Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns
Year: 2017
Type: article
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.
Source: npj Microgravity
Authors: Jennifer Barrila, Jiseon Yang, Aurélie Crabbé, Shameema F. Sarker, Yulong Liu, C. Mark Ott, Mayra A. Nelman-Gonzalez, Simon J. Clemett, Seth D. Nydam, Rebecca J. Forsyth, Richard R. Davis, Brian E. Crucian, Heather Quiriarte, Kenneth L. Roland, Karen Brenneman, Clarence Sams, Christine Loscher, Cheryl A. Nickerson
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,690
Awards: Enterovirus Infection of Polarized Intestinal Cells, A MAJOR CHALLENGE IN MITIGATING INFECTIOUS DISEASE RISKS DURING SPACEFLIGHT IS TO UNDERSTAND MODEL AND INTEGRATE THE COMBINED ACTION OF CELLULAR MOLECULAR AND BIOCHEMICAL NETWORKS IN THE HOST THAT POTENTIATE TRANSITION TO DISEASE IN RESPONSE TO INFECTION BY PATHOGENS CULTURED INMICROGRAVITY. THIS IS A CRITICAL ISSUE TO ADDRESS AS SPACEFLIGHT NEGATIVELY IMPACTS CREW IMMUNE FUNCTION ANDALTERS MICROBIAL VIRULENCE GENE EXPRESSION AND ANTIMICROBIAL RESISTANCE STRONGLY SUGGESTING AN INCREASED RISK FOR IN-FLIGHT INFECTIOUS DISEASE. HEREIN WE PROPOSE TO APPLY OUR 3-D INTESTINAL CO-CULTURE MODELS AS HUMAN SURROGATES TO CHARACTERIZE THEIR TRANSCRIPTOMIC (VIA RNA-SEQ) METABOLOMIC (VIA MASS SPECTROMETRY) AND INFLAMMATORY RESPONSES TO INFECTION WITH SPACEFLIGHT ANALOGUE AND CONTROLCULTURED SALMONELLA TYPHIMURIUM. TO FACILITATE A PRACTICAL INTEGRATION OF THESE SYSTEMS THE COMBINED ANALYSIS PROPOSED IN THIS STUDY WILL FOCUS ON THE METABOLOMIC SUBSET ASSOCIATED WITH HOST OXIDATIVE STRESS REDOX AND INFLAMMATORY RESPONSES. THESE FACTORS ARE OF KEYIMPORTANCE IN THE INFECTION PROCESS AND MAY CONTRIBUTE TO REPORTED ABNORMALITIES AND DYSFUNCTION IN THE CREWS' IMMUNE SYSTEM DURING FLIGHT. WE WILL USE SYSTEMS MODELING APPROACHES BASED ON BOTH QUANTITATIVE GENE TRANSCRIPT AND BIOCHEMICAL METABOLITE CORRELATION AND ALL OF THEIR KNOWN/INFERABLE RELATIONSHIPS BETWEEN GENES PROTEINS PATHWAYS AND BIOCHEMISTRY THAT WILL ALLOW US TO FORM NEW BRIDGES AND A GENERAL FRAMEWORK FOR MAKING THESE LINKAGES BETWEEN GENE EXPRESSION (RNA-SEQ) AND BIOCHEMICAL (METABOLOMIC) DATA. THIS APPROACH WILL ALLOW FOR NEW INSIGHTS INTO RELATIONSHIPS BETWEEN REDOX FUNCTIONS OXIDATIVE STRESS INFLAMMATION AND INFECTION THAT EITHERAPPROACH ALONE WOULD NOT BE ABLE TO ACHIEVE. BY EXPLORING INTERCONNECTIONS BETWEEN THESE SYSTEMS OVER DIFFERENT KINETIC TIMEPOINTS BEFORE AND AFTER INFECTION THIS SYSTEMATIC APPROACH WILL PROVIDE AN UNPARALLELED LEVEL OF SENSITIVITY AND RESOLUTION OF THE DYNAMICS OF THE HOST RESPONSE TO A MICROGRAVITY-ANALOGUE CULTURED PATHOGEN WHICH MAY LEAD TO IDENTIFLCATION OF NOVEL INFECTION MECHANISMS AND STRATEGIES FOR PREVENTION AND CONTROL., G5520316217, G9000071016
OpenAlex ID: W2593740498