate for 3 days. Supernatants were analyzed for the presence of IL-5, IL-13 and IL-33 by ELISA (invitrogen).Flow cytometry analysis on lung mononuclear cellsLung mononuclear cells were stained with V450-conjugated anti-CD45 (clone 30F11), PerCp anti-CD3e (clone 145-2C11), FITC-conjugated anti-CD4 (clone RM4-5), PE-Cy7 -conjugated anti-ICOS (clone FITC-conjugated anti-ST2 (clone U29-93), anti B220 (clone RA3-6B2), anti FcεRI (clone MAR-1), anti CD11b (clone M1/70), anti Siglec-F (clone E50-2440) and Fixable Viability Dye eFluor™ 780 (eBioscience). After washing, cells were permeabilized for 20 min with cytofix/cytoperm kit (BD Biosciences) and stained with, eFluor 660-conjugated anti-IL13 (clone eBio13A, eBiosciences) and PE-conjugated anti-IL-5 (clone All antibodies used in this were from BD Biosciences, unless otherwise specified. Data were acquired using FACS Canto II flow cytometer and analyzed using Diva and FlowJo analysisData were analyzed using Prism version 5 (Graphpad Software, San Diego, USA). The parametric one-way ANOVA test with multiple Bonferroni’s comparison test was used. Values are expressed as mean ± SEM. Statistical significance was defined at a p-value < ReferencesAccordini, S. et al. The cost of persistent asthma in Europe: an international population-based study in adults. International archives of allergy and immunology 160, 93–101, (2013).Article PubMed Google Scholar Barnett, S. B. & Nurmagambetov, T. A. Costs of asthma in the United States: 2002–2007. 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The authors are grateful to Dieudonnée Togbé for helpful discussions and suggestions. This work was supported by ANR (ANR-GUI-AAP-06-Coliforlife), le Centre National de la Recherche Scientifique, the University of Orléans, la Région Centre (2013-00085470), European funding in Region Centre-Val de Loire (FEDER N° 2016-00110366), le Ministère de l’Education Nationale, de la Recherche et de la Technologie to RA as PhD fellowship, l’Institut National de la Santé et de la Recherche Médicale to ACM as a postdoctoral informationAuthor notesThomas SecherPresent address: INSERM, UMR 1100, Research Center for Respiratory Diseases, and University of Tours, Tours, FranceAuthors and AffiliationsIRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, FranceThomas Secher, Michèle Boury & Eric OswaldCNRS, UMR7355, Experimental and Molecular Immunology and Neurogenetics, Orleans, FranceIsabelle Maillet, Claire Mackowiak, Jessica Le Bérichel, Amandine Philippeau, Corinne Panek, Francois Erard, Marc Le Bert, Valérie Quesniaux, Aurélie Couturier-Maillard & Bernhard RyffelCHU Toulouse, Hôpital Purpan, Service de Bactériologie-Hygiène, Toulouse, FranceEric OswaldCentre de Physiopathologie de Toulouse Purpan (CPTP), Université de Toulouse, UPS, Inserm, CNRS, Toulouse, FranceAbdelhadi SaoudiUniversity of Orleans, Orleans, FranceValérie Quesniaux & Bernhard RyffelUniversity of Cape Town, IDM, Cape Town, Republic of South AfricaBernhard RyffelAuthorsThomas SecherYou can also search for this author in PubMed Google ScholarIsabelle MailletYou can also search for this author in PubMed Google ScholarClaire MackowiakYou can also search for this author in PubMed Google ScholarJessica Le BérichelYou can also search for this author in PubMed Google ScholarAmandine PhilippeauYou can also search for this author in PubMed Google ScholarCorinne PanekYou can also search for this author in PubMed Google ScholarMichèle BouryYou can also search for this author in PubMed Google ScholarEric OswaldYou can also search for this author in PubMed Google ScholarAbdelhadi SaoudiYou can also search for this author in PubMed Google ScholarFrancois ErardYou can also search for this author in PubMed Google ScholarMarc Le BertYou can also search for this author in PubMed Google ScholarValérie QuesniauxYou can also search for this author in PubMed Google ScholarAurélie Couturier-MaillardYou can also search for this author in PubMed Google ScholarBernhard RyffelYou can also search for this author in PubMed Google ScholarContributionsConceived and designed the experiments: and Performed the experiments: and Analyzed the data: Wrote the paper: and authorsCorrespondence to Thomas Secher or Bernhard declarations Competing Interests The authors declare no competing interests. 