III. ABSTRACTS AND POSTERS / ORAL PRESENTATIONS
6. Mice deficient in interferon regulatory factor 4 (IRF4) are more susceptible to infection with mouse-adapted influenza A/Aichi/2/68
3.5.7. Quantitative Real-Time PCR to detect for gene expression
Levels of expressed genes at the mRNA level will change under a variety of conditions such as those investigated in this project such as the presence of an infectious agent. The analysis of gene expression requires a sensