A.J.W. Hoekman

Gene expression patterns associated with chicken jejunal development.

Jejunal development occurs in a spatio-temporal pattern and is characterized by morphological and functional changes. To investigate jejunal development at the transcriptomic level, we performed microarray studies in 1-21-day-old chickens. Nine gene clusters were identified, each with a specific gene expression pattern. Subsequently, groups of genes with similar functions could be identified. Genes involved in morphological and functional development were highly expressed immediately after hatch with declining expression patterns afterwards. Immunological development can be roughly divided based on expression patterns into three processes over time; first innate response and immigration of immune cells, secondly differentiation and specialization, and thirdly maturation and immune regulation. We conclude that specific gene expression patterns coincide with the immunological, morphological, and functional development as measured by other methods. Our data show that transcriptomic approaches provide more detailed information on the biological processes underlying jejunal development.

Early host gene expression responses to a Salmonella infection in the intestine of chickens with different genetic background examined with cDNA and oligonucleotide microarrays.

So far the responses of chickens to Salmonella have not been studied in vivo on a whole genome-wide scale. Furthermore, the influence of the host genetic background on gene expression responses is unknown. In this study gene expression profiles in the chicken (Gallus gallus) intestine of two genetically different chicken lines were compared, 24 h after a Salmonella enteritidis inoculation in 1-day-old chicks. The two chicken lines differed in the severity of the systemic infection. For gene expression profiles, a whole genome oligonucleotide array and a cDNA microarray were used to compare both platforms. Genes upregulated in both chicken lines after the Salmonella infection had a function in the innate immune system or in wound healing. Genes regulated after the Salmonella infection in one chicken line encoded proteins involved in inflammation, or with unknown functions. In the other chicken line upregulated genes encoded proteins involved in acute phase response, the fibrinogen system, actin polymerisation, or with unknown functions. Some of the host gene responses found in this study are not described before as response to a bacterial infection in the intestine. The two chicken lines reacted with different intestinal gene responses to the Salmonella infection, implying that it is important to use chickens with different genetic background to study gene expression responses.

Effects of Salmonella on spatial-temporal processes of jejunal development in chickens

To study effects of Salmonella enteritidis on morphological and functional changes in chicken jejunal development, we analysed gene expression profiles at seven points post-infection in 1-21 day-old broiler chickens. Nine clusters with different gene expression patterns were identified, and the genes in each cluster were further analyzed by a functional annotation clustering method (DAVID). Functional and morphological developmental processes dominated in all the nine clusters. Salmonella infection caused delays in several intestinal-morphological processes, whereas functional metabolic processes occurred in a similar spatial-temporal frame compared to normal jejunum development. A clear difference between normal developing- and Salmonella disturbed jejunum was the higher expression of genes involved in cell turn-over at early stages in the infected jejunum. Surprisingly, we found no clustered immune related processes in the infected birds. To compare the immunological processes between control and Salmonella infected chickens, the gene expression data was superimposed on known immunological KEGG pathways. Furthermore an in-depth analysis on the immune gene level was performed. As expected, we did find immunological processes in the Salmonella infected jejunum. Several of these processes could be verified by immunohistochemistry measurements of different immunological cell types. However, the well-ordered spatial-temporal development of the immune system, as observed in control non-infected animals, was completely abolished in the infected animals. Several immunological processes started much earlier in time, whereas other processes are disorganised. These data indicate that normal morphological and immunological development of jejunum is changed dramatically by a disturbance due to Salmonella infection. Due to the disturbance, the well-organized spatial-temporal development of morphological processes are delayed, those of the immunological development are scattered, whereas metabolic functional processes are almost not affected. This demonstrates the flexibility of developmental processes in the broiler chicken intestine.

A Pathway Analysis Tool for Analyzing Microarray Data of Species with Low Physiological Information

Pathway information provides insight into the biological processes underlying microarray data. Pathway information is widely available for humans and laboratory animals in databases through the internet, but less for other species, for example, livestock. Many software packages use species-specific gene IDs that cannot handle genomics data from other species. We developed a species-independent method to search pathways databases to analyse microarray data. Three PERL scripts were developed that use the names of the genes on the microarray. (1) Add synonyms of gene names by searching the Gene Ontology (GO) database. (2) Search the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database for pathway information using this GO-enriched gene list. (3) Combine the pathway data with the microarray data and visualize the results using color codes indicating regulation. To demonstrate the power of the method, we used a previously reported chicken microarray experiment investigating line-specific reactions to Salmonella infection as an example.

Differences in the early response of hatchlings of different chicken breeding lines to Salmonella enterica serovar Enteritidis infection

Poultry products are the major source of food-borne Salmonella infection in humans. Broiler lines selected to be more resistant to Salmonella could reduce the transfer of Salmonella to humans. To investigate differences in the susceptibility of newly hatched chicks to oral infection with Salmonella enterica serovar Enteritidis, 3 commercial broiler lines (A, B, and C) were infected immediately after hatch and compared to healthy controls at 0.33, 1, and 2 d postinfection. Weight, bacteriological examination, and the jejunal influx of CD4, CD8, TCRαβ, TCRγδ, and KUL01 (macrophages and dendritic cells) cells that are positive was investigated. In addition, the jejunal transcriptional response was analyzed using whole-genome chicken cDNA arrays. Salmonella colony-forming unit counts from cecal content and liver revealed that Salmonella enterica entered the body at 0.33 d postinfection. Broiler line A appeared most susceptible to intestinal colonization and the systemic spread of Salmonella. In addition, the Salmonella-induced jejunal influx of macrophages in this line showed a clear increase in time, which is in contrast to lines B and C. On the other hand, all lines showed a peak of CD4(+) cells at 1 d postinfection when infected chicks were compared to control chicks. The transcriptional response of line A clearly differed from the responses in lines B and C. Functional analysis indicated that the majority of the differentially expressed genes at 0.33 d postinfection in line A were involved in cell-cycle functions, whereas at 2 d postinfection the majority of the differentially expressed genes could be assigned to inflammatory disorder, differentiation and proliferation of (T) lymphocytes. These data indicate that hatchlings of different broiler lines differ in their systemic spread of Salmonella and suggest that intestinal barrier functions, as well as immunological responses, may be the underlying factors. We hypothesize that the differences between genetic chicken lines divergent in their response to Salmonella infection at a young age include developmental differences of the gut.

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa

BackgroundGene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are frequently used to modulate animal performance or therapeutically for monogastric livestock. In this study, changes in gene expression induced by these three interventions in cultured “Intestinal Porcine Epithelial Cells” (IPEC-J2) recorded after a short-term period of 2 and 6 hours, were compared to the in vivo gene expression profiles in order to evaluate the capability of this in vitro bioassay in predicting in vivo responses.MethodsLists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature.ResultsFor all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye.ConclusionsShort-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by “early” signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed.