The gastrointestinal wall of an ovine intestine is comprised of four anatomically distinct layers intended for digestion, absorption, secretion and protection. The ovine intestinal epithelial cells (OIECs) are the first line of defense against pathogens and underneath, a thin layer of connective tissue- lamina propria rich in immune cells. Initiation of innate immune response is triggered by recognition of pathogen-associated molecular patterns (PAMPs) by pathogen-recognition receptors (PRRs) such as Toll-like receptors (TLR). The goal is to understand the role of ovine epithelial cells in innate immunity.
Primary OIECs from a 3-day lamb will be used to generate cell line. To sustain the OIECs for research, cell death must be prevented by immortalization using Human Telomerase Reverse Transcriptase gene. Immortalized cells will be compared to primary OIECs. Immunohistochemistry will be used to characterize specific surface markers on these cells by exploiting the principle of antibodies binding to antigens.
Lectins binding assay with flow cytometry will confirm the expression for lectin binding profile by selectively identifying antigens in cell tissue. This procedure will provide important information on how OIECs detect and interact with pathogens. Eventually, we will achieve an OIEC line, characterize these cells and examine their role in ovine innate immunity.
Development and Characterization of primary and immortalized ovine intestinal epithelial cells (OIECs); analyze the lectin binding profile on these cells and the role in innate immunity.
Background & Primary Literature Comment by Microsoft Office User: Minimum of 1 page and 3 peer reviewed journal articles.Context:Provides clear awareness of the ‘big picture’; Why is this proposed question important/interesting in this field? What are the knowns that relate directly to the question? How does this experiment address the question?Accuracy and Relevance Content knowledge is accurate, relevant, and provides appropriate background for reader including defining critical terms. Comment by Adaeze Ifesi:
While the gastrointestinal tract epithelium must be accessible for nutrient absorption, ingestion of food is a potential source of pathogenic influx (Shipra and Miwako 2011). Presence of so many bacteria triggers potential immune system response. As food is swept along the digestive tract, epithelium cells of the mucosal membrane acts as the first line of defense by producing a range of antimicrobial factors including antimicrobial peptides (Wells 2011).
Enteric pathogens pose a threat to ovine livestock and can lead to animal death and money loss (Connor and Li 2010). Knowledge of how ovine intestinal epithelial cells (OIECs) develop, differentiate and fight off pathogens can provide insight on mechanisms to enhance nutrient uptake and protect against pathogens in ruminants. OIECs interact with enteric pathogens and display a significant role in mediating mucosal immune responses. Epithelia cells of the intestine do not only act as a barrier, interactions between antigen-presenting cells and OIECs regulate B and T cell responses to pathogens (Uprety 2018).
The gastrointestinal wall of ovine is similar to that of humans. Currently, ovine intestinal epithelial cultures are not available while intestinal epithelial cultures for bovine, guinea pigs and humans are available. Because of the difficulty in preserving primary OIECs for a long period of time, these cells must be immortalized.
Toll-like receptors (TLRs) are pattern recognition receptors that detect conserved molecular structures of microorganisms and instigate immune response (Shipra and Miwako 2011). In the presence of many bacteria, intestinal myofibroblasts are activated expressing toll-like receptors. To further research on the role of OIECs in innate immunity, Lectins are proteins or glycoproteins that bind complex sugar with high affinity. Lectins play a significant role in nonself recognition and immune responses (Nilsson 2016). With the use of lectins, the immune system achieves basic functions such as cell to cell adherence, programmed cell death and inflammatory modulation (Andrews 2014). Establishing and characterizing lectin binding profile of ovine intestinal epithelial cells (OIECs) can provide significant information on infectivity and tropism of various enteric pathogens.
Hypothesis Comment by Microsoft Office User: Testable and consider alternatives: Hypothesis is clearly stated, testable, and consider plausible alternative explanationsScientific merit: Hypothesis has scientific merit Ovine intestinal epithelial cells (OIECs) express various sugar moieties kin to initiate innate immune response. Immortalized cells express the same phenotypical characteristics as primary cultured cell lines. Lectin binding profile of OIECs provide important infectivity information.
Controls and Replication Comment by Microsoft Office User: Controls and replication: Appropriate controls (including appropriate replications) are clearly explained. Primary cell line of OIEC would serve as control. Immortalized cells using immunohistochemistry and lectin binding assay will be compared for phenotypic expression, lectin binding profile in pathogenic exposure and activity of pattern of recognition reception such as toll-like receptors. Experimental Design Comment by Microsoft Office User: Experimental design is likely to produce salient and fruitful results (tests the hypotheses posed).
Primary cultured cell lines are obtained from the ileum of a 3-day lamb. Collagenase digestion of cells has already been performed. The ovine intestinal epithelial cell will be grown in a mixture of Dulbecco’s Modified Essential Medium (DMEM) and high glucose media with 10% fetal calf serum, 1% antibiotics and 1% NEAA (Non-Essential Amino Acid). This would be incubated at 37 and 5% and supplemented with growth nutrients. The cells are already immortalized. Immortalization was done using Human Telomerase Reverse Transcriptase gene (hTERT) to prevent shortening of telomere important for cell longevity. After the establishment of primary cell lines, the cells are further characterized by biochemical and immunological techniques using immunohistochemistry.
Primary OIECs are compared to immortalized cells for TLR gene expression, growth time rate and lectin binding profile in innate immunity. The role of pathogen-associated molecular patterns (PAMPs) in innate immunity would be studied by identifying some conserved nonself recognition molecules such as Lectin. Flow cytometry would be used to test for lectin ability to bind to specific sugar expressed by OIECs. We will use Inhibition assay to confirm lectin binding specificity for accuracy.
Data selection Comment by Microsoft Office User: Proposed data collection is comprehensive, accurate, and relevant. Data will include the percentage of OIECs binding to lectin in vitro and percentage that did not. ICC/IHC imagery of both primary and immortalized cell lines will be included. Data analysis Comment by Microsoft Office User: Proposed data analysis is appropriate for hypothesis tested and appears to be the correct analysis with proposed data Data will be analyzed using two-tailed t-test at 5% significant level.
