Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Veterinary Large Animal Clinical Sciences
Non Technical Summary
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently administeredpharmaceuticals in the world. Due to the frequency of use of this class of medications, they aregenerally considered very safe by both the public and prescribing veterinarians. There is strongevidence, however, in both people and animals that routine clinical usage of these drugs haveprofound deleterious effects on the gastrointestinal (GI) tract of both people and animals. Amajor contributor to the lack of appreciation for the negative effects of these drugs on the GItract is related to difficulties diagnosing their effects of the GI tract. Fulminant, severe NSAIDenteropathy in the horse has been well-described but the less severe effects of these drugs on theequine GI tract likely remain under-recognized similar to the "silent epidemic" of NSAIDenteropathy reported in people. Importantly, in both human and veterinary medicine there are noeffective treatment or prevention strategies to manage the deleterious effects of NSAIDs on thelower GI tract (i.e., NSAID enteropathy). In addition to lack of treatment very little is knownabout the pathophysiology of NSAID enteropathy in both people and animals. One aspect of thepathophysiology that is gaining increasing recognition is the role of the microbiota. Themicrobiota has been shown to influence NSAID enteropathy but exactly how this occurs remainsunclear. Thus, given the importance of the equine microbiota in both health and disease, there isa critical need to more thoroughly investigate the effects of NSAIDs on the equine GI tract andon the equine microbiota. In the absence of such information, a mechanistic frameworknecessary for the subsequent development of novel strategies to treating or preventing NSAID inducedenteropathy will remain elusive. Therefore, our long-term goals are to more completelyunderstand how NSAIDs injure the equine GI tract, and how the microbiota influences theseeffects, in order to derive more effective prevention and treatment strategies. We have strongpreliminary evidence suggesting routine use of NSAIDs does, in fact, result in both dysbiosis andmucosal injury to the equine GI tract. In addition, we have validated the use of a novel approachto non-invasively examine the intestinal transcriptome in the context of NSAID enteropathy.Based on the critical clinical need for such information and our strong preliminary data wepropose the following objectives.Objective 1: Characterize the gene expression profile (i.e., transcriptome) in exfoliatedintestinal epithelial cells from NSAID-treated horses as compared with control horses.Objective 2: Document NSAID-induced dysbiosis and link these changes to alterations inthe function of the microbiota as determined by alterations in the metagenome of themicrobiota and alterations in the fecal metabolome.This proposal involves a novel approach to non-invasively examining the intestinaltranscriptome of horses in the context of complications from one of the most commonlyadministered classes of medications in the world. Should we achieve our objectives then we willhave validated a technique to non-invasively examine the intestinal transcriptome of the horse.This approach will have broad application in equine intestinal disorders in terms of biomarkerdiscovery and potential diagnostic modality. Additionally, upon completion of this work we willhave described the metagenome and metabolome of the equine microbiota AND described howNSAIDs alter this critically important system. Documenting NSAID-induced alterations in thesesystems will be the foundation of identifying potential effective prevention and treatmentstrategies for NSAID enteropathy and other intestinal mucosal disorders of the horse.
Animal Health Component
10%
Research Effort Categories
Basic
50%
Applied
10%
Developmental
40%
Goals / Objectives
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently administeredpharmaceuticals in the world. Due to the frequency of use of this class of medications, they aregenerally considered very safe by both the public and prescribing veterinarians. There is strongevidence, however, in both people and animals that routine clinical usage of these drugs haveprofound deleterious effects on the gastrointestinal (GI) tract of both people and animals. Amajor contributor to the lack of appreciation for the negative effects of these drugs on the GItract is related to difficulties diagnosing their effects of the GI tract. Fulminant, severe NSAIDenteropathy in the horse has been well-described but the less severe effects of these drugs on theequine GI tract likely remain under-recognized similar to the "silent epidemic" of NSAIDenteropathy reported in people. Importantly, in both human and veterinary medicine there are noeffective treatment or prevention strategies to manage the deleterious effects of NSAIDs on thelower GI tract (i.e., NSAID enteropathy). In addition to lack of treatment very little is knownabout the pathophysiology of NSAID enteropathy in both people and animals. One aspect of thepathophysiology that is gaining increasing recognition is the role of the microbiota. Themicrobiota has been shown to influence NSAID enteropathy but exactly how this occurs remainsunclear. Thus, given the importance of the equine microbiota in both health and disease, there isa critical need to more thoroughly investigate the effects of NSAIDs on the equine GI tract andon the equine microbiota. In the absence of such information, a mechanistic frameworknecessary for the subsequent development of novel strategies to treating or preventing NSAIDinducedenteropathy will remain elusive. Therefore, our long-term goals are to more completelyunderstand how NSAIDs injure the equine GI tract, and how the microbiota influences theseeffects, in order to derive more effective prevention and treatment strategies. We have strongpreliminary evidence suggesting routine use of NSAIDs does, in fact, result in both dysbiosis andmucosal injury to the equine GI tract. In addition, we have validated the use of a novel approachto non-invasively examine the intestinal transcriptome in the context of NSAID enteropathy.Based on the critical clinical need for such information and our strong preliminary data wepropose the following objectives and hypotheses:Objective 1: Characterize the gene expression profile (i.e., transcriptome) in exfoliatedintestinal epithelial cells from NSAID-treated horses as compared with control horses.Hypothesis 1: Exfoliated cell transcriptomes will differ significantly between control andNSAID-treated horses revealing a specific gene expression signature that willdiscriminate NSAID treated horses from control horses and be informative about thepathophysiology of NSAID enteropathy.Objective 2: Document NSAID-induced dysbiosis and link these changes to alterations inthe function of the microbiota as determined by alterations in the metagenome of themicrobiota and alterations in the fecal metabolome.Hypothesis 2: NSAID administration will induce a characteristic dysbiosis that willcorrelate to specific changes in both the metagenome of the microbiota and the fecalmetabolome thus linking specific microb
