Human Organ Culture: Updating the Approach to Bridge the Gap from In Vitro to In Vivo in Inflammation, Cancer, and Stem Cell Biology

Human studies, critical for developing new diagnostics and therapeutics, are limited by ethical and logistical issues, and preclinical animal studies are often poor predictors of human responses. Standard human cell cultures can address some of these concerns but the absence of the normal tissue microenvironment can alter cellular responses. Three-dimensional cultures that position cells on synthetic matrices, or organoid or organ-on-a-chip cultures, in which different cell spontaneously organize contacts with other cells and natural matrix only partly overcome this limitation. Here, we review how human organ cultures (HOCs) can more faithfully preserve in vivo tissue architecture and can better represent disease-associated changes. We will specifically describe how HOCs can be combined with both traditional and more modern morphological techniques to reveal how anatomic location can alter cellular responses at a molecular level and permit comparisons among different cells and different cell types within the same tissue. Examples are provided involving use of HOCs to study inflammation, cancer, and stem cell biology.The authors would like to express their gratitude to The National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (RA-L, JB)

Caractérisation des propriétés d'adhésion de Streptococcus thermophilus LMD-9 aux cellules épithéliales intestinales: 1. Rôle des protéines de surface dans la résistance aux sels biliaires et dans l’adhésion2. Impact de l’adhésion sur l’expression des gènes eucaryotes et bactériens

Lactic acid bacteria are of great economic interest because of their use in the food industry. Among them, Streptococcus thermophilus (ST) is of major interest since it is the most used after Lactococcus lactis, for the manufacture of fermented dairy products and cheese. In addition, this bacterium is the only streptococcus to benefit from GRAS status (Generally Recognized As Safe). Beside its interest in the dairy industry, ST has beneficial effects on human intestinal health. Although these effects are widely documented, the probiotic status of ST remains to be consolidated. Therefore, studies are currently being conducted in order to select strains of ST with a high probiotic potential. Among criteria that are important to select ST strains include their ability to survive stress the drastic conditions of the digestive tract (DT) and their ability to adhere to intestinal cells. In this context, the aim of this thesis was to investigate firstly the in vitro adhesion capacity of the ST LMD-9 strain to different human intestinal cell lines and to evaluate the survival of this strain to bile salt stress. In order to highlight the potential role of some surface proteins in these two processes, three mutants derived from this strain and inactivated in the genes prtS (parietal protease), srtA (sortase A) and mucBP (Mucin Binding-protein) were also included in this study. Secondly, the impact of LMD-9 adhesion was analyzed, on one hand on the expression of some mucin-encoding genes in eukaryotic cells, and on the other hand on the expression of genes that would be specifically induced during adhesion process using the R-IVET (Recombinase-based In Vivo Expression Technology) approach. The results obtained demonstrated the ability of LMD-9 to survive up to the concentration of 3 mM of bile salts and that the PrtS, SrtA and MucBP surface proteins would be involved in the resistance to this stress. Our results also showed that the LMD-9 strain was capable of adhering to three cell lines used suggesting that this strain could interact with different mucins that may encounter in the DT. Moreover, the involvement of some surface proteins in the adhesion of LMD-9 has been found to be dependent on the surface characteristics of these cell lines, whether they are enterocytic (Caco-2) or mucus-secreting cells (HT29-MTX and HT29-CL.16E). Regarding the impact of LMD-9 adhesion on MUC2 and MUC5AC gene expression, no effect has been observed on the transcript level under our experimental conditions. Furthermore, for the first time, our results allowed us to identify genes specifically induced in the LMD-9 strain during adhesion process to epithelial cells. We have thus shown that the LMD-9 adhesion does not depend solely on surface proteins, but other functions and metabolic pathways are also involved. This thesis work contributes thus to new knowledge related to (i) the choice of the cellular model in in vitro bacterial adhesion studies, (ii) the ability of LMD-9 to survive bile salt stress by involving some surface proteins and (iii) understanding the molecular mechanisms of LMD-9 adhesion to epithelial cellsLes bactéries lactiques présentent un grand intérêt économique de par leur large utilisation dans l’industrie agroalimentaire. Parmi elles, Streptococcus thermophilus (ST) est d’une importance majeure puisqu’elle est la plus utilisée après Lactococcus lactis, pour la fabrication de produits laitiers fermentés et de fromages. De plus, cette bactérie est le seul streptocoque à bénéficier du statut GRAS (Generally Recognized As Safe). Outre son intérêt en industrie laitière, ST présente des effets bénéfiques sur la santé intestinale de l’Homme. Bien que ces effets soient largement documentés, le statut probiotique de ST reste encore à conforter. C’est pourquoi des études sont actuellement menées afin de sélectionner des souches de ST à fort potentiel probiotique. Parmi les critères importants figurent leur capacité à survivre aux conditions drastiques du tube digestif (TD) et leur capacité à adhérer aux cellules intestinales. Dans cette optique, les objectifs de cette thèse étaient d’étudier, dans un premier temps, la capacité d’adhésion in vitro de la souche ST LMD-9 à différentes lignées cellulaires intestinales d’origine humaine et d’évaluer la survie de cette souche au stress biliaire. Afin de mettre en évidence un rôle potentiel de certaines protéines de surface dans ces deux processus, trois mutants issus de cette souche et inactivés dans les gènes prtS (protéase pariétale), srtA (sortase A) et mucBP (protéine de liaison aux mucines), ont été inclus dans cette étude. Dans un second temps, l’impact de l’adhésion de LMD-9 a été analysé, d’une part sur l’expression de gènes codant certaines mucines dans les cellules eucaryotes, et d’autre part sur l’expression de gènes qui seraient spécifiquement induits durant le processus d’adhésion, ceci en utilisant la technologie R-IVET (Recombinase-based In Vivo Expression Technology). Les résultats obtenus ont permis de montrer que la souche LMD-9 était capable de survivre jusqu’à une concentration de 3 mM en sels biliaires et que les protéines de surface PrtS, SrtA et MucBP seraient impliquées dans la résistance à ce stress. Nos résultats ont également montré que LMD-9 adhérait aux trois différentes lignées cellulaires, suggérant ainsi que la souche pourrait interagir avec les différentes mucines qu’elle peut rencontrer dans le TD. De plus, l’implication de certaines protéines de surface dans l’adhésion de LMD-9 s’est avérée dépendante des caractéristiques de ces lignées, qu’il s’agisse de cellules entérocytaires (Caco-2) ou productrices de mucus (HT29-MTX et HT29-CL.16E). Concernant l’impact de l’adhésion de la souche LMD-9 sur l’expression des gènes MUC2 et MUC5AC, aucun effet sur le taux de transcrits n’a été observé dans nos conditions expérimentales. Par ailleurs, nos résultats ont permis, pour la première fois, d’identifier les gènes spécifiquement induits dans la souche LMD-9 durant l’adhésion aux cellules épithéliales. Nous avons ainsi montré que l’adhésion de la souche LMD-9 ne dépend pas uniquement des protéines de surface, mais d’autres fonctions et voies métaboliques seraient également impliquées. Ce travail de thèse contribue ainsi à apporter de nouvelles connaissances liées (i) au choix du modèle cellulaire dans les études d’adhésion bactérienne in vitro, (ii) à l’aptitude de la souche LMD-9 à survivre au stress biliaire en faisant intervenir certaines protéines de surface et (iii) à la compréhension des mécanismes moléculaires de l’adhésion de LMD-9 aux cellules épithéliales intestinale

Characterization of adhesion properties of Streptococcus thermophilus LMD-9 to intestinal epithelial cells : 1. Role of surface proteins in bile salt resistance and adhesion, 2. Impact of adhesion on the expression of eukaryotic and bacterial genes

Les bactéries lactiques présentent un grand intérêt économique de par leur large utilisation dans l’industrie agroalimentaire. Parmi elles, Streptococcus thermophilus (ST) est d’une importance majeure puisqu’elle est la plus utilisée après Lactococcus lactis, pour la fabrication de produits laitiers fermentés et de fromages. De plus, cette bactérie est le seul streptocoque à bénéficier du statut GRAS (Generally Recognized As Safe). Outre son intérêt en industrie laitière, ST présente des effets bénéfiques sur la santé intestinale de l’Homme. Bien que ces effets soient largement documentés, le statut probiotique de ST reste encore à conforter. C’est pourquoi des études sont actuellement menées afin de sélectionner des souches de ST à fort potentiel probiotique. Parmi les critères importants figurent leur capacité à survivre aux conditions drastiques du tube digestif (TD) et leur capacité à adhérer aux cellules intestinales. Dans cette optique, les objectifs de cette thèse étaient d’étudier, dans un premier temps, la capacité d’adhésion in vitro de la souche ST LMD-9 à différentes lignées cellulaires intestinales d’origine humaine et d’évaluer la survie de cette souche au stress biliaire. Afin de mettre en évidence un rôle potentiel de certaines protéines de surface dans ces deux processus, trois mutants issus de cette souche et inactivés dans les gènes prtS (protéase pariétale), srtA (sortase A) et mucBP (protéine de liaison aux mucines), ont été inclus dans cette étude. Dans un second temps, l’impact de l’adhésion de LMD-9 a été analysé, d’une part sur l’expression de gènes codant certaines mucines dans les cellules eucaryotes, et d’autre part sur l’expression de gènes qui seraient spécifiquement induits durant le processus d’adhésion, ceci en utilisant la technologie R-IVET (Recombinase-based In Vivo Expression Technology). Les résultats obtenus ont permis de montrer que la souche LMD-9 était capable de survivre jusqu’à une concentration de 3 mM en sels biliaires et que les protéines de surface PrtS, SrtA et MucBP seraient impliquées dans la résistance à ce stress. Nos résultats ont également montré que LMD-9 adhérait aux trois différentes lignées cellulaires, suggérant ainsi que la souche pourrait interagir avec les différentes mucines qu’elle peut rencontrer dans le TD. De plus, l’implication de certaines protéines de surface dans l’adhésion de LMD-9 s’est avérée dépendante des caractéristiques de ces lignées, qu’il s’agisse de cellules entérocytaires (Caco-2) ou productrices de mucus (HT29-MTX et HT29-CL.16E). Concernant l’impact de l’adhésion de la souche LMD-9 sur l’expression des gènes MUC2 et MUC5AC, aucun effet sur le taux de transcrits n’a été observé dans nos conditions expérimentales. Par ailleurs, nos résultats ont permis, pour la première fois, d’identifier les gènes spécifiquement induits dans la souche LMD-9 durant l’adhésion aux cellules épithéliales. Nous avons ainsi montré que l’adhésion de la souche LMD-9 ne dépend pas uniquement des protéines de surface, mais d’autres fonctions et voies métaboliques seraient également impliquées. Ce travail de thèse contribue ainsi à apporter de nouvelles connaissances liées (i) au choix du modèle cellulaire dans les études d’adhésion bactérienne in vitro, (ii) à l’aptitude de la souche LMD-9 à survivre au stress biliaire en faisant intervenir certaines protéines de surface et (iii) à la compréhension des mécanismes moléculaires de l’adhésion de LMD-9 aux cellules épithéliales intestinalesLactic acid bacteria are of great economic interest because of their use in the food industry. Among them, Streptococcus thermophilus (ST) is of major interest since it is the most used after Lactococcus lactis, for the manufacture of fermented dairy products and cheese. In addition, this bacterium is the only streptococcus to benefit from GRAS status (Generally Recognized As Safe). Beside its interest in the dairy industry, ST has beneficial effects on human intestinal health. Although these effects are widely documented, the probiotic status of ST remains to be consolidated. Therefore, studies are currently being conducted in order to select strains of ST with a high probiotic potential. Among criteria that are important to select ST strains include their ability to survive stress the drastic conditions of the digestive tract (DT) and their ability to adhere to intestinal cells. In this context, the aim of this thesis was to investigate firstly the in vitro adhesion capacity of the ST LMD-9 strain to different human intestinal cell lines and to evaluate the survival of this strain to bile salt stress. In order to highlight the potential role of some surface proteins in these two processes, three mutants derived from this strain and inactivated in the genes prtS (parietal protease), srtA (sortase A) and mucBP (Mucin Binding-protein) were also included in this study. Secondly, the impact of LMD-9 adhesion was analyzed, on one hand on the expression of some mucin-encoding genes in eukaryotic cells, and on the other hand on the expression of genes that would be specifically induced during adhesion process using the R-IVET (Recombinase-based In Vivo Expression Technology) approach. The results obtained demonstrated the ability of LMD-9 to survive up to the concentration of 3 mM of bile salts and that the PrtS, SrtA and MucBP surface proteins would be involved in the resistance to this stress. Our results also showed that the LMD-9 strain was capable of adhering to three cell lines used suggesting that this strain could interact with different mucins that may encounter in the DT. Moreover, the involvement of some surface proteins in the adhesion of LMD-9 has been found to be dependent on the surface characteristics of these cell lines, whether they are enterocytic (Caco-2) or mucus-secreting cells (HT29-MTX and HT29-CL.16E). Regarding the impact of LMD-9 adhesion on MUC2 and MUC5AC gene expression, no effect has been observed on the transcript level under our experimental conditions. Furthermore, for the first time, our results allowed us to identify genes specifically induced in the LMD-9 strain during adhesion process to epithelial cells. We have thus shown that the LMD-9 adhesion does not depend solely on surface proteins, but other functions and metabolic pathways are also involved. This thesis work contributes thus to new knowledge related to (i) the choice of the cellular model in in vitro bacterial adhesion studies, (ii) the ability of LMD-9 to survive bile salt stress by involving some surface proteins and (iii) understanding the molecular mechanisms of LMD-9 adhesion to epithelial cell

Importance of digestive mucus and mucins for designing new functional food ingredients

International audienceThe mucus, mainly composed of the glycoproteins mucins, is a rheological substance that covers the intestinal epithelium and acts as a protective barrier against a variety of harmful molecules, microbial infection and varying lumen environment conditions. Alterations in the composition or structure of the mucus could lead to various diseases such as inflammatory bowel disease or colorectal cancer. Recent studies revealed that an exogenous intake of probiotic bacteria or other dietary components (such as bioactive peptides and probiotics) derived from food influence mucus layer properties as well as modulate gene expression and secretion of mucins. Therefore, the use of such components for designing new functional ingredients and then foods, could constitute a novel approach to preserve the properties of mucus. After presenting some aspects of the mucus and mucins in the gastrointestinal tract as well as mucus role in the gut health, this review will address role of dietary ingredients in improving mucus/mucin production and provides new suggestions for further investigations of how dietary ingredients/probiotics based functional foods can be developed to maintain or improve the gut health

Importance of digestive mucus and mucins for designing new functional food ingredients

International audienc

Adhesion Properties of Probiotic <i>Lactobacillus</i> Strains Isolated from Tunisian Sheep and Goat Milk

International audienceFour hundred strains isolated from Tunisian sheep and goat raw milks were initially screened for their ability to survive the GastroIntestinal Tract (GIT). Forty-three among the four hundred bacteria were resistant to pepsin, pH 2, pancreatin and bile salts at 0.3%, even after 5 hours of incubation. Identification using 16S rRNA gene sequencing was established and we obtained as a species Lactobacillus plantarum (29 isolates from sheep milk and 11 from goat milk) and Lactobacillus pentosus (2 isolates from sheep milk and 1 from goat milk). We showed the ability for auto-aggregation and/or hydrophobicity properties. Finally, both M63 and C78 strains showed an important level of adhesion to three intestinal epithelial cells Caco-2 TC7, HT29-MTX, and HT29-CL.16E. Taken together, these properties allow the lactobacilli strains to be considered promising beneficial strains for developing functional foods for consumers

Surface proteins involved in the adhesion of Streptococcus salivarius to human intestinal epithelial cells

International audienceThe adhesion properties of 14 Streptococcus salivarius strains to mucus (HT29-MTX) and non-mucus secreting (Caco-2/TC7) human intestinal epithelial cells were investigated. Ability to adhere to these two eukaryotic cell lines greatly differs between strains. The presence of mucus played a major factor in adhesion, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface. Only one S. salivarius strain (F6-1), isolated from the feces of a healthy baby, was found to strongly adhere to HT-29 MTX cells at a level comparable to that of Lactobacillus rhamnosus GG, a probiotic strain considered to be highly adherent. By sequencing the genome of F6-1, we were able to identify 36 genes encoding putative surface proteins. Deletion mutants were constructed for six of them and their adhesion abilities on HT-29 MTX cells were checked. Our study confirmed that four of these genes encode adhesins involved in the adhesion of S. salivarius to host cells. Such adhesins were also identified in other S. salivarius strains

Streptococcus macedonicus strains isolated from traditional fermented milks: resistance to gastrointestinal environment and adhesion ability

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Structure and function of the Leptospira interrogans peroxide stress regulator (PerR), an atypical PerR devoid of a structural metal-binding site

International audiencePeroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress