SCIENTIFIC/TECHNICAL PROJECT DESCRIPTION RNA Deep Sequencing and Metabolomic Profiling of Microgravity-Induced Regulation of the Host-Pathogen Interaction: An Integrated Systems Approach 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 in microgravity. We propose to exploit advances in high throughput metabolomics, transcriptomics (RNA-Seq) and integrative data modeling of these systems, in combination with organotypic 3-D tissue models as human surrogates of the infection process, to provide a systems-level understanding of interconnections between this information at the host-pathogen interface, which holds tremendous potential to unveil previously unreported host responses to infection. Specifically, this project will be the first to integrate the biochemical (metabolomic) and biological (transcriptomic) responses of the host to infection with a microgravity analogue-cultured pathogen. 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 key importance in the infection process and may contribute to reported abnormalities and dysfunction in the crews immune system during flight [1-7]. By exploring interconnections between these systems over different kinetic timepoints, 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 identification of novel infection mechanisms and strategies for control. This is a critical issue to address, as spaceflight negatively impacts crew immune function and alters microbial virulence, gene expression and antimicrobial resistance, strongly suggesting an increased risk for in-flight infectious disease [2, 8-25]. The rational for the proposed study is strongly supported by previous studies by the PI team, and others, indicating that spaceflight-analogue culture using the Rotating Wall Vessel (RWV) and/or true spaceflight culture alter expression of key genes and proteins in both prokaryotic and eukaryotic cells that are important for the infectious disease process, including those mediating cellular redox, oxidative stress, and inflammatory responses [2, 8-10, 26-31, 62, 117] We were first to report that spaceflight and RWV culture of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) globally alters its virulence, stress response, transcriptomic and proteomic profiles [9, 10, 15], including oxidative stress and redox responses. We, and others, further showed that when cultured in spaceflight and/or the RWV, human intestinal epithelial cells and macrophages, which are key cell types targeted by Salmonella during enteric infection, also exhibit alterations in oxidative/redox functions and cytokine production [2, 16-18, 27, 29, 33, 117]. We also use the RWV for engineering three-dimensional (3-D) organotypic models of human intestinal epithelium that closely mimic the differentiated structure-function of the parental tissue and which respond to infection with S. Typhimurium in key ways that mimic in vivo responses, including production of inflammatory cytokines/chemokines [29, 32, 33]. This includes our immunocompetent 3-D intestinal co-culture models that contain both epithelial cells and functional phagocytic macrophages. Hypothesis: We hypothesize that an integrated multi-systems analysis of transcriptomic and metabolomic profiles of 3-D immunocompetent intestinal models in response to infection with S. Typhimurium cultured under either spaceflight-analogue or control conditions will unveil 1 - Nickerson previously unreported relationships between redox responses, oxidative stress, inflammation and infection that either approach alone would not be able to achieve. Herein, we propose to apply our 3-D intestinal co-culture models as human surrogates to characterize their transcriptomic, metabolomic and inflammatory responses to infection with RWV and control-cultured S. Typhimurium. As for our previous studies, we will use the NASAdesigned RWV as our microgravity analogue culture system in which cells experience low fluid shear culture conditions similar to those encountered during spaceflight, which our lab has designated as low shear modeled microgravity (LSMMG) . While the response of either intestinal epithelial or macrophage cells to Salmonella infection in vitro is among the best studied host-pathogen interactions and has yielded critical insight into pathogenic mechanisms, all previous studies have been performed using conventional culture of the pathogen (i.e. not under LSMMG conditions), with most of these studies having used in vitro models composed of single cell types of either intestinal epithelial cells or macrophages cultured as flat monolayers (which do not reflect the multicellular complexity and 3-D architecture of the parental tissue) [119-120]. These previous studies have yielded important, but still limited views of in vivo mechanisms determining infection outcome in humans that could be improved using next generation, high throughput, extremely sensitive technologies. Specifically, we propose to: AIM 1: Define high-resolution transcriptomic profiles (using RNA-Seq) of an immunocompetent 3-D intestinal co-culture model when infected by S. Typhimurium cultured in LSMMG as compared to control conditions. This data will be integrated in the context of metabolomic signatures (Aim 2) to unveil coordinated behavior between these networks, especially as they relate to redox potential, oxidative stress and inflammatory responses over different kinetic timepoints before and after infection. We anticipate that the implicated and interlinked biological networks elicited by the host in response to infection with LSMMG cultured Salmonella will reveal unique genomic signatures and metabolic changes that are reflective of alterations in oxidative, redox and inflammatory profiles. AIM 2: Define metabolomic signatures (using mass spectrometry) of an immunocompetent 3-D intestinal co-culture model when infected by S. Typhimurium cultured in LSMMG as compared to control conditions. We will focus on host metabolomic profiling of products associated with alterations in oxidative stress, redox, and inflammatory responses to show the power of the proposed interface between the biochemical (metabolomics) data generated in this Aim with the RNA-Seq data (Aim 1) and the cytokine data (Aim 3). We will profile the same kinetic timepoints before and after infection as Aim 1. AIM 3: Define inflammatory responses (using bead array detection of secreted inflammatory cytokines via flow cytometry) of an immunocompetent 3-D intestinal coculture model when infected by S. Typhimurium cultured in LSMMG as compared to control conditions. Our previous animal infection studies suggested that LSMMG culture of S. Typhimurium may decrease the expression of surface components that are recognized by the host immune system. Thus, we will profile expression of inflammatory cytokines in 3-D models following infection with LSMMG (as compared to control) -grown Salmonella at the same kinetic timepoints before and after infection as Aim 1, using a panel of cytokines known to be elicited in vivo in response to Salmonella infection. Since inflammatory processes are 2 - Nickerson accompanied by increased oxidative stress, which can impair the epithelial barrier, we will profile tight junction integrity as phenotypic validation of cytokine responses.
|Effective start/end date||7/1/13 → 9/30/17|
- NASA: Ames Research Center: $701,433.00