Publications by authors named "Hang Thi Thu Nguyen"

45 Publications

Colibactin-Producing Induce the Formation of Invasive Carcinomas in a Chronic Inflammation-Associated Mouse Model.

Cancers (Basel) 2021 Apr 24;13(9). Epub 2021 Apr 24.

M2iSH, UMR 1071 Inserm, Université Clermont Auvergne, INRAE USC 2018, CRNH, 63001 Clermont-Ferrand, France.

Background: producing the genotoxin colibactin (CoPEC or colibactin-producing ) abnormally colonize the colonic mucosa of colorectal cancer (CRC) patients. We previously showed that deficiency of autophagy in intestinal epithelial cells (IECs) enhances CoPEC-induced colorectal carcinogenesis in mice. Here, we tested if CoPEC trigger tumorigenesis in a mouse model lacking genetic susceptibility or the use of carcinogen.

Methods: Mice with autophagy deficiency in IECs () or wild-type mice () were infected with the CoPEC 11G5 strain or the mutant 11G5∆clbQ incapable of producing colibactin and subjected to 12 cycles of DSS treatment to induce chronic colitis. Mouse colons were used for histological assessment, immunohistochemical and immunoblot analyses for DNA damage marker. : 11G5 or 11G5∆clbQ infection increased clinical and histological inflammation scores, and these were further enhanced by IEC-specific autophagy deficiency. 11G5 infection, but not 11G5∆clbQ infection, triggered the formation of invasive carcinomas, and this was further increased by autophagy deficiency. The increase in invasive carcinomas was correlated with enhanced DNA damage and independent of inflammation. : CoPEC induce colorectal carcinogenesis in a CRC mouse model lacking genetic susceptibility and carcinogen. This work highlights the role of (i) CoPEC as a driver of CRC development, and (ii) autophagy in inhibiting the carcinogenic properties of CoPEC.
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http://dx.doi.org/10.3390/cancers13092060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123153PMC
April 2021

Deciphering the Relationship Between Cycloheximides Structures and Their Different Biological Activities.

Front Microbiol 2021 7;12:644853. Epub 2021 Apr 7.

Department of Agricultural Chemistry, College of Agriculture and Life Science, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju, South Korea.

species are the most important sources of antibacterial, antifungal, and phytotoxic metabolites. In this study, cycloheximide (CH) and acetoxycycloheximide (ACH) were isolated from the fermentation broth of sp. JCK-6092. The antifungal and phytotoxic activities of the two compounds (CH and ACH) and a cycloheximide derivative, hydroxycycloheximide (HCH), were compared. CH exhibited the strongest antagonistic activity against all the true fungi tested, followed by ACH and HCH. However, both CH and ACH displayed similar mycelial growth inhibitory activities against several phytopathogenic oomycetes, and both were more active than that of HCH. Disparate to antifungal ability, ACH showed the strongest phytotoxic activity against weeds and crops, followed by HCH and CH. ACH caused chlorophyll content loss, leaf electrolytic leakage, and lipid peroxidation in a dose-dependent manner. Its phytotoxicity was stronger than that of glufosinate-ammonium but weaker than that of paraquat in the experiments. CH and its derivatives are well-known protein synthesis inhibitors; however, the precise differences between their mechanism of action remain undiscovered. A computational study revealed effects of CHs on the protein synthesis of (oomycetes), (true fungus), and (plant) and deciphered the differences in their biological activities on different targets. The binding energies and conformation stabilities of each chemical molecule correlated with their biological activities. Thus, molecular docking study supported the experimental results. This is the first comparative study to suggest the ribosomal protein alteration mechanisms of CHs in plants and fungi and to thus show how the protein inhibitory activities of the different derivatives are altered using molecular docking. The correlation of structures features of CHs in respect to bond formation with desired protein was revealed by density functional theory. Overall collective results suggested that CHs can be used as lead molecules in the development of more potent fungicides and herbicides molecules.
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http://dx.doi.org/10.3389/fmicb.2021.644853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058199PMC
April 2021

Yersiniabactin Siderophore of Crohn's Disease-Associated Adherent-Invasive Is Involved in Autophagy Activation in Host Cells.

Int J Mol Sci 2021 Mar 29;22(7). Epub 2021 Mar 29.

M2iSH (Microbes, Intestin, Inflammation and Susceptibility of the Host), Inserm U1071, INRAE USC 2018, Université Clermont Auvergne, CRNH, 63001 Clermont-Ferrand, France.

Background: Adherent-invasive (AIEC) have been implicated in the etiology of Crohn's disease. The AIEC reference strain LF82 possesses a pathogenicity island similar to the high pathogenicity island of spp., which encodes the yersiniabactin siderophore required for iron uptake and growth of the bacteria in iron-restricted environment. Here, we investigated the role of yersiniabactin during AIEC infection.

Methods: Intestinal epithelial T84 cells and CEABAC10 transgenic mice were infected with LF82 or its mutants deficient in yersiniabactin expression. Autophagy was assessed by Western blot analysis for p62 and LC3-II expression.

Results: Loss of yersiniabactin decreased the growth of LF82 in competitive conditions, reducing the ability of LF82 to adhere to and invade T84 cells and to colonize the intestinal tract of CEABAC10 mice. However, yersiniabactin deficiency increased LF82 intracellular replication. Mechanistically, a functional yersiniabactin is necessary for LF82-induced expression of HIF-1α, which is implicated in autophagy activation in infected cells.

