Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species
2013; Springer Nature; Volume: 32; Issue: 23 Linguagem: Inglês
10.1038/emboj.2013.224
ISSN1460-2075
AutoresRheinallt M. Jones, Liping Luo, Courtney Ardita, Arena N. Richardson, Young Man Kwon, Jeffrey W. Mercante, Ashfaqul Alam, Cymone Gates, Huixia Wu, Phillip A. Swanson, J. David Lambeth, Patricia W. Denning, Andrew S. Neish,
Tópico(s)Infections and bacterial resistance
ResumoArticle18 October 2013free access Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species Rheinallt M Jones Corresponding Author Rheinallt M Jones Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Liping Luo Liping Luo Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Courtney S Ardita Courtney S Ardita Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Arena N Richardson Arena N Richardson Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Young Man Kwon Young Man Kwon Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Jeffrey W Mercante Jeffrey W Mercante Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Ashfaqul Alam Ashfaqul Alam Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Cymone L Gates Cymone L Gates Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Huixia Wu Huixia Wu Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Phillip A Swanson Phillip A Swanson Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author J David Lambeth J David Lambeth Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Patricia W Denning Patricia W Denning Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Andrew S Neish Andrew S Neish Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Rheinallt M Jones Corresponding Author Rheinallt M Jones Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Liping Luo Liping Luo Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Courtney S Ardita Courtney S Ardita Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Arena N Richardson Arena N Richardson Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Young Man Kwon Young Man Kwon Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Jeffrey W Mercante Jeffrey W Mercante Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Ashfaqul Alam Ashfaqul Alam Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Cymone L Gates Cymone L Gates Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Huixia Wu Huixia Wu Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Phillip A Swanson Phillip A Swanson Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author J David Lambeth J David Lambeth Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Patricia W Denning Patricia W Denning Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Andrew S Neish Andrew S Neish Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Author Information Rheinallt M Jones 1, Liping Luo1, Courtney S Ardita1, Arena N Richardson2, Young Man Kwon1, Jeffrey W Mercante1, Ashfaqul Alam1, Cymone L Gates1, Huixia Wu1, Phillip A Swanson1, J David Lambeth1, Patricia W Denning2 and Andrew S Neish1 1Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 2Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA *Corresponding author. Department of Pathology, Emory University School of Medicine, 135C Whitehead Building, 615 Michael Street, Atlanta, GA 30322, USA. Tel.:+1 404 712 2816; Fax:+1 404 727 8538; E-mail: [email protected] The EMBO Journal (2013)32:3017-3028https://doi.org/10.1038/emboj.2013.224 There is a Have you seen? (November 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic–eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential. Introduction It is becoming increasingly evident that an optimal metazoan gut microbiota serves beneficial functions for the host that includes energy extraction, stimulation of immune development, and competitive exclusion of pathogenic microorganisms (Neish, 2009). In addition, experiments in germ-free animals have demonstrated a physiological role for the microbiota in regulation of epithelial homeostasis, as well as host immunity and metabolism (Hooper et al, 2012; Nicholson et al, 2012). Consistently, abnormalities (‘dysbiosis’) in the intestinal microbiota may be sufficient to provoke intestinal inflammation as seen in inflammatory bowel disease (IBD), and quantitative and/or qualitative abnormalities of the microbiota have been associated with other allergic, metabolic, systemic immune, and infectious disorders (Sartor, 2008). Indeed, therapeutic administration of exogeneous viable bacteria, termed probiotics, has also been reported to dampen inflammation, improve barrier function, and promote intestinal reparative responses in response to inflammatory and developmental disorders of the intestinal tract (Park and Floch, 2007). Eukaryotes have evolved dedicated perception and signalling systems for monitoring potential pathogens, and necessarily, their own symbiotic microbiota. These pathways allow for recognition of microbes via pattern recognition receptors (PRRs) and activation of signal transduction cascades such as the MAPK and NF-κB pathways. While generally considered pro-inflammatory, basal low level PRR signalling has been implicated in normal homeostatic maintenance in the gut (Rakoff-Nahoum et al, 2004). Additionally, our research group has reported that the microbiota in the intestinal lumen modulates host redox biochemistry to limit pro-inflammatory signalling and activate reparative responses (Kumar et al, 2007, 2009; Wentworth et al, 2010; Swanson et al, 2011; Wentworth et al, 2011). Thus, there is increasing evidence that the gut microbiota is perceived by the host, and influences a wide range of physiological processes. However, little is known of how the microbiota mechanistically influences gut biology. Moreover, delineation of the molecular mechanisms that underlies host and microbe interactions would be instrumental in understanding this important symbiotic relationship, its role in heath and disease, and therapeutic the exploitation of beneficial bacteria. The gut epithelium of both mammals and invertebrates is a highly adapted tissue that has evolved for both digestive and absorptive functions as well as providing a vital mechanical and immunological barrier against gut luminal contents. The epithelia of both are a two-dimensional, single cell sheet of enterocytes interspersed with pluripotent stem cell niches that serve as sources of cellular renewal (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; van der Flier and Clevers, 2009). In mammals, the stem cell compartment is at the base of the three-dimensional epithelial invaginations forming the crypt niche. The daughter progeny of mammalian stem cells further proliferate and migrate luminally defining the adjacent transient amplifying compartment prior to terminal differentiation into absorptive, mucus secreting, and neuroendocrine epithelial cells. It is estimated that the dynamic renewal of murine epithelia occurs within 4–5 days (van der Flier and Clevers, 2009). In Drosophila, the epithelial cells of the larval fly gut are initially supplied by dispersed single intestinal stem cells (ISCs) that proliferate into a multicellular niche over the course of larval life. The stem cells and progeny represent adult midgut precursors (AMPs) that serve as the primordia of the adult intestinal epithelium that forms during pupal metamorphosis. The adult Drosophila midgut comprises an enterocyte monolayer interspersed with hormone-producing enteroendocrine cells. The adult midgut enterocytes are continuously replenished by ISCs that adjoin the intestinal basement membrane (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Together, these systems form an attractive and genetically tractable target system for the study of gut stem cell dynamics and the role of environmental influences in this process (Mathur et al, 2010). Reactive oxygen species (ROS) are short-lived molecules derived from incomplete reduction of oxygen metabolites that at high levels have a microbicidal function in professional phagocytes. However, a lower ‘physiological’ level of ROS is increasingly recognized in the mediation of intracellular signalling events in a wide variety of cell types (Hernandez-Garcia et al, 2010). Importantly, ROS are induced in response to bacteria in virtually all forms of multicellular life ranging from plants and social amoebae to humans, thus representing a primordial form of microbial perception and control (Ha et al, 2005b; Kotchoni et al, 2006). Significantly, ROS are also increasingly recognized as mediators of cellular proliferation and differentiation in disparate biological systems such as plant root hair development (Tsukagoshi et al, 2010), and Drosophila haematopoiesis (Owusu-Ansah and Banerjee, 2009). Recently, we reported that some species of human gut bacteria can induce rapid, physiological generation of ROS that has potent regulatory effects on host immune function, intracellular signalling, and cytoskeletal dynamics (Kumar et al, 2007, 2009; Swanson et al, 2011; Wentworth et al, 2011). Cellular ROS are often produced via the catalytic action of NADPH oxidases. The archetypal member of this family, Nox2, was first identified in neutrophils, and was shown to play an important role in phagocyte microbicidal ROS generation in response to bacteria (oxidant burst). Subsequently, paralogues of Nox2 were identified in non-phagocytic tissues, including Nox1 and Duox2, which are strongly expressed in colonic intestinal epithelia of both flies and mice (Lambeth, 2004; Bedard and Krause, 2007; Ogier-Denis et al, 2008). Herein, we show that the commensal Lactobacillus spp. are potent inducers of endogenous ROS generation, and of ROS-dependent cellular proliferation within intestines of two metazoan models, namely the fruitfly Drosophila melanogaster and the mouse. In addition, we show that Lactobacillus-induced ROS generation and cell proliferation is dependent on a functional Nox1 enzyme in intestinal epithelial cells. ROS production was absent in germ-free animals and was associated with suppressed epithelial growth. Together, these data indicate that bacteria-induced activation of an ROS generating enzyme in enterocytes influences cellular proliferation. Results Colonization of the Drosophila midgut by Lactobacillus plantarum induces cellular ROS generation We previously demonstrated that contact with the human commensal (and commonly used probiotic) Lactobacillus rhamnosus GG with cultured epithelial cells induces the endogeneous generation of cellular ROS (Wentworth et al, 2011). Here, we assessed the ability of diverse strains of bacteria to elicit this response in viable Drosophila gut epithelia. To undertake these analyses, and as an improvement on previous techniques that used ROS detection dyes that were prone to auto-oxidation or photobleaching, we employed a new class of hydrocyanine dyes (hereafter referred to as hydro-Cy3) that exhibit far greater specificity and stability, enabling detection of ROS generation in tissue compartments in vivo (Kundu et al, 2009). Whereas the mammalian gut microbiota includes several hundred distinct species of bacteria, the Drosophila gut microbiota is markedly less complex (Wong et al, 2011). We isolated and cultured six distinct bacteria (three Gram negative and three Gram positive) from the luminal content of adult Drosophila (Supplementary Table S1). Pure cultures of the isolated bacteria were mixed with sterile media, and germ-free Drosophila embryos introduced into the vial. Thus, emerged first-instar larvae gnotobiotically ingested sterile media supplemented with pure cultures of the respective bacteria. Culture-based quantification revealed that each first-instar larvae typically ingested a total of about 103–104 CFUs (see Materials and methods). Fluorescent imaging revealed that gnotobiotic ingestion of L. plantarum potently induced the rapid generation of ROS in midgut enterocytes within 30 min, as detected by the oxidation of the hydro-Cy3 dye, and activation of the ROS-responsive gstD1-gfp reporter element (Sykiotis and Bohmann, 2008) (Figure 1). Ingestion of pure cultures of other members of the microbiota, particularly Gram-negative bacteria, elicited nearly undetectable levels of cellular ROS generation (Figure 1), and no ROS generation was detected when emerged germ-free larvae consumed sterilized food (Figure 1), although minor fragments of auto-fluorescent particles were visible. In addition to first-instar larvae, L. plantarum ingestion by germ-free third-instar larvae also induced the generation of cellular ROS, and ROS-responsive genetic elements within 30 min of feeding (Figures 2A and B). Importantly, ingestion of B. cereus isolated from the Drosophila gut, or the Drosophila pathogen Erwinia carotovora, did not induce ROS generation at up to 4 h post ingestion (Figures 2C and D). These results were recapitulated in adult Drosophila where ingestion of L. plantarum but not E. carotovora (detected in the figure by GFP activity) induced ROS generation after 4 h (Figure 2E). Figure 1.Ingestion of Lactobacillus plantarum by first-instar Drosophila larvae induces cellular ROS generation. Detection of ROS generation following the ingestion of indicated bacteria by germ-free newly emerged gstD1-gfp first-instar larvae for 30 min with the indicated Gram-positive or Gram-negative bacteria isolated from Drosophila midguts (Supplementary Table S1). Germ-free gstD1-gfp embryos were placed in a vial containing sterilized Drosophila growth media inoculated with 1 × 108 cfu of the indicated bacteria. ROS were detected by oxidation of the hydrocyanin ROS-sensitive dye (upper panels), that is present in the larval food. Larvae used also harbour an ROS inducible gstD1-gfp reporter gene (green lower panels). (A′) Cartoon of first-instar midgut. Enterocyte (EC), intestinal stem cell (ISC), luminal contents (LCs). (A″) Tissue orientation control by staining of first-instar midgut stained for DNA. (A′′′) Exploded view of the interface between the ECs and the LC in larvae fed L. plantarum. Numbers of bacteria ingested by larva were quantified and results are presented in Materials and methods. Download figure Download PowerPoint Figure 2.Ingestion of Lactobacillus plantarum by Drosophila induces cellular ROS generation in the midgut. (A) ROS generation following the ingestion of L. plantarum by germ-free third-instar larvae over 1 h. ROS were detected by oxidation of the hydrocyanin ROS-sensitive dye also included in the media. (B) Microscopic analysis at × 4 magnification of larval midgut dissected from (A). (C) ROS generation in the third-instar midgut following the ingestion of L. plantarum, Bacillus cereus or Erwinia carotovora for up to 4 h. (D) Densitometric analysis of larval midguts described in (C). Results are an average for 5 dissected midguts from each assay. (E) ROS generation in the gstD1-gfp adult Drosophila midgut following the ingestion of L. plantarum, or Erwinia carotovora-GFP for up to 4 h. Note Hydro-Cy3 fluorescence and expression of GFP in enterocytes following the L. plantarum ingestion. Also note GFP fluorescence detected in the midgut following the ingestion of E. carotovora-GFP. (F) ROS generation following the ingestion of L. plantarum in adult Drosophila midguts of the indicated genotypes for 1 h. (G) Densitometric analysis of larval midguts described in (F). Results are an average for five dissected midguts from each assay. All histograms report densitometric analysis (arbitrary units) of hydro-Cy3 oxidation, using the ImageJ software. Ten identically sized areas within an image were measured. n=50. **P<0.01, ***P<0.0001. Download figure Download PowerPoint As stated in the introduction, bacterial-induced ROS generation occurs in phagocytes via the enzymatic activity of the Nox2 NADPH oxidase. Indeed, it is well established that NADPH oxidases function in the anti-microbial response in mammalian phagocytes, and in epithelial cells of the Drosophila gut (Quinn and Gauss, 2004; Ha et al, 2005a). However, the function of ROS generated in response to colonization with commensal bacteria is not understood. We thus examined the extent to which the dNox or dDuox (the sole NADPH oxidases in Drosophila) function in L. plantarum-induced ROS generation within enterocytes. We expressed RNAi constructs against dNox and dDuox under the enterocyte-specific myoIA-GAL4 driver fly (Morgan et al, 1994). We confirm that the stock used significantly reduced the expression of dnox and dduox respectively compared to w1118 or gal4IR control flies (Supplementary Figure S1). Our analyses show that depletion in the levels of dNox but not dDuox markedly dampened induced ROS generation following the L. plantarum ingestion (Figure 2F and G). We conclude that initial ingestion of L. plantarum induces the rapid generation of ROS by a dNox-dependent mechanism. Lactobacillus induces ROS-dependent cellular proliferation in the Drosophila intestine The Drosophila larvae midgut enterocytes are large polyploid cells that form the interface with the gut luminal contents. The stem cells are interspersed between enterocytes, and the stem cells expand over the larval life to form proliferative stem cell niches. These niches harbour adult midgut progenitors (AMPs), from which the adult intestinal epithelium is derived during pupal metamorphosis (Mathur et al, 2010), whereas the adult midgut enterocytes are continuously replenished by pluripotent ISCs (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). We assessed the number of proliferative niche cells in the midguts of conventionally raised and germ-free Drosophila larvae. Germ-free third-instar larvae had significantly fewer numbers of EdU-positive cells in the midgut stem cell niches, compared to conventionally raised larvae (Figure 3A and B), as well as fewer GFP-positive cells in germ-free esg-GAL4 UAS-gfp, a genetic marker of midgut niche stem cells and their progeny (Micchelli and Perrimon, 2006), compared to isogenic conventionally raised flies (Supplementary Figure S2). Strikingly, colonization of germ-free third instar Drosophila larvae with L. plantarum for 2 h significantly increased the number of proliferating niche cells in the midgut (Figure 3A and B). As stated in Introduction, ROS have been shown to function in cell proliferation within diverse tissues (Owusu-Ansah and Banerjee, 2009; Tsukagoshi et al, 2010). We corroborate these reports by showing that supplementing the N-acetylcysteine (NAC) (a glutathione precursor and direct antioxidant) into the fly media for 12 h before bacterial ingestion significantly decreased L. plantarum-induced cell proliferation (Figure 3A and B). We also detected similar responses in adult Drosophila. Germ-free adult Drosophila had significantly fewer EdU-positive ISCs and progeny than conventionally raised flies (Figure 3C and D). Similar to the response in larvae, L. plantarum ingestion for 12 h resulted in a significant increase in the number of EdU-positive ISCs and their progeny in the adult midgut while E. carotovora ingestion did not (Figure 3C and D). Figure 3.Ingestion of Lactobacillus plantarum induces ROS-dependent cellular proliferation in the Drosophila intestine. (A) EdU-positive cells in the midgut of w1118 germ-free third-instar larvae, or germ-free larvae fed with 1 × 108 cfu L. plantarum for 4 h. Where indicated, the media also contained 1 mM N-acetylcysteine (NAC). (A′) Cartoon of en face third-instar midgut. Enterocyte (EC) in grey, adult midgut progenitors (AMPs) in red. Note some large enterocytes are EdU positive due to endonuclear DNA replication in maturing larval. (B) Number of EdU-positive cells under conditions described in (A) n=20, ***P<0.001. (C) Detection of EdU-positive cells in the midgut of adult conventionally raised, germ-free, or germ-free adult Drosophila following the ingestion of L. plantarum or Erwinia carotovora for 12 h. (D) Number of EdU-positive cells under conditions described in (C) n=20, ***P<0.001. (E) Detection of EdU-positive cells in the midgut of adult where the levels of Nox or Duox are diminished under the enterocyte-specific myoIA-GAL4 driver. Full genotypes myoIA-GAL4;UAS-dnox-RNAi and myoIA-GAL4;UAS-dduox-RNAi, and myoIA-GAL4;UAS-gal4-RNAi. (F) Number of EdU-positive cells in (A). n=10, ***P<0.001. Download figure Download PowerPoint As shown in Figure 2, the NADPH oxidase, dNox is required for L. plantarum-induced ROS generation in enterocytes. Here, we show that depletion of dNox, but not dDuox levels also significantly reduced the numbers of proliferating cells in conventionally raised larval midgut (Supplementary Figure S3), and in the 5-day-old adult midgut (Figure 3E and F). Consequentially, examination of the DNA counter stain in Figure 3E indicated changes in midgut histological architecture, and we detected significantly shorter lifespan in Drosophila expressing enterocyte-specific dnoxIR compared to control flies (Supplementary Figure S4). Examination of the adult midgut at 10, 20, and 30 days following the eclosure reveals only few detectable EdU-positive cells and marked observable changes in midgut histological architecture (Supplementary Figure S5). Similarly, using a GFP marker for enterocyte cells (Jiang et al, 2009), markedly altered enterocyte histological architecture was detected in dnoxIR compared to control flies (Supplementary Figure S6). Intriguingly, we also examined the influence of diminishing dNox levels in midgut ISCs using the ISC-specific escargot-GAL4 