Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Bos taurus mucosal derived ab and gd T cells during Salmonella enterica serovar typhimurium enterocolitis

ABSTRACT: gd T cells have an important yet incompletely defined role in inflammation associated with a variety of infectious and autoimmune conditions. To better understand the precise roles of gd T cells relative to ab T cells in a specific infection, we utilized Salmonella Enterica Serovar Typhimurium (S. typhimurium) infection in cattle as it is a leading cause of disease in cattle and closely approximates S. typhimurium-induced enterocolitis in humans. To best represent phenotype and gene expression changes occurring in the gut mucosa early in S. typhimurium infection, gd and ab T cells were collected directly from the mesenteric lymphatic ducts and analyzed by FACS or immediately sorted and processed for microarray analysis. Gene expression profiles were compared at intervals during infection within T cell subsets. The majority of gene expression changes in both subsets occurred 48 hours after infection. In response to S. typhimurium infection there was an increase in expression of several genes in gd T cells which were indicative of activation, proliferation and innate function, whereas in ab T cells gene expression changes suggested a lack of S. typhimurium-specific response. This work represents the first focus on gene expression trends in tissue-derived T lymphocytes in an in vivo model that is highly relevant to human S. typhimurium-induced enterocolitis. Experiment Overall Design: For one mock infection (calf 156) and two experimental S. typhimurium infections (calves 112 and 162), gd and ab lymphatic T cells were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. Sorted gd and ab T cells were collected directly into TRIzol reagent (Invitrogen; calf 112) and lysed or suspended in Buffer RLT (Qiagen; calves 156 and 162) and lysed using Qiashredder columns, then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for Trizol (Invitrogen) extraction, or RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified either using Affymetrix Two-cycle (calf 112) target labeling protocol with 100 ng total RNA or the One-cycle protocol (calves 156 and 162) with approximately 1.6 micrograms of total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (June 2004). Hybridizations to GenechipÂ® Bovine Genome Arrays (Affymetrix) were performed with 15 micrograms biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) was used for data collection. Experiment Overall Design: Table I represents annotated genes of potential interest that changed 2 fold or greater in expression between 0 and 48 hours post-Salmonella infection (calves 112 and 162) or mock-infection (calf 156) in T cell subsets.

ORGANISM(S): Bos taurus

SUBMITTER: Jodi Fern Hedges

PROVIDER: E-GEOD-3439 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha.

Hedges Jodi F JF Buckner Diana L DL Rask Kerri M KM Kerns Hannah M M HM Jackiw Larissa O LO Trunkle Theresa C TC Pascual David W DW Jutila Mark A MA

Cellular immunology 20070301 1

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expr ...[more]

PMID: 17574223

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Project description:We have begun to approach gd T cells more as prospective innate cells than as conventional T cells. Recent results indicated that purified gd T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. In microarray and real time PCR analyses of RNA derived from bovine and human gd T cells, transcripts encoding Nod2 were repeatedly amplified. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), a subunit of PGN, functions in regulating innate activities, and was thought to be expressed primarily in APCs. Given our repeated detection of Nod2 transcripts in gd T cells, the specific direct response of gd T cells to MDP was analyzed by microarray, real time PCR, proteome array and in a functional priming assay. The results indicate a subtle activation in response to MDP akin to priming, and suggest a unique mechanism for differential gene expression. Experiment Overall Design: PBLs from calf 129 were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. PBLs from calves 150 and 151 were stained with GD3.8, and anti-Mouse IgG magnetic beads and sorted with MACS magnetic bead system (Miltenyi Biotec) as previously described to a purity of >98%. Sorted cells were rested overnight, stimulated for 4 hours then lysed in Buffer RLT and genomic DNA sheared using Qiashredder columns then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified using the Affymetrix One-cycle protocol with approximately 1.7micrograms of total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (June 2004). Hybridizations to GenechipÂ® Bovine Genome Arrays (Affymetrix) were performed with 15 mg biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) 19;20 was used for data collection.

Project description:Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis in humans and calves characterized by diarrhea and polymorphonuclear cell (PMN) influx to the intestinal mucosa. The Salmonella Type III Secretion System encoded at Pathogenicity Island I (SPI-1) translocates the Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into the host epithelial cell cytoplasm. These five effector proteins act in concert to induce fluid secretion and transcription of C-X-C chemokines, which serve to recruit PMNs to the intestine. While the individual molecular interactions of these Salmonella proteins with cultured host cells have been extensively characterized, their combined role in the generation of fluid secretion and inflammation is less well understood. A bovine ligated ileal loop model was used in conjunction with a custom bovine microarray to determine intestinal response to acute S. Typhimurium infection in the calf. Gene expression responses to both wild type S. Typhimurium a delta sipA, sopABDE2 mutant were measured at seven times during the initial 12 hours of infection. Microarray analysis confirmed increased expression of genes encoding proteins previously associated with immune response to Salmonella spp. infection. Gene expression changes were mapped to molecular interaction pathways and changes in expression of mechanistic genes, which are defined as perturbed genes identified by Bayesian genetic network modeling, were strongly involved in the mechanisms of the host immune response. In addition to correctly identifying known effects of wild type S. Typhimurium on host (bovine) gene expression, Bayesian genetic network modeling identified novel effects of S. Typhimurium on several molecular interaction pathways. Novel effects impacted gene regulation in the following pathways: adipocytokine signaling, insulin signaling, complement and coagulation cascades, axon guidance, gap junction, neuroactive ligand-receptor interaction, long-term depression, long-term potentiation, melanogenesis, and natural killer cell mediated cytotoxicity. Known effects were observed in the following pathways: regulation of actin cytoskeleton, apoptosis, cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), MAPK signaling, calcium signaling, Jak-STAT signaling, leukocyte transendothelial migration, adherens junction, tight junction, and ECM-receptor interactions, phosphatidylinositol signaling system, and antigen processing and presentation. Quantitative real-time PCR was used to verify the expression of some of these mechanistic genes. Microarrays were used to examine the transcriptional profiles of bovine intestinal epithelia infected with wild type Salmonella enterica serotype Typhimurium (control and wild type or delta sipA sopABDE2 mutant infected) across seven time points (15 min, 30 min, 1, 2, 4, 8, and 12 hours). Experiments were performed in quadruplicate (bovine ligated ileal loops surgeries were performed with four calves), generateing a total of 84 arrays.

Project description:gd T cells recognize unprocessed or non-peptide antigens, respond rapidly to infection, and localize to mucosal surfaces. We have hypothesized that the innate functions of gd T cells may be more similar to those of cells of the myeloid lineage than to other T cells. To begin to test this assumption, we have analyzed the direct response of cultured human and peripheral blood bovine gd T cells to pathogen associated molecular patterns (PAMPs) in the absence of APCs using microarray, real time RT-PCR, proteome array, and chemotaxis assays. Our results indicate that purified gd T cells respond directly to PAMPs by increasing expression of chemokine and activation related genes. The response was distinct from that to known gd T cell antigens and different from the response of myeloid cells to PAMPs. In addition, we have analyzed the expression of a variety of PAMP receptors in gd T cells. Freshly purified bovine gd T cells responded more robustly to PAMPs than did cultured human cells and expressed measurable mRNA encoding a variety of PAMP receptors. Our results suggest that rapid response to PAMPs through the expression of PAMP receptors may be another innate role of gd T cells. Experiment Overall Design: Sorted cells from 4 human gd T cell cultures were treated with either PBS or phLPS for 4 hours, RNA was extracted with TRIzolÂ® Reagent (Invitrogen Life Technologies) according to the manufacturerâs instructions, pooled and used to probe Affymetrix GenechipÂ® Human Genome U133A 2.0 Arrays (Cat. # 900471 Affymetrix, Santa Clara, CA) that represent 14,500 human genes. cDNA amplification and synthesis of biotin-labeled cRNA was performed with the Alternative One-cycle target labeling protocol with 10 micrograms total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (March 2004). Hybridization was performed with 10 micrograms biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) was used for data collection.

Dataset Information

Transcription profiling of Bos taurus mucosal derived ab and gd T cells during Salmonella enterica serovar typhimurium enterocolitis

Publications

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha.

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