11 research outputs found

    Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

    Get PDF
    Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection

    Analysis of Interactions of Salmonella Type Three Secretion Mutants with 3-D Intestinal Epithelial Cells

    Get PDF
    The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms

    New Insights into the Bacterial Fitness-Associated Mechanisms Revealed by the Characterization of Large Plasmids of an Avian Pathogenic E. coli

    Get PDF
    Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI(2))]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes

    Organotypic 3D cell culture models : using the rotating wall vessel to study host–pathogen interactions

    No full text
    Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed

    A: Immunohistochemical profiling of decellularized lung scaffolds recellularized with MSCs in static and bioreactor conditions for 14 days.

    No full text
    <p>Profiling of whole normal mouse lung tissue and MSC monolayers was performed as well. Cell nuclei are labeled in blue; markers of interest are labeled in green and are Fsp1 (panel A to E), collagen I (panel a to e), and osteopontin (panel aa to ee). White arrows point to multilayered cell aggregates, observed in static recellularization conditions and for this test condition profiling for both aggregates (A, a, aa) and single cells (B, b, bb) is presented. Since collagen I-positive cells showed higher signal intensity compared to that of the collagen I-positive scaffold, the background scaffold signal in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126846#pone.0126846.g005" target="_blank">Fig 5Ab and 5Ac</a> was removed for clarity. To demonstrate the collagen I-positive scaffolds, the background signal is only shown in Fig 5Aa. Magnifications are 400x or 630x. B: Alizarin red staining of decellularized lung scaffolds recellularized with MSCs in static (panels A, B) and bioreactor (panel C) conditions for 14 days. As a negative control, decellularized lungs that were not seeded with cells are presented (panel D). MSC monolayers differentiated along the osteoblastic lineage are included as positive control (panel E). Magnification is 200x or 630x. For each condition, images are representative of the entire lung.</p

    Hematoxylin-eosin staining of decellularized lungs recellularized with (A) MSCs in static (panels A to F) and bioreactor (panels a to f) conditions for 7, 14, and 28 days, and (B) with C10 cells in static (panels A to D) or bioreactor (panels a to d) conditions for 11 and 14 days.

    No full text
    <p>For each condition, a low (100x) and high magnification (630x) are shown (e.g., A is low magnification, B is high magnification). Insets are included to show phenotypes at the single cell level. Black arrows point to MSC cell aggregation observed in static recellularization conditions. Blue arrows point to cytoplasmic vacuoles indicative of cell stress. Airways are labelled. For each condition, images are representative of the entire lung, with the exception of panels AA and AB, which reflect a region with high cell density whereas some regions were devoid of cells (not shown).</p

    Overview of experimental set-up used to recellularize decellularized lung scaffolds with MSCs or C10 cells in static and bioreactor conditions.

    No full text
    <p>MSCs or C10 cells were introduced in the decellularized lung scaffolds through the cannulated trachea. Next, lungs were statically incubated for 4 days, regardless of the subsequent test condition. Culture medium for MSCs was IMDM and for C10 cells GTSF-2. Different time points were tested to assess recellularization with MSCs (3, 10, 24 days) or C10 cells (7, 10 days) in static or bioreactor conditions.</p

    Recellularization of Decellularized Lung Scaffolds Is Enhanced by Dynamic Suspension Culture

    No full text
    <div><p>Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.</p></div
    corecore