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Gene expression profiling in heterophils with Salmonella enteritidis challenge using a chicken 44K Agilent microarray

ABSTRACT: To determine the transcriptional response to Salmonella enterica serovar Enteritidis (SE) infection, a newly developed chicken 44K Agilent array was used to analyze total RNA of heterophils from SE-resistant (line A) and SE–susceptible chickens (line B), treated with in vitro infection of SE (I) or non SE medium (N) for 1 hr. A dual-color balanced design was used to provide a direct comparison between SE-infected and non-infected groups (AI/AN, BI/BN) and between line A and line B (AN/BN, AI/BI). Data retrieved from 426 immunologically important genes were used for functional analysis. The results indicated that: In the comparisons of SE infection with non-infection, 39 genes were found differentially expressed (P < 0.05 with at least 2-fold) after Salmonella infection, while 21 genes were found differentially expressed between different lineages. Numerous of members in Toll-like receptor (TLR) signaling pathway were identified which include receptors: MD-2, TLR-4, -5, -15; adaptors: TLR adaptor molecule1 (TICAM1/TRIF), MAP kinase kinase 3 (MKK3) and NFkB-1, as well as cytokines, chemokines and costimulatory molecule: interleukin (IL)-1β, IL-6, IL-8, IL-12, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 4, CCL 5, and CD80. Most of immune genes were up-regulated after SE infection, and the magnitude of up-regulation was higher in line A than line B in general. The results suggested that a similar TLR regulatory network exists in both lines with strong response in resistant line. This finding has laid the foundation for further study of molecular and cellular modulation of SE infection in chickens. Keywords: disease state analysis

ORGANISM(S): Gallus gallus

PROVIDER: GSE9416 | GEO | 2008/09/01

SECONDARY ACCESSION(S): PRJNA103153

REPOSITORIES: GEO

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Project description:To determine the transcriptional response to Salmonella enterica serovar Enteritidis (SE) infection, a newly developed chicken 44K Agilent array was used to analyze total RNA of heterophils from SE-resistant (line A) and SEâsusceptible chickens (line B), treated with in vitro infection of SE (I) or non SE medium (N) for 1 hr. A dual-color balanced design was used to provide a direct comparison between SE-infected and non-infected groups (AI/AN, BI/BN) and between line A and line B (AN/BN, AI/BI). Data retrieved from 426 immunologically important genes were used for functional analysis. The results indicated that: In the comparisons of SE infection with non-infection, 39 genes were found differentially expressed (P < 0.05 with at least 2-fold) after Salmonella infection, while 21 genes were found differentially expressed between different lineages. Numerous of members in Toll-like receptor (TLR) signaling pathway were identified which include receptors: MD-2, TLR-4, -5, -15; adaptors: TLR adaptor molecule1 (TICAM1/TRIF), MAP kinase kinase 3 (MKK3) and NFkB-1, as well as cytokines, chemokines and costimulatory molecule: interleukin (IL)-1Î², IL-6, IL-8, IL-12, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 4, CCL 5, and CD80. Most of immune genes were up-regulated after SE infection, and the magnitude of up-regulation was higher in line A than line B in general. The results suggested that a similar TLR regulatory network exists in both lines with strong response in resistant line. This finding has laid the foundation for further study of molecular and cellular modulation of SE infection in chickens. Keywords: disease state analysis A dual color, balanced design was carried on for all of heterophils samples from sixteen chickens. Each sample type (AI, AN, BI and BN) includes four biological replication for labeling. A Dye swap was used in each pair of comparisons including AI/AN, BI/BN, and AI/BI, and AN/BN. Background subtracted signal intensity were collected from 16 arrays and normalized for data analysis.

Project description:The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria. Increased levels of G0S2 might decrease the availability of fatty acids to mitochondria. In non-professional phagocytes such as CEFs, this may lead to the dysfunction of mitochondria, apoptosis of CEFs and release of intracellular Salmonella. In professional phagocytes, G0S2 might be involved in the control of mitochondrial respiration, resulting in a decrease of reactive oxygen species as respiration by-products and lower damage to tissue. In this study we were interested whether chicken embryo fibroblast (CEFs) respond to S. Enteritidis infection and to what extent their response differs from that of other cells and caecal tissue. To address this, we characterised the gene expression of CEFs after infection with two different wild-type S. Enteritidis strains of poultry origin - SE 147 and SE 11 - using Agilen custom 8×15K microarrays. In total, 13,681 probes were designed to characterise the expression of ~9,000 transcripts of Gallus gallus.

Project description:Campylobacter jejuni (C. jejuni) is a zoonotic pathogen that causes human diarrhea worldwide. Chickens are a natural reservoir of C. jejuni. Understanding the host response to C. jejuni infection at the molecular level will lay the foundation to control human campylobacterosis by reducing food contamination. Two distinct genetic lines, resistant (line A) and susceptible (line B) to C. jejuni colonization, were utilized to profile the host response to C. jejuni infection using an Agilent chicken 44K microarray. Day-old chickens were challenged orally with C. jejuni and spleens collected for total RNA 7 days post-challenge. Twenty infected samples with highest (a) or lowest bacterial number (b) in cecal content and twenty non-infected (c) in each line were randomly pooled into four biological replicates. The pair comparisons among these three groups within each line were analyzed. The signal intensity of each gene was normalized using LOWESS method. A mixed model was used to identify differentially expressed genes by SAS (P < 0.001). This was opposite to previous cecal tonsil microarray result. There were 468, 743, and 939 genes differentially expressed between groups a and c, groups a and b, and groups b and c in line A, respectively, and 201, 37, 126 genes in line B, respectively. More differentially expressed genes in spleen in line A than in line B were found. The results indicated that significantly different response to C. jejuni infection occurred between resistant and susceptible chicken lines, and the effects of interaction between genetics and tissue should be considered. Chickens in two broiler lines were inoculated with 10^5 cfu C. jejuni on one day after hatch. The cecal content and cecal tonsil was collected and bacterial number in cecal content was counted on day 7 after inoculation. Twenty samples were separated into 3 groups (high burden, low burden, and control) based on bacterial burden of cecal content in each line, 5 samples were mixed randomly into one pool. A dual color, balanced design was carried on for all samples. Three comparisons were used in each line, non-infected/susceptible, susceptible/resistant, resistant/non-infected, totally, four biological replicates in each line. A Dye swap was used in each pair of comparisons including AN/AS, AS/AR, AR/AN; BN/BS, BS/BR, and BR/BN. Background subtracted signal intensity were collected from 24 arrays and normalized for data analysis.

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Gene expression profiling in heterophils with Salmonella enteritidis challenge using a chicken 44K Agilent microarray

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