Macrophages from Susceptible and Resistant Chicken Lines have Different Transcriptomes following Marek’s Disease Virus Infection

Figures

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  Figure 1. (a) The in vitro infection of macrophages from two inbred lines with Marek’s disease virus (MDV). Chicken bone marrow-derived macrophages (BMDMs) from lines 61 and 72 were cultured with Colony stimulating factor 1 (CSF-1) for 4 days. On the day of infection, pre-sorted eGFP+ CD45- chick embryo fibroblasts (CEF)s were co-cultured with BMDMs at a ratio of 1:5 (CEF:BMDM). After 1 day in culture, flow cytometric characterization of in vitro-infected BMDMs was performed for the surface expression of KUL01 and CD45. Data shown are representative of three independent experiments. Distribution of cells: Q1, infected CEFs; Q2, infected BMDMs; Q3, uninfected CEFs; Q4, uninfected BMDMs. Panel A and B: staining with isotype control for line 61 and 72; Panel C and D: staining with KUL01 for line 61 and 72; Panel E and F: staining with CD45 for line 61 and 72. (b) Graph showing means and the respective standard error of mean (SEM) of the percentages of infected cells found within the three biological replicates from lines 61 and 72 BMDM during cell sorting experiments.

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  Figure 2. Quantification of pro-inflammatory cytokines by qRT-PCR. The mRNA expression levels of IL6 and IL18 between control and flow sort purified infected BMDMs of lines 61 and 72 were measured on 1 dpi. Pairwise statistical comparisons between means in two groups (n = 3 in each group) of the two inbred chicken lines were carried out with a two-sample t-test (95% confidence interval) using Minitab 18 software. Con = control; In = infected. Means that share the same letter are significantly different (p < 0.05).

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  Figure 3. Differentially expressed genes in pathways responding to MDV infection as identified by iPathwayGuide. Up- and downregulated genes in (A) MD-resistant line 61 and in (B) MD-susceptible line 72 BMDMs are associated with focal adhesion and cytokine–cytokine receptor interaction pathways, respectively. Blue = upregulated; red = downregulated.

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  Figure 4. Functional analysis of DE genes using IPA (ingenuity pathway analysis). IPA showing the comparison of the most highly represented canonical pathways due to MDV infection in line 61 and line 72 BMDMs. MAC = macrophage; HR = host response.

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  Figure 5. Over-representation analysis of genes with their involvement in biological processes as determined by Expander software. (a) The GO (gene ontology) processes involved with genes downregulated only in line 61 infected BMDM and (c) the GO process involved with genes upregulated only in line 72 infected BMDM. The frequency of genes of a functional class within the examined set is described as a percentage of the total. (b) Significantly enriched ZNF42 transcription factor (TF) binding sites in genes downregulated in line 61 infected BMDM. The frequency ratio (frequency of the set divided by the frequency of the background) was 1.24, which means presence of 1.24 times as many ZNF42 sites than would be expected by chance.