Project Methods
Study Design: Twenty horses will be randomly selected and put into matched pairs based on age (+/- 2 years), breed, sex, and weight (+/- 100 pounds) for a total of 10 pairs. One horse from each pair will be assigned randomly to be in the control orphenylbutazone groups. Fecal samples will be collected by rectal palpation using 1 rectal sleeve per animal every 10 days for 20 days beginning on day 0. Samples will be stored in sterile containers and frozen at -80ºC immediately after collection. NSAID administration will begin on day 1 and continue for 10 days; based on the group to which they are assigned horses will be administered phenylbutazone (4.4 mg/kg q 24 hrs) or vehicle control (base paste of phenylbutazone).Confirmation of NSAID-induced intestinal injury: Prior to examining the exfoliome, we will document that 10 days of NSAID therapy has induced GI inflammation via the following assays with which we have extensive experience; fecal MPO, plasma Il-1B, and plasma TNF-α.Specifically, we will perform these assays on samples collected on days 0, 10, and 20 in order to document the changes in these parameters over time. Fecal MPO will be performed following a commercially available sandwich ELISAPlasma Il-1β and TNF- α concentration will be determined using a commercially available ELISA (R&D Systems) designed and validated for equine samples.RNA isolation and sequencing from exfoliated cells: Following documentation of NSAIDinduced intestinal injury we will then characterize the gene expression profile (i.e., transcriptome) found in exfoliated intestinal epithelial cells from NSAID-treated horses ascompared with control horses from samples collected on day 10. Fecal samples utilized in this aim will be homogenized in RNA shield (Zymo research) prior to freezing at -80°C. PolyA+ RNA will be isolated from stool samples from horses as previously described in ratsand humans. Briefly, RNA will be extracted using a commercially available kit and quantified using a Nanodrop spectrophotometer (FisherThermoscientific) and quality will be assessed using the Nano6000 chip on a Bioanalyzer 2100(Agilent Technologies, Santa Clara, CA). Each sample will be processed with the NuGEN Ovation 3′-DGE kit (San Carlos, CA) to convert RNA into cDNA followed by NuGEN Encore Rapid Library kit to create Illumina libraries, as per manufacturer's instructions. Sequencing on Illumina HiSeq 2500 platforms will be performed using Illumina protocols.Data Analysis to determine differentially expressed genes, pathway analysis, and a subset of genes that classifies NSAID-treated from control animals: Sequencing data will be demultiplexed and assessed for quality using FastQC. Reads will be aligned using SplicedTranscripts Alignment to a Reference (STAR) software with default parameters and referenced against the genome of the horse (EquCab2). Differentially expressed genes will be determined using the R package EdgeR based on the matrix of gene counts 34. Gene pathway involvement and intersections will be analyzed using QIAGEN's Ingenuity ® Pathway Analysis (IPA, QIAGEN, Redwood City, CA) by uploading appropriate gene lists with fold change and false discovery rate (FDR) P-values. Finally, we will determine, within the differentially expressed genes, which gene (1-feature), pairs of genes (2-feature), or triplets of genes (3-feature) accurately discriminate NSAID treated animals from control animals.Microbial DNA isolation from feces: DNA will be extracted from fecal samples as previously described. Briefly, genomic DNA will be isolated using a commercially available fecal DNA extraction kit according to manufacturer's protocol with slight modifications. Briefly, 200 mg of frozen feces will be homogenized with 0.1- and 0.5-mm silica zirconium beads for 90 seconds at 6.5 m/sec with FastPrep FP120 cell disrupter prior to following the manufacturer's protocol.Whole genome sequencing: DNA quality will be determined by an Agilent DNA 12000 kit on the 2100 Bioanalyzer instrument. Libraries will be constructed with NEBNext ultra DNA library prep kit for illumina according to manufactures protocol . Sequencing will be performed on the Illumina Hiseq platform.Downstream processing: Species level taxonomic binning and function (i.e., metagenome) will be determined from these data with a whole genome sequencing pipeline that encompasses several open source software packages and online interfaces. Specifically we will use the tool MEtaGenomeANalyzer (MEGAN) and Metagenomic Phylogenetic Analysis (MetaPhlAn v2.0) to analyze these data along with dependent software packages.Metabolome: Frozen fecal samples will be sent to a metabolomics core facility. Biochemical extraction of all polar and non-polar molecules will be performed. All samples will be run on 2 independent platforms, including ultra-high pressure liquid chromatography/tandem mass spectrometry for positively charged ions and ultra-high pressure liquid chromatography/ tandem mass spectrometry for negatively charged ions. Each platform will produce between 10,000 and 15,000 ion features. Detection, identification, integration, and clustering of all ion features into individual compounds will be performed through use of their library of conserved chemical standards. Quality control measurements will be used to check all compounds to assure concentrations are accurate. Analyses of these data will be performed with open source software package MetaboAnalyst (v3.0) to determine which metabolites are altered by treatment.