Conclusion: Our study highlights a novel role for yersiniabactin siderophore in AIEC-host interaction. Indeed, yersiniabactin, which is an advantage for AIEC to growth in a competitive environment, could be a disadvantage for the bacteria as it activates autophagy, a key host defense mechanism, leading to bacterial clearance.
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http://dx.doi.org/10.3390/ijms22073512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037853PMC
March 2021

Differential miRNA-Gene Expression in M Cells in Response to Crohn's Disease-Associated AIEC.

Microorganisms 2020 Aug 7;8(8). Epub 2020 Aug 7.

M2iSH, UMR 1071 Inserm, Université Clermont Auvergne, INRAE USC 2018, CRNH, 63001 Clermont-Ferrand, France.

Adherent-invasive (AIEC), which abnormally colonize the ileal mucosa of Crohn's disease (CD) patients, are able to invade intestinal epithelial cells (IECs) and translocate through M cells overlying Peyer's patches. The levels of microRNA (miRNA) and gene expression in IECs and M cells upon AIEC infection have not been investigated. Here, we used human intestinal epithelial Caco-2 monolayers and an in vitro M-cell model of AIEC translocation to analyze comprehensive miRNA and gene profiling under basal condition and upon infection with the reference AIEC LF82 strain. Our results showed that AIEC LF82 translocated through M cells but not Caco-2 monolayers. Both differential gene expression and miRNA profile in M cells compared to Caco-2 cells were obtained. In addition, AIEC infection induces changes in gene and miRNA profiles in both Caco-2 and M cells. In silico analysis showed that certain genes dysregulated upon AIEC infection were potential targets of AIEC-dysregulated miRNAs, suggesting a miRNA-mediated regulation of gene expression during AIEC infection in Caco-2, as well as M cells. This study facilitates the discovery of M cell-specific and AIEC response-specific gene-miRNA signature and enhances the molecular understanding of M cell biology under basal condition and in response to infection with CD-associated AIEC.
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http://dx.doi.org/10.3390/microorganisms8081205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466023PMC
August 2020

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn's disease-associated adherent-invasive .

Gut Microbes 2020 11 25;11(6):1677-1694. Epub 2020 Jun 25.

M2iSH, UMR 1071 Inserm, Université Clermont Auvergne , Clermont-Ferrand, France.

Adherent-invasive (AIEC), which abnormally colonize the intestinal mucosa of Crohn's disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic K12 strain (Exo-K12) or the commensal HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.
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http://dx.doi.org/10.1080/19490976.2020.1771985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524154PMC
November 2020

Emerging Role of Exosomes in Diagnosis and Treatment of Infectious and Inflammatory Bowel Diseases.

Cells 2020 04 30;9(5). Epub 2020 Apr 30.

M2iSH, UMR 1071 Inserm, Université Clermont Auvergne, INRAE USC 2018, CRNH, 63001 Clermont-Ferrand, France.

To communicate with each other, cells release exosomes that transfer their composition, including lipids, proteins and nucleic acids, to neighboring cells, thus playing a role in various pathophysiological processes. During an infection with pathogenic bacteria, such as adherent-invasive (AIEC) associated with Crohn disease, exosomes secreted by infected cells can have an impact on the innate immune responses of surrounding cells to infection. Furthermore, inflammation can be amplified via the exosomal shuttle during infection with pathogenic bacteria, which could contribute to the development of the associated disease. Since these vesicles can be released in various biological fluids, changes in exosomal content may provide a means for the identification of non-invasive biomarkers for infectious and inflammatory bowel diseases. Moreover, evidence suggests that exosomes could be used as vaccines to prime the immune system to recognize and kill invading pathogens, and as therapeutic components relieving intestinal inflammation. Here, we summarize the current knowledge on the role of exosomes in bacterial infections and highlight their potential use as biomarkers, vaccines and conveyers of therapeutic molecules in inflammatory bowel diseases.
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http://dx.doi.org/10.3390/cells9051111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290936PMC
April 2020

Immunization with the H5N1 Recombinant Vaccine Candidate Induces High Protection in Chickens against Vietnamese Highly Pathogenic Avian Influenza Virus Strains.

Vaccines (Basel) 2020 Apr 2;8(2). Epub 2020 Apr 2.

Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi 100000, Vietnam.

Vietnam is one of the countries most affected worldwide by the highly pathogenic avian influenza (HPAI) virus, which caused enormous economic loss and posed threats to public health. Over nearly two decades, with the antigenic changes in the diversified H5Ny viruses, the limited protective efficacy of the available vaccines was encountered. Therefore, it is necessary to approach a technology platform for the country to accelerate vaccine production that enables quick response to new influenza subtypes. This study utilized a powerful reverse genetics technique to successfully generate a recombinant H5N1 vaccine strain (designated as IBT-RG02) containing two surface proteins (haemagglutinin (HA) and neuraminidase (NA)) from the HPAI H5N1 (A/duck/Vietnam/HT2/2014(H5N1)) of the dominant clade 2.3.2.1c in Vietnam during 2012-2014. Importantly, the IBT-RG02 vaccine candidate has elicited high antibody titres in chickens (geometric mean titre (GMT) of 6.42 and 6.92, log on day 14 and day 28 p.i., respectively). To test the efficacy, immunized chickens were challenged with the circulating virulent strains. As results, there was a high protection rate of 91.6% chickens against the virulent A/DK/VN/Bacninh/NCVD-17A384/2017 of the same clade and a cross-protection of 83.3% against A/duck/TG/NAVET(3)/2013 virus of clade 1.1. Our promising results showed that we can independently master the reverse genetics technology for generation of highly immunogenic vaccine candidates, and henceforth, it is a timely manner to reformulate avian influenza virus vaccines against variable H5 clade HPAI viruses in Vietnam.
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http://dx.doi.org/10.3390/vaccines8020159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348806PMC
April 2020

Establishment of a Standardized Vaccine Protocol for the Analysis of Protective Immune Responses During Experimental Trypanosome Infections in Mice.

Methods Mol Biol 2020 ;2116:721-738

Biomedical Research Centre, Department of Environmental Technology, Food Technology and Molecular Biotechnology, Ghent University, Incheon, South Korea.

To date, trypanosomosis control in humans and animals is achieved by a combination of parasitological screening and treatment. While this approach has successfully brought down the number of reported T. b. gambiense Human African Trypanosomosis (HAT) cases, the method does not offer a sustainable solution for animal trypanosomosis (AT). The main reasons for this are (i) the worldwide distribution of AT, (ii) the wide range of insect vectors involved in transmission of AT, and (iii) the existence of a wildlife parasite reservoir that can serve as a source for livestock reinfection. Hence, in order to control livestock trypanosomosis the only viable long-term solution is an effective antitrypanosome vaccination strategy. Over the last decades, multiple vaccine approaches have been proposed. Despite repeated reports of promising experimental approaches, none of those made it to a field applicable vaccine format. This failure can in part be attributed to flaws in the experimental design that favor a positive laboratory result. This chapter provides a vaccine protocol that should allow for a proper outcome prediction in experimental anti-AT vaccine approaches.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_42DOI Listing
February 2021

Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in Apc Mice.

Gastroenterology 2020 04 7;158(5):1373-1388. Epub 2020 Jan 7.

M2iSH (Microbes, intestine, inflammation and Susceptibility of the Host), UMR 1071 Inserm, University of Clermont Auvergne, Clermont-Ferrand, France. Electronic address:

Background & Aims: Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC.

Methods: Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. Apc mice and Apc mice with IEC-specific disruption of Atg16l1 (Apc/Atg16l1) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction.

Results: Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, Apc/Atg16l1 mice developed fewer and smaller colon tumors than Apc mice. However, after infection with 11G5, Apc/Atg16l1 mice developed more and larger tumors, with a significant increase in mean histologic score, than infected Apc mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected Apc/Atg16l1 mice vs 11G5-infected Apc mice.

Conclusion: Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected Apc mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.
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http://dx.doi.org/10.1053/j.gastro.2019.12.026DOI Listing
April 2020

New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD.

Autophagy 2020 01 9;16(1):38-51. Epub 2019 Jul 9.

M2iSH, UMR 1071 Inserm, INRA USC 2018, CRNH, University of Clermont Auvergne, Clermont-Ferrand, France.

One of the most significant challenges of inflammatory bowel disease (IBD) research is to understand how alterations in the symbiotic relationship between the genetic composition of the host and the intestinal microbiota, under impact of specific environmental factors, lead to chronic intestinal inflammation. Genome-wide association studies, followed by functional studies, have identified a role for numerous autophagy genes in IBD, especially in Crohn disease. Studies using and models, in addition to human clinical studies have revealed that autophagy is pivotal for intestinal homeostasis maintenance, gut ecology regulation, appropriate intestinal immune responses and anti-microbial protection. This review describes the latest researches on the mechanisms by which dysfunctional autophagy leads to disrupted intestinal epithelial function, gut dysbiosis, defect in anti-microbial peptide secretion by Paneth cells, endoplasmic reticulum stress response and aberrant immune responses to pathogenic bacteria. A better understanding of the role of autophagy in IBD pathogenesis may provide better sub-classification of IBD phenotypes and novel approaches for disease management. AIEC: adherent-invasive ; AMPK: AMP-activated protein kinase; ATF6: activating transcription factor 6; ATG: autophagy related; mice: mice with depletion specifically in intestinal epithelial cells; mice: mice hypomorphic for expression; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; CALCOCO2: calcium binding and coiled-coil domain 2; CASP: caspase; CD: Crohn disease; CGAS: cyclic GMP-AMP synthase; CHUK/IKKA: conserved helix-loop-helix ubiquitous kinase; CLDN2: claudin 2; DAPK1: death associated protein kinase 1; DCs: dendritic cells; DSS: dextran sulfate sodium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK: eukaryotic translation initiation factor 2 alpha kinase; ER: endoplasmic reticulum; ERBIN: Erbb2 interacting protein; ERN1/IRE1A: ER to nucleus signaling 1; FNBP1L: formin binding protein 1-like; FOXP3: forkhead box P3; GPR65: G-protein coupled receptor 65; GSK3B: glycogen synthase kinase 3 beta; IBD: inflammatory bowel disease; IECs: intestinal epithelial cells; IFN: interferon; IL: interleukin; IL10R: interleukin 10 receptor; IRGM: immunity related GTPase M; ISC: intestinal stem cell; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; LRRK2: leucine-rich repeat kinase 2; MAPK: mitogen-activated protein kinase; MHC: major histocompatibility complex; MIF: macrophage migration inhibitory factor; MIR/miRNA: microRNA; MTMR3: myotubularin related protein 3; MTOR: mechanistic target of rapamycin kinase; MYD88: myeloid differentiation primary response gene 88; NLRP3: NLR family, pyrin domain containing 3; NOD2: nucleotide-binding oligomerization domain containing 2; NPC: Niemann-Pick disease type C; NPC1: NPC intracellular cholesterol transporter 1; OMVs: outer membrane vesicles; OPTN: optineurin; PI3K: phosphoinositide 3-kinase; PRR: pattern-recognition receptor; PTPN2: protein tyrosine phosphatase, non-receptor type 2; PTPN22: protein tyrosine phosphatase, non-receptor type 22 (lymphoid); PYCARD/ASC: PYD and CARD domain containing; RAB2A: RAB2A, member RAS oncogene family; RELA: v-rel reticuloendotheliosis viral oncogene homolog A (avian); RIPK2: receptor (TNFRSF)-interacting serine-threonine kinase 2; ROS: reactive oxygen species; SNPs: single nucleotide polymorphisms; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; Th: T helper 1; TIRAP/TRIF: toll-interleukin 1 receptor (TIR) domain-containing adaptor protein; TLR: toll-like receptor; TMEM173/STING: transmembrane protein 173; TMEM59: transmembrane protein 59; TNF/TNFA: tumor necrosis factor; Treg: regulatory T; TREM1: triggering receptor expressed on myeloid cells 1; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type; XBP1: X-box binding protein 1; XIAP: X-linked inhibitor of apoptosis.
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http://dx.doi.org/10.1080/15548627.2019.1635384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6984609PMC
January 2020

AIEC infection triggers modification of gut microbiota composition in genetically predisposed mice, contributing to intestinal inflammation.

Sci Rep 2018 08 17;8(1):12301. Epub 2018 Aug 17.

M2iSH (Microbes, intestine, inflammation and Susceptibility of the Host), UMR 1071 Inserm, Université Clermont Auvergne, INRA USC 2018, Clermont-Ferrand, 63001, France.

A high prevalence of adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn's disease patients has been shown. AIEC colonize the intestine and induce inflammation in genetically predisposed mouse models including CEABAC10 transgenic (Tg) mice expressing human CEACAM6-receptor for AIEC and eif2ak4 mice exhibiting autophagy defect in response to AIEC infection. Here, we aimed at investigating whether gut microbiota modification contributes to AIEC-induced intestinal inflammation in these mouse models. For this, eif2ak4 and eif2ak4 mice or CEABAC10 Tg mice invalidated for Eif2ak4 gene (Tg/eif2ak4) or not (Tg/eif2ak4) were infected with the AIEC reference strain LF82 or the non-pathogenic E. coli K12 MG1655 strain. In all mouse groups, LF82 colonized the gut better and longer than MG1655. No difference in fecal microbiota composition was observed in eif2ak4 and eif2ak4 mice before infection and at day 1 and 4 post-infection. LF82-infected eif2ak4 mice exhibited altered fecal microbiota composition at day 14 and 21 post-infection and increased fecal lipocalin-2 level at day 21 post-infection compared to other groups, indicating that intestinal inflammation developed after microbiota modification. Similar results were obtained for LF82-infected Tg/eif2ak4 mice. These results suggest that in genetically predisposed hosts, AIEC colonization might induce chronic intestinal inflammation by altering the gut microbiota composition.
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http://dx.doi.org/10.1038/s41598-018-30055-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098085PMC
August 2018

Microbiota, Inflammation and Colorectal Cancer.

Int J Mol Sci 2017 Jun 20;18(6). Epub 2017 Jun 20.

M2iSH, UMR 1071 Inserm, University of Clermont Auvergne, INRA USC 2018, Clermont-Ferrand 63001, France.

Colorectal cancer, the fourth leading cause of cancer-related death worldwide, is a multifactorial disease involving genetic, environmental and lifestyle risk factors. In addition, increased evidence has established a role for the intestinal microbiota in the development of colorectal cancer. Indeed, changes in the intestinal microbiota composition in colorectal cancer patients compared to control subjects have been reported. Several bacterial species have been shown to exhibit the pro-inflammatory and pro-carcinogenic properties, which could consequently have an impact on colorectal carcinogenesis. This review will summarize the current knowledge about the potential links between the intestinal microbiota and colorectal cancer, with a focus on the pro-carcinogenic properties of bacterial microbiota such as induction of inflammation, the biosynthesis of genotoxins that interfere with cell cycle regulation and the production of toxic metabolites. Finally, we will describe the potential therapeutic strategies based on intestinal microbiota manipulation for colorectal cancer treatment.
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http://dx.doi.org/10.3390/ijms18061310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486131PMC
June 2017

[Regulation of immunity and inflammation by autophagy: « All is well, all is fine, all goes as well as possible»].

Med Sci (Paris) 2017 03 3;33(3):305-311. Epub 2017 Apr 3.

CIRI, Centre international de recherche en infectiologie, équipe autophagie infection immunité, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, 21, avenue Tony Garnier, F-69007, Lyon, France.

Autophagy is a lysosomal degradation mechanism which helps to control intracellular infections and contributes to the regulation of innate and adaptive immune responses. Defects in autophagy lead to exacerbated proliferation of microorganisms and/or to excessive immune responses which are both highly deleterious. Thus, infectious and chronic inflammatory human diseases, such as Crohn's disease, are often associated with inappropriate modulation of autophagy, which is mainly linked to autophagy-associated gene polymorphisms. In this review, we highlight the current understanding of role of autophagy in infections and immunity.
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http://dx.doi.org/10.1051/medsci/20173303018DOI Listing
March 2017

Exosomes: From Functions in Host-Pathogen Interactions and Immunity to Diagnostic and Therapeutic Opportunities.

Rev Physiol Biochem Pharmacol 2016 ;172:39-75

University of Clermont Auvergne, M2iSH, UMR 1071 INSERM/University of Auvergne, Clermont-Ferrand, 63001, France.

Since their first description in the 1980s, exosomes, small endosomal-derived extracellular vesicles, have been involved in innate and adaptive immunity through modulating immune responses and mediating antigen presentation. Increasing evidence has reported the role of exosomes in host-pathogen interactions and particularly in the activation of antimicrobial immune responses. The growing interest concerning exosomes in infectious diseases, their accessibility in various body fluids, and their capacity to convey a rich content (e.g., proteins, lipids, and nucleic acids) to distant recipient cells led the scientific community to consider the use of exosomes as potential new diagnostic and therapeutic tools. In this review, we summarize current understandings of exosome biogenesis and their composition and highlight the function of exosomes as immunomodulators in pathological states such as in infectious disorders. The potential of using exosomes as diagnostic and therapeutic tools is also discussed.
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http://dx.doi.org/10.1007/112_2016_7DOI Listing
June 2017

Analysis of microRNA Levels in Intestinal Epithelial Cells.

Methods Mol Biol 2016 ;1422:89-99

UMR 1071 Inserm, University of Auvergne, 28 Place Henri Dunant, Clermont-Ferrand, 63001, France.

The field of microRNA (miRNA) research is expanding rapidly with the crucial role of miRNAs in almost every biological process and their implication in many diseases. The role of miRNAs in modulating inflammatory responses in the gut has attracted many research groups including us. Here, we first briefly summarize our current understanding of the role of miRNAs in maintaining and regulating gut physiopathology and in inflammatory bowel diseases. We then describe in detail our techniques to analyze miRNA levels with notes that we have collected and summarized during our experiments.
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http://dx.doi.org/10.1007/978-1-4939-3603-8_9DOI Listing
December 2017

Activation of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway triggers autophagy response to Crohn disease-associated adherent-invasive Escherichia coli infection.

Autophagy 2016 05 17;12(5):770-83. Epub 2016 Mar 17.

a University of Clermont Auvergne, M2iSH, UMR 1071 INSERM/University of Auvergne , Clermont-Ferrand , France.

The intestinal mucosa of Crohn disease (CD) patients is abnormally colonized by adherent-invasive E. coli (AIEC). Upon AIEC infection, autophagy is induced in host cells to restrain bacterial intracellular replication. The underlying mechanism, however, remains unknown. Here, we investigated the role of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway in the autophagic response to AIEC infection. We showed that infection of human intestinal epithelial T84 cells with the AIEC reference strain LF82 activated the EIF2AK4-EIF2A-ATF4 pathway, as evidenced by increased phospho-EIF2AK4, phospho-EIF2A and ATF4 levels. EIF2AK4 depletion inhibited autophagy activation in response to LF82 infection, leading to increased LF82 intracellular replication and elevated pro-inflammatory cytokine production. Mechanistically, EIF2AK4 depletion suppressed the LF82-induced ATF4 binding to promoters of several autophagy genes including MAP1LC3B, BECN1, SQSTM1, ATG3 and ATG7, and this subsequently inhibited transcription of these genes. LF82 infection of wild-type (WT), but not eif2ak4(-/-), mice activated the EIF2AK4-EIF2A-ATF4 pathway, inducing autophagy gene transcription and autophagy response in enterocytes. Consequently, eif2ak4(-/-) mice exhibited increased intestinal colonization by LF82 bacteria and aggravated inflammation compared to WT mice. Activation of the EIF2AK4-EIF2A-ATF4 pathway was observed in ileal biopsies from patients with noninflamed CD, and this was suppressed in inflamed CD, suggesting that a defect in the activation of this pathway could be one of the mechanisms contributing to active disease. In conclusion, we show that activation of the EIF2AK4-EIF2A-ATF4 pathway upon AIEC infection serves as a host defense mechanism to induce functional autophagy to control AIEC intracellular replication.
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http://dx.doi.org/10.1080/15548627.2016.1156823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854551PMC
May 2016

Exosomes Released from Cells Infected with Crohn's Disease-associated Adherent-Invasive Escherichia coli Activate Host Innate Immune Responses and Enhance Bacterial Intracellular Replication.

Inflamm Bowel Dis 2016 Mar;22(3):516-28

*UMR 1071 Inserm, University of Auvergne, Clermont-Ferrand, France; ‡INRA USC 2018, Clermont-Ferrand, France; and §Centre Hospitalier Universitaire, Clermont-Ferrand, France.

Background: Crohn's disease is a chronic inflammatory bowel disease, of which the etiology involves environmental, genetic, and microbial factors. A high prevalence of adherent-invasive Escherichia coli, named AIEC, has been reported in the intestinal mucosa of patients with Crohn's disease. Exosomes are extracellular vesicles that function in intercellular communication and have been implicated in host responses to intracellular pathogens. We investigated the potential involvement of exosomes in host response to AIEC infection.

Methods: Human intestinal epithelial T84 cells, THP-1 macrophages, and CEABAC10 transgenic mice were infected with the AIEC reference strain LF82 or the nonpathogenic E. coli K-12 MG1655 strain. Exosomes were purified using the ExoQuick reagent.

Results: LF82 infection induced the release of exosomes by T84 and THP-1 cells. Compared with exosomes released from the uninfected or MG1655-infected T84 cells, those released from LF82-infected cells activated nuclear factor-kappa B, mitogen-activated protein kinases p38, and c-Jun N-terminal kinase and increased the secretion of proinflammatory cytokines in naive THP-1 macrophages. LF82 infection of THP-1 macrophages also induced the release of exosomes that triggered a proinflammatory response in recipient THP-1 cells. Importantly, stimulation of T84 or THP-1 cells with exosomes released from LF82-infected cells increased LF82 intracellular replication compared with stimulation with exosomes secreted by uninfected cells. Exosomes purified from intestinal lumen of CEABAC10 transgenic mice infected with LF82 increased proinflammatory responses in murine RAW 264.7 macrophages compared with those from uninfected or MG1655-infected mice.

Conclusions: Exosomes are new mediators of host-AIEC interaction with their capacity to activate innate immune responses and subvert the control of AIEC replication.
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http://dx.doi.org/10.1097/MIB.0000000000000635DOI Listing
March 2016

The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli.

Cell Microbiol 2016 May 23;18(5):617-31. Epub 2015 Nov 23.

Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France.

The aetiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are receiving increased attention because in studies of mucosa-associated microbiota, they are more prevalent in CD patients than in healthy subjects. AIEC are associated both with ileal and colonic disease phenotypes. In this study, we reported a protease called Vat-AIEC from AIEC that favours the mucosa colonization. The deletion of the Vat-AIEC-encoding gene resulted in an adhesion-impaired phenotype in vitro and affected the colonization of bacteria in contact with intestinal epithelial cells in a murine intestinal loop model, and also their gut colonization in vivo. Furthermore, unlike LF82Δvat-AIEC, wild-type AIEC reference strain LF82 was able to penetrate a mucus column extensively and promoted the degradation of mucins and a decrease in mucus viscosity. Vat-AIEC transcription was stimulated by several chemical conditions found in the ileum environment. Finally, the screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Overall, this study revealed a new component of AIEC virulence that might favour their implantation in the gut of CD patients.
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http://dx.doi.org/10.1111/cmi.12539DOI Listing
May 2016

Infectious etiopathogenesis of Crohn's disease.

World J Gastroenterol 2014 Sep;20(34):12102-17

Jessica Carrière, Arlette Darfeuille-Michaud, Hang Thi Thu Nguyen, Clermont Université, M2iSH, UMR 1071 Inserm/University of Auvergne, 63001 Clermont-Ferrand, France.

Important advances during the last decade have been made in understanding the complex etiopathogenesis of Crohn's disease (CD). While many gaps in our knowledge still exist, it has been suggested that the etiology of CD is multifactorial including genetic, environmental and infectious factors. The most widely accepted theory states that CD is caused by an aggressive immune response to infectious agents in genetically predisposed individuals. The rise of genome-wide association studies allowed the identification of loci and genetic variants in several components of host innate and adaptive immune responses to microorganisms in the gut, highlighting an implication of intestinal microbiota in CD etiology. Moreover, numerous independent studies reported a dysbiosis, i.e., a modification of intestinal microbiota composition, with an imbalance between the abundance of beneficial and harmful bacteria. Although microorganisms including viruses, yeasts, fungi and bacteria have been postulated as potential CD pathogens, based on epidemiological, clinicopathological, genetic and experimental evidence, their precise role in this disease is not clearly defined. This review summarizes the current knowledge of the infectious agents associated with an increased risk of developing CD. Therapeutic approaches to modulate the intestinal dysbiosis and to target the putative CD-associated pathogens, as well as their potential mechanisms of action are also discussed.
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http://dx.doi.org/10.3748/wjg.v20.i34.12102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161797PMC
September 2014

Crohn's disease-associated adherent invasive Escherichia coli modulate levels of microRNAs in intestinal epithelial cells to reduce autophagy.

Gastroenterology 2014 Feb 19;146(2):508-19. Epub 2013 Oct 19.

M2iSH, UMR 1071 Inserm/University of Auvergne, Clermont-Ferrand, France; INRA USC 2018, Clermont-Ferrand, France; Centre Hospitalier Universitaire, Clermont-Ferrand, France. Electronic address:

Background & Aims: Levels of microRNAs are altered in intestinal tissues of patients with Crohn's disease (CD). The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with CD, adhere to and invade intestinal epithelial cells. We investigated the mechanism by which AIEC infection alters the expression of microRNAs and the host immune response.

Methods: Levels of microRNAs in human intestinal epithelial T84 cells and in mouse enterocytes were measured using quantitative reverse-transcription polymerase chain reaction. Luciferase assays were used to measure binding of microRNAs to the 3'-untranslated region of messenger RNA targets. Binding of nuclear factor-κB to promoters of genes encoding microRNAs was assessed by chromatin immunoprecipitation assays. Autophagy was measured by immunoblot analyses and immunofluorescent labeling of LC3. Anti-microRNAs were transferred to mice using ileal loops. Biopsy specimens from the terminal ileum of patients with ulcerative colitis (n = 20), CD (n = 20), or individuals without inflammatory bowel disease undergoing surveillance colonoscopies (controls, n = 13) were collected during endoscopic examination.

Results: AIEC infection up-regulated levels of microRNA (MIR) 30C and MIR130A in T84 cells and in mouse enterocytes by activating nuclear factor-κB. Up-regulation of these microRNAs reduced the levels of ATG5 and ATG16L1 and inhibited autophagy, leading to increased numbers of intracellular AIEC and an increased inflammatory response. In ileal biopsy samples of patients with CD, there was an inverse correlation between levels of MIR30C and MIR130A and those of ATG5 and ATG16L1, supporting in vitro findings. Inhibition of MIR30C and MIR130A in cultured intestinal epithelial cells and in mouse enterocytes blocked AIEC-induced inhibition of ATG5 and ATG16L1 expression and restored functional autophagy. This resulted in more effective clearance of intracellular AIEC and reduced AIEC-induced inflammation.

Conclusions: Infection with AIEC up-regulates microRNAs to reduce expression of proteins required for autophagy and autophagy response in intestinal epithelial cells. Ileal samples from patients with CD have increased levels of these same microRNAs and reduced levels of ATG5 and ATG16L1.
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http://dx.doi.org/10.1053/j.gastro.2013.10.021DOI Listing
February 2014

Role of microRNAs in the immune system, inflammation and cancer.

World J Gastroenterol 2013 May;19(20):2985-96

University of Auvergne, 63000 Clermont-Ferrand, France.

MicroRNAs, a key class of gene expression regulators, have emerged as crucial players in various biological processes such as cellular proliferation and differentiation, development and apoptosis. In addition, microRNAs are coming to light as crucial regulators of innate and adaptive immune responses, and their abnormal expression and/or function in the immune system have been linked to multiple human diseases including inflammatory disorders, such as inflammatory bowel disease, and cancers. In this review, we discuss our current understanding of microRNAs with a focus on their role and mode of action in regulating the immune system during inflammation and carcinogenesis.
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http://dx.doi.org/10.3748/wjg.v19.i20.2985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662938PMC
May 2013

Autophagy and Crohn's disease.

J Innate Immun 2013 15;5(5):434-43. Epub 2013 Jan 15.

UMR 1071 Inserm/Université d'Auvergne, Paris, France.

Advances in genetics have shed light on the molecular basis of Crohn's disease (CD) predisposition and pathogenesis, via linkage disequilibrium analysis to genome-wide association studies. The discovery of genetic variants of NOD2, an intracellular pathogen molecular sensor, as risk factors for CD has paved the way for further research on innate immunity in this disease. Remarkably, polymorphisms in autophagy genes, such as ATG16L1 and IRGM, have been identified, allowing the pivotal role of autophagy in innate immunity to be uncovered. In this review, we summarize recent studies on the CD-associated NOD2, ATG16L1 and IRGM risk variants and their contribution to the autophagy functions that have most influenced our understanding of CD pathophysiology.
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http://dx.doi.org/10.1159/000345129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6741541PMC
November 2013

Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice.

Lab Invest 2012 Aug 28;92(8):1203-12. Epub 2012 May 28.

Department of Biology, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-β-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.
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http://dx.doi.org/10.1038/labinvest.2012.83DOI Listing
August 2012

Homeostatic and innate immune responses: role of the transmembrane glycoprotein CD98.

Cell Mol Life Sci 2012 Sep 30;69(18):3015-26. Epub 2012 Mar 30.

M2iSH, UMR 1071 Inserm, University of Auvergne, Clermont-Ferrand, 63000, France,

The transmembrane glycoprotein CD98 is a potential regulator of multiple functions, including integrin signaling and amino acid transport. Abnormal expression or function of CD98 and disruption of the interactions between CD98 and its binding partners result in defects in cell homeostasis and immune responses. Indeed, expression of CD98 has been correlated with diseases such as inflammation and tumor metastasis. Modulation of CD98 expression and/or function therefore represents a promising therapeutic strategy for the treatment and prevention of such pathologies. Herein, we review the role of CD98 with focus on its functional importance in homeostasis and immune responses, which could help to better understand the pathogenesis of CD98-associated diseases.
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http://dx.doi.org/10.1007/s00018-012-0963-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402973PMC
September 2012

The PepT1-NOD2 signaling pathway aggravates induced colitis in mice.

Gastroenterology 2011 Oct 14;141(4):1334-45. Epub 2011 Jul 14.

Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322, USA.

Background & Aims: The human di/tripeptide transporter human intestinal H-coupled oligonucleotide transporter (hPepT1) is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1.

Methods: Transgenic mice were generated in which hPepT1 expression was regulated by the β-actin or villin promoters; colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) and the inflammatory responses were assessed. The effects of NOD2 deletion in the hPepT1 transgenic mice also was studied to determine the involvement of the PepT1-NOD2 signaling pathway.

Results: TNBS and DSS induced more severe levels of inflammation in β-actin-hPepT1 transgenic mice than wild-type littermates. Intestinal epithelial cell-specific hPepT1 overexpression in villin-hPepT1 transgenic mice increased the severity of inflammation induced by DSS, but not TNBS. Bone marrow transplantation studies showed that hPepT1 expression in intestinal epithelial cells and immune cells has an important role in the proinflammatory response. Antibiotics abolished the effect of hPepT1 overexpression on the inflammatory response in DSS-induced colitis in β-actin-hPepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2-/-, β-actin-hPepT1 transgenic/Nod2-/-, and villin-hPepT1 transgenic/Nod2-/- littermates had similar levels of susceptibility to DSS-induced colitis, indicating that hPepT1 overexpression increased intestinal inflammation in a NOD2-dependent manner.

Conclusions: The PepT1-NOD2 signaling pathway is involved in aggravation of DSS-induced colitis in mice.
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http://dx.doi.org/10.1053/j.gastro.2011.06.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186842PMC
October 2011

Microbiota modulate host gene expression via microRNAs.

PLoS One 2011 Apr 29;6(4):e19293. Epub 2011 Apr 29.

Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3'-untranslated regions (3'-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3'-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019293PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084815PMC
April 2011

CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice.

J Clin Invest 2011 May 1;121(5):1733-47. Epub 2011 Apr 1.

Department of Medicine, Emory University, Atlanta, Georgia, USA.

Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.
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http://dx.doi.org/10.1172/JCI44631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083801PMC
May 2011

MicroRNA-92b regulates expression of the oligopeptide transporter PepT1 in intestinal epithelial cells.

Am J Physiol Gastrointest Liver Physiol 2011 Jan 28;300(1):G52-9. Epub 2010 Oct 28.

Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta GA 30322, USA.

MicroRNAs (miRNAs), which are noncoding RNAs that posttranscriptionally inhibit expression of target genes, have recently emerged as important regulators of many cellular functions such as cell differentiation. The epithelial di/tripeptide membrane transporter PepT1 is expressed in highly differentiated cells (the villous tip) but not in undifferentiated cells (the crypt) of the small intestine. Here, we investigated the regulation of PepT1 expression by miRNAs and its functional consequences. We observed a reverse correlation between the expression levels of PepT1 and mature miRNA-92b (miR-92b) during the differentiation of intestinal epithelial Caco2-BBE cells, suggesting a miR-92b-mediated regulation of PepT1 expression. We demonstrate that miR-92b suppressed PepT1 expression at both mRNA and protein levels, with subsequent reduced PepT1 transport activity, in Caco2-BBE cells by directly targeting the PepT1 3'-untranslated region. In addition, miR-92b suppresses bacterial peptide-induced proinflammatory responses in intestinal epithelial cells by inhibiting PepT1 expression. Altogether, our study provides for the first time evidence for the regulation of PepT1 expression at a posttranscriptional level by miRNAs in intestinal epithelial cells during pathophysiological states.
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http://dx.doi.org/10.1152/ajpgi.00394.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025505PMC
January 2011

MicroRNAs determine human intestinal epithelial cell fate.

Differentiation 2010 Sep-Oct;80(2-3):147-54. Epub 2010 Jul 17.

Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that post-transcriptionally regulate gene expression. Evidence has shown that miRNAs play important roles in various cellular processes, including proliferation, differentiation and survival. The intestinal epithelium is regenerated throughout life, and enterocytes undergo differentiation during migration along the crypt/villus axis. Our study aimed at establishing the expression profiles of miRNAs during intestinal epithelial cell (IEC) differentiation and determining a miRNA "signature" that distinguishes between small and large IECs. MiRNA arrays were employed to profile miRNA expression in two IEC models: the enterocyte-like Caco2-BBE and the colonocyte-like HT29-Cl.19A cell lines. Microarray data showed that in both cell lineages, the differentiated stage exhibited a different miRNA expression profile from undifferentiated stage. Interestingly, Caco2-BBE cells were distinguished from HT29-Cl.19A cells by their unique miRNA expression profile. Notably, HT29-Cl.19A cells exhibited down-regulation of miR-1269 and up-regulation of miR-99b and miR-125a-5p compared with Caco2-BBE cells. Most importantly, transfection of Caco2-BBE cells with mature miR-99b, mature miR-125a-5p and antisense of mature miR-1269 decreased growth rate and trans-epithelial resistance of the cells, indicating their shift toward HT29-Cl.19A cell phenotype. In conclusion, our study shows that miRNAs might play a role in determining the unique physiological characteristics of IECs.
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http://dx.doi.org/10.1016/j.diff.2010.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943016PMC
January 2011

PepT1 mediates transport of the proinflammatory bacterial tripeptide L-Ala-{gamma}-D-Glu-meso-DAP in intestinal epithelial cells.

Am J Physiol Gastrointest Liver Physiol 2010 Sep 17;299(3):G687-96. Epub 2010 Jun 17.

Department of Medicine, Emory University, Atlanta, GA 30322, USA.

PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-gamma-d-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-kappaB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.
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http://dx.doi.org/10.1152/ajpgi.00527.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950691PMC
September 2010