PP-54
Uncovering the hidden effects of antibiotics on microbiota and immune defense system
MohammadHeidari and Huanmin Zhang
USDA-ARS, Avian Disease and Oncology Laboratory (ADOL)
Background:Intestinal microbiota is an essential player in stabilizing homeostasis of GI tract and priming the immune system. Antibiotic-mediated alteration in the composition and functions of microbiota can produce deleterious effect on the host innate and adaptive immune systems. Intestinal microbiota provides protection for animals by competing for space, attachment sites, nutrients, as well as production of antimicrobial peptides. Antibiotic-mediated disturbances in intestinal microbiota negatively affects the immune homeostasis that leads to infection and disease. Use of antibiotics in humans and animals induces dysregulation in the normal composition of microbiota and consequently, loss of metabolites production by microbiota, loss of bacterial signals (TLRs and NOD-like receptors), reduction in antimicrobial peptides (defensins, cathelicidins), downregulation of MHC I and MHC II, decrease in production of pro-inflammatory cytokines (IL-1b, IL-8), macrophage inflammatory protein, neutrophil migration, and destruction of intestinal epithelial cells.
Problem:Antibiotic-mediated dysregulation of microbiota disrupts the normal functions of the gut immune responses, induces alteration in global gene expression, inhibits the biological function of lymphocytes, and impairs the viability of mitochondria and intestinal epithelial cells. Antibiotics administration dysregulates the normal function of microbiota with adverse effect on the host health and immune defenses. Testing strategies to counteract the negative impact of antibiotics use in animal production is of critical importance. The alternative use of probiotics outlined in this proposal is one such strategy to replace the unnecessary overuse of antibiotics in poultry and other meat animal agriculture.
Proposal: To determine the comparative effects of antibiotics and probiotics use in poultry on:
1. Global gene expression profiling: RNAseq-based comparative global gene expression profiling in the spleen and cecal tonsils of antibiotic and probiotic fed chickensat different time points post treatment. 2. Cytokine gene expression and Th1/Th2/Th17 paradigm:determine the expression pattern of pro- anti-inflammatory cytokines, as well as cytokines associated with activation of Th1/Th2/Th17 cells (IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IL-23, IFN-, IFN-, TNF-, and TGF-). 3. NK cell function: characterization of NK cells isolated from ilium and duodenum sections of the treated and control birds by expressionanalysis of CD107a, an activation marker for NK cells, using flow cytometry. 4. Lymphocyte proliferation: mononuclear cells isolated from blood of treated and control birds will be treated with concanavalin A or RPMI-1640 medium (negative control) and water-soluble tetrazolum salt (colorimetric substrate for cell proliferation). The color change will be measured at 450 nM by a colorimetric plate reader. 5. Mitochondria and intestinal epithelial cell viability: cell suspension isolated fromsmall sections of ilium and duodenum will be stained with anti-CD45 antibody (Exclusion of hematopoietic cells) and Annexin (Detection of translocation of phosphatidylserine, a hallmark of apoptosis) and used in flow cytometry in the presence of 7AAD for cell viability and Mitotracker Green for detection of mitochondria. 6. Small RNAseq analyses: the total RNA samples of the spleen tissues will be used to conduct global small RNAseq to determine small RNA expression changes in response to antibiotic versus probiotic treatments. Changes in small RNA expressions would provide insights to explain gene expression changes through small RNA target gene analysis and to advance the basic understanding on how antibiotics and probiotics differentially affect varied biological functions including innate and adaptive immune system in chicken.