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To view a copy of this license, visit Reprints and PermissionsAbout this articleCite this articleSecher, T., Maillet, I., Mackowiak, C. et al. The probiotic strain Escherichia coli Nissle 1917 prevents papain-induced respiratory barrier injury and severe allergic inflammation in mice. Sci Rep 8, 11245 (2018). citationReceived: 12 September 2017Accepted: 16 July 2018Published: 26 July 2018DOI: CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Review Escherichia coli Nissle 1917 in Ulcerative Colitis Treatment: Systematic Review and Meta-analysis Giuseppe Losurdo et al. J Gastrointestin Liver Dis. 2015 Dec. Free article Abstract Background and aims: Escherichia coli Nissle 1917 (EcN) has been recommended as a therapeutic tool for ulcerative colitis (UC) treatment. However, to date, no meta-analysis has been performed on this topic. Methods: We performed a literature search on PubMed, MEDLINE, Science Direct and EMBASE. We evaluated success rates for induction of remission, relapse rates and side effects, expressed as Intention-To-Treat. Odd ratios (OR), pooled OR and 95% confidence intervals (CI) were calculated, based on the Mantel-Haenszel method. Heterogeneity was assessed by using the χ2 and I2 statistics and, if present, a random-effects model was adopted. Results: We selected six eligible trials, with 719 patients, 390 assigned to the study group and 329 to the control group. EcN induced remission in of cases, while in the control group (mesalazine) the remission was achieved in of cases, with a mean difference of The pooled OR was (95% CI p= A single study showed a better performance of EcN than the placebo. A relapse of the disease occurred in in the EcN group and in in the control group (mesalazine), with a mean difference of OR= with a 95% CI of (p= Side effects were comparable (OR= 95% CI p= Conclusions: EcN is equivalent to mesalazine in preventing disease relapse, thus confirming current guideline recommendations. EcN seems to be as effective as controls in inducing remission and therefore, its use cannot be recommended as in one study the comparison was performed against placebo. Further studies may be helpful for this subject. Similar articles Role and mechanisms of action of Escherichia coli Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update. Scaldaferri F, Gerardi V, Mangiola F, Lopetuso LR, Pizzoferrato M, Petito V, Papa A, Stojanovic J, Poscia A, Cammarota G, Gasbarrini A. Scaldaferri F, et al. World J Gastroenterol. 2016 Jun 28;22(24):5505-11. doi: World J Gastroenterol. 2016. PMID: 27350728 Free PMC article. Review. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT. Rembacken BJ, et al. Lancet. 1999 Aug 21;354(9179):635-9. doi: Lancet. 1999. PMID: 10466665 Clinical Trial. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Kruis W, Fric P, Pokrotnieks J, Lukás M, Fixa B, Kascák M, Kamm MA, Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. Kruis W, et al. Gut. 2004 Nov;53(11):1617-23. doi: Gut. 2004. PMID: 15479682 Free PMC article. Clinical Trial. [Maintaining remission of ulcerative colitis with the probiotic Escherichia Coli Nissle 1917 is as effective as with standard mesalazine]. Adam B, Liebregts T, Holtmann G. Adam B, et al. Z Gastroenterol. 2006 Mar;44(3):267-9. doi: Z Gastroenterol. 2006. PMID: 16514573 German. No abstract available. Probiotics for maintaining remission of ulcerative colitis in adults. Do VT, Baird BG, Kockler DR. Do VT, et al. Ann Pharmacother. 2010 Mar;44(3):565-71. doi: Epub 2010 Feb 2. Ann Pharmacother. 2010. PMID: 20124461 Review. Cited by Efficacy and Safety of Probiotics Combined With Traditional Chinese Medicine for Ulcerative Colitis: A Systematic Review and Meta-Analysis. Hu Y, Ye Z, She Y, Li L, Wu M, Qin K, Li Y, He H, Hu Z, Yang M, Lu F, Ye Q. Hu Y, et al. Front Pharmacol. 2022 Mar 7;13:844961. doi: eCollection 2022. Front Pharmacol. 2022. PMID: 35321324 Free PMC article. Review. Comment on Depoorter, L.; Vandenplas, Y. Probiotics in Pediatrics. A Review and Practical Guide. Nutrients 2021, 13, 2176. von Bünau R, Erhardt A, Stange E. von Bünau R, et al. Nutrients. 2022 Feb 9;14(4):724. doi: Nutrients. 2022. PMID: 35215374 Free PMC article. Review. A Probiotic Friend. Dubbert S, von Bünau R. Dubbert S, et al. mSphere. 2021 Dec 22;6(6):e0085621. doi: Epub 2021 Dec 22. mSphere. 2021. PMID: 34935447 Free PMC article. No abstract available. MicroRNA and Gut Microbiota: Tiny but Mighty-Novel Insights into Their Cross-talk in Inflammatory Bowel Disease Pathogenesis and Therapeutics. Casado-Bedmar M, Viennois E. Casado-Bedmar M, et al. J Crohns Colitis. 2022 Jul 14;16(6):992-1005. doi: J Crohns Colitis. 2022. PMID: 34918052 Free PMC article. Review. Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation. Seco EM, Fernández LÁ. Seco EM, et al. Microb Biotechnol. 2022 May;15(5):1374-1391. doi: Epub 2021 Nov 9. Microb Biotechnol. 2022. PMID: 34755474 Free PMC article. Publication types MeSH terms Substances LinkOut - more resources Full Text Sources Iuliu Hatieganu Medical Publishing House Other Literature Sources The Lens - Patent Citations Medical Genetic Alliance MedlinePlus Health Information

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