Performing Department
Veterinary Pathology
Non Technical Summary
Porcine gastroenteritides including porcine postweaning diarrhea, swine dysentery, porcine epidemic diarrhea virus infection, and Clostridial enteritides result in significant clinical disease burden with substantial health and economic loss culminating in porcine quality of life, reduced feed efficiency, and a predisposing factor for chronic inflammatory enteritides. The enteric villus-crypt axis is a platform and unique niche for persistent microbial interactions and microbiome ecosystems that can result in metabolic dysbiosis that favors the replication of extracellular and intracellular organisms that alter enteric homeostasis with resultant pathology. Tuft cells are unique endodermal derived specialized cells found on mucosal surfaces of the gastrointestinal, pancreatobiliary, and pulmonary tracts. The role of tuft cells, a specialized chemosensory and immunomodulatory cell that regulates innate mucosal immunity, modulates enteric mucosal immunity, and induces goblet cell hyperplasia has not been explored and investigated in porcine enteric diseases. There is a dearth of knowledge on the role of this unique cell in villus-crypt axis pathology in farm and companion animals.The goals of this proposal and project are set to advance the frontiers of porcine enteric pathology with emphasis on providing insights on the relationship of tuft cells and enteric dysbiosis and in addition to its role in the pathogenesis and pathology of porcine gastrointestinal disease. The results generated from this project will provide data for a comprehensive research program on porcine enteric pathology and the role of tuft cells in the pathogenesis of postweaning diarrhea, Brachyspira hyodysenteriae infection, and Clostridiodes difficile infection. These results will provide a platform and context to advance our understanding of porcine enteric pathogenesis and pathology and potentially identify and develop therapeutics to modulate enteric chemosensory and immunomodulatory effectors to prevent mortalities associated with porcine enteric diseases with comparative applications for human health.
Animal Health Component
45%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Goals / Objectives
The goals of this project are designed and structured to expand the frontiers of porcine enteric pathology and fulfill the objectives listed below.Define and determine the distribution, prevalence, histomorphometry, and ultrastructural features of porcine tuft cellsUnderstand the homeostatic regulatory mechanisms and dynamics of porcine enteric tuft cells during a management and metabolite associated disease; porcine postweaning diarrhea, viral disease; porcine epidemic diarrhea virus infection, enteric bacterial disease; Brachyspiral dysentery and Clostridial enteritisDefine the significance and relationship of Succinate receptor 1 (SUCNR1) and CD300lf expression in porcine tuft cell homeostasisUnderstand the role of porcine enteric microbiome and dysbiosis in the regulation of porcine tuft cell function and its role in enteric pathologyElucidate the role of porcine tuft cell in the modulation of porcine enteric mucosal innate and adaptive immunityDefine how porcine tuft cells mediate enteric innate lymphoid cell 2 (ILC-2) through the expression of Th2 cytokines such as interleukin 25 (IL-25), interleukin 5 (IL-5), and interleukin 13 (IL-13)
Project Methods
The prevalence, distribution, histomorphology, and biochemical identification of porcine enteric tuft cells will be determined and evaluated by a combination of light microscopy, immunohistochemistry, immunofluorescence, and electron microscopy. To define and quantify the distribution of tuft cells in porcine stomach, small intestine, large intestine, pancreas, and gallbladder, we intend to collect fresh enteric samples fixed in 10% formalin-fixed and 2.5% glutaraldehyde. Paraffin-embedded formalin-fixed samples will be sectioned at 5µm for hematoxylin and eosin-stained slides. Samples for Immunohistochemistry (IHC) and immunofluorescence (IF) will be sectioned at 5µm and 4µm respectively. IHC samples will be processed for antigen retrieval with deparaffinization and subsequent incubation with primary antibodies for tuft cell markers (DCLK1, POU2F3, GFI1B) and appropriate secondary antibodies for chromogen detection. IF samples will be deparaffinized, rehydrated, and boiled for 20 mins in 10Mm sodium carbonate buffer for antigen retrieval. The sections will be permeabilized in PBS 0.5% Triton X-100. The sections will be incubated with tuft cell-specific primary antibodies and appropriate secondary antibodies after washing. Hematoxylin and eosin sections in addition to the IHC sections will be analyzed by Olympus BX43 microscope and the IF slides with analyzed by an Olympus immunofluorescence and confocal microscope. Each segment of the small intestine, large intestine, pancreas, and gallbladder will be evaluated by digital pathology with a quantification software such as the HALO program or Aiforia digital pathology software to quantify tuft cell marker expressing cells. Tuft cell marker transcripts will be assessed by molecular biology and molecular pathology techniques such as real-time polymerase chain reaction, reverse transcription-polymerase chain reaction, and in-situ hybridization (RNAscope). Similar methods as stated above will be applied to evaluate porcine innate lymphoid cells 2 (ILC2), IL-25, IL-13, and IL-5 and succinate receptor 1 (SUCNR1) to evaluate the role porcine in the modulation of innate mucosal immunity and the regulation of succinate receptor 1 (SUCNR1) expression on porcine tuft cells. Glutaraldehyde fixed enteric samples will be assessed for tuft cells ultrastructural features and morphology by preparation of the semi-thin plastic sections with Toluidine blue for light microscopy to identify enterocytes with classic tuft cell-specific apical microvilli. Electron microscopy will be concluded with grid preparation, staining, and image capture.