driver fly. We detected less influence on the numbers of EdU-positive cells in the midgut, although subtle changes in cell arrangement were seen (Supplementary Figure S7), suggesting that dNox-mediated pro-proliferative ROS production occurs predominantly in enterocytes. Together, these observations demonstrate that Lactobacillus-induced ROS generation promotes cell proliferation in the Drosophila intestine, by a mechanism dependent on dNox activity in enterocytes. Contact of lactobacilli with cultured mammalian cells induces ROS generation To determine whether the specific influence of ROS-inducing lactobacilli is conserved in mammalian systems, we assessed the ability of diverse strains of mammalian commensal bacteria to elicit this response in cultured Caco-2 cells. Cells were grown to confluency and pre-loaded with hydro-Cy3 for 30 min, before contact with 1 × 108 cfu of the candidate bacteria. Consistent with previous experiments, lactobacilli rapidly induced the generation of cellular ROS within minutes of contact (Figure 4A and B). Other Lactobacillus species tested, including L. acidophilus or L. casei, as well as the Gram-positive intestinal bacteria Bifidobacteria bifidum and Lactococcus lactis also induced the generation of cellular ROS, albeit to a lower extent than L. rhamnosus (Figure 4A and B). Importantly, L. rhamnosus-induced ROS generation was abolished in the presence of the superoxide dismutase (SOD) mimetic TEMPOL (Figure 4B). In contrast, Bacteroides thetaiotaomicron, a well-known enteric Gram-negative anaerobic microbe, as well as Gram-negative Escherichia coli or the mammalian pathogen Salmonella typhimurium could not induce cellular ROS generation. In addition, contact of cultured cells with the luminal contents isolated from conventionally raised mice also induced the generation of cellular ROS, whereas similar fecal preparations from germ-free mice could not (Figure 4C). These data confirm the conserved ability of members of the lactobacilli to induce the epithelial ROS generation across distant metazoan phyla. Figure 4.Contact of cultured cells with lactobacilli induces the generation of cellular reactive oxygen species (ROS). (A) Bacterial-induced ROS in cells contacted by the indicated bacteria for up to 40 min. Caco-2 cells seeded in a 96-well format were preloaded with 100 μM hydro-Cy3, then contacted with 3 × 108/100 μl viable bacteria for the indicated times. Cells were then washed three times with PBS before fluoromometric analysis at 575 nm. (B) Bacterial-induced ROS in cells treated as described in (A) detected by confocal microscopy at 585 nm. In some experiments, 5 mM TEMPOL (a membrane-permeable oxygen radical scavenger) was also included. (C) ROS generation in response to contact of Caco-2 cells as described in (A) by the cecal contents of a conventionally raised BL6, or of germ-free BL6 mice. ROS was detected by fluoromometric analysis at 575 nm. Download figure Download PowerPoint Ingestion of Lactobacillus induces Nox1-dependent generation of cellular ROS in murine enterocytes To examine the extent to which bacterially stimulated and Nox1-mediated ROS generation following the ingestion of lactobacilli can be detected in vivo in the murine model, we assessed the effects of feeding L. rhamnosus GG in 6-week-old and 2-day-old wild-type mice, and in intestinal epithelial cell-specific Nox1-deficient (B6.Nox1ΔIEC) animals. We recently described the generation of B6.Nox1ΔIEC mice and the intestinal epithelial expression pattern of Nox1 (Leoni et al, 2013). In addition, previous publications have reported strong expression of nox1 in colonic tissue (Suh et al, 1999; Geiszt et al, 2003). We confirm these findings, and also detect Nox1 activity in the distal small intestine (Supplementary Figure S8). To assess ROS generation, wild-type and B6.Nox1ΔIEC littermates were administered hydro-Cy3 by IP injection 30 min before oral gavage feeding with preparations of 1 × 109 cfu L. rhamnosus or E. coli. Preliminary experiments indicated the bacterial gavage contents reached the colon within 1 h. Examination of colonic tissues at 1 h post feeding revealed that L. rhamnosus, but not E. coli ingestion resulted in the generation of ROS within colonic and sma
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