Tissue and time specific expression pattern of interferon regulated genes in the chicken

Abstract

Background: Type I interferons are major players against viral infections and mediate their function by the induction of Interferon regulated genes (IRGs). Recently, it became obvious that these cytokines have a multitude of additional functions. Due to the unique features of the chickens' immune system, available data from mouse models are not easily transferable; hence we performed an extensive analysis of chicken IRGs.

Results: A broad database search for homologues to described mammalian IRGs (common IRGs, cIRGs) was combined with a transcriptome analysis of spleen and lung at different time points after application of IFNα. To apply physiological amounts of IFN, half-life of IFN in the chicken was determined. Interestingly, the calculated 36 min are considerably shorter than the ones obtained for human and mouse. Microarray analysis revealed many additional IRGs (newly identified IRGs; nIRGs) and network analysis for selected IRGs showed a broad interaction of nIRGs among each other and with cIRGs. We found that IRGs exhibit a highly tissue and time specific expression pattern as expression quality and quantity differed strongly between spleen and lung and over time. While in the spleen for many affected genes changes in RNA abundance peaked already after 3 h, an increasing or plateau-like regulation after 3, 6 and 9 h was observed in the lung.

Conclusions: The induction or suppression of IRGs in chickens is both tissue and time specific and beside known antiviral mechanisms type I IFN induces many additional cellular functions. We confirmed many known IRGs and established a multitude of so far undescribed ones, thus providing a large database for future research on antiviral mechanisms and additional IFN functions in non-mammalian species.

Keywords: Chicken; Expression profile; Half-life; IFNα; IRG.

Fig. 1

Pharmacokinetic profile of recombinant ChIFNα after i.v. injection. Three chickens were treated i.v. with a single dose of recChIFNα (1x10⁷Units for the biggest bird and a direct proportional weight adjusted dose for the others). Plasma was taken at the indicated time points, and the amount of biological active type I IFN was determined using an IFN reportergene assay

Fig. 2

IFN treatment for the microarray experiment. a Four groups of chickens (n = 6 per group) received recChIFNα as indicated in the experimental scheme. b At the indicated time points animals were sacrificed and the plasma concentration of biologically active type I IFN was measured using an IFN reporter gene assay. Shown are group mean and individual IFN concentrations

Fig. 3

Microarray analysis after IFN injection. a “R” based heat map of spleen and lung tissue: Probes are labeled by group membership and animal number (1-4). Data sets with an unusual assignment which deviates from the rest of the group are boxed. b Venn diagram for the comparison of microarray and in silico analysis: Shown are the intersections of significantly regulated genes 3 h after IFN injection between spleen (red), lung (blue) tissue and the “common IRGs” (green), identified by database analysis. c Intersections of IRGs in spleen and lung at different time points after IFN injection: Numbers indicate array-based significantly regulated genes; numbers in brackets represent “common IRGs”

Fig. 4

IRGs exhibit different expression profiles. a MEV analysis of genes with differential expression after 3 h identified nine different groups of expression profiles over the three sampling time points. Shown are characteristic examples for each group with gene name and tissue (S for spleen, L for lung) for which the profile was found. b Comparison of profiles for Mx, OAS and PKR in spleen (black symbols) and lung (grey symbols)

Fig. 5

Validation of array results. a Comparison of detected fold changes for IL6 mRNA from the microarray experiment and qRT-PCR on identical samples. b Amount of IL6 protein in plasma samples of IFN treated and untreated animals

Fig. 6

Gene ontology of differentially expressed genes. Differentially expressed genes with an FC of at least ± 3 were subjected to Panther gene ontology. Shown are identified biological processes and their percental distribution between genes with higher (+) and lower (-) mRNA abundance after IFN injection in spleen and lung

Fig. 7

Multilayer interaction of differentially expressed genes. IPA network analysis demonstrating gene interactions with the upstream regulators PTX3 (a) and Albumin (b). Genes with higher mRNA abundance in the IFN treated animals are shown in red, genes with lower mRNA abundance in the treated animals in green. The small diagrams next to each differentially expressed gene display expression (FC) at the different time points. The magnification shows the expression course for IL6

Fig. 8

Tissue and time specific expression profiles for chemokines and cytokines. Fold expression of differentially expressed chemokines (a + b) and cytokines (c + d) after IFN injection in the spleen (a + c) and the lung (b + d) in relation to control birds