Abstract
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.
Highlights
In vitro cell culture models comprised solely of either epithelial cells or macrophages cultured as monolayers are often used to study Salmonella enterica infections, as both cell types play critical roles in the infection process.[1, 2] Following ingestion, Salmonella actively invade and replicate within intestinal epithelial cells and are engulfed by macrophages upon crossing the epithelial barrier, where they exploit phagocytes as a preferred niche for replication and transport.[3]
Development of a novel 3-D co-culture model Our previous 3-D model of HT-29 colonic epithelial cells[7, 16, 17] was advanced in this study by the inclusion of U937 monocytes that were pre-differentiated into macrophages by PMA (Figs. 1–2)
We found that the addition of U937 cells to a partially or fully established 3-D epithelial model did not result in adequate incorporation of the U937 cells into the model, nor did it allow for the U937 cells to be incorporated beneath or within the epithelium, as would be relevant to the in vivo scenario
Summary
In vitro cell culture models comprised solely of either epithelial cells or macrophages cultured as monolayers are often used to study Salmonella enterica infections, as both cell types play critical roles in the infection process.[1, 2] Following ingestion, Salmonella actively invade and replicate within intestinal epithelial cells and are engulfed by macrophages upon crossing the epithelial barrier, where they exploit phagocytes as a preferred niche for replication and transport.[3] While classic two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding the interactions between Salmonella and host tissues during enteric infection, they lack the multicellular complexity and three-dimensional (3-D) architecture that are important for the differentiated structure and function of the in vivo parental tissue.[4,5,6,7] Synergism between epithelial cells and macrophages is important for driving responses of both cell types.[5, 8, 9] Not surprisingly, monotypic (single cell type) cultures of either epithelial cells or macrophages respond differently to challenge with pathogens or their toxins as compared to coculture models containing both of these cell types, the latter of which demonstrate a synergistic phenotype that better reflect the in vivo response.[5, 10] it is widely recognized there is an urgent need for advanced in vitro cell culture models that mimic the complex 3-D architecture, multicellular complexity and phenotypic characteristics of in vivo tissues for use in predictive human disease modeling, including infectious disease.[4, 5, 11,12,13] achieving a deeper understanding of host–pathogen interactions at the intestinal mucosa requires cell culture models that incorporate 3-D architecture, differentiation and multicellular complexity to characterize the interaction between epithelial cells and macrophages during enteric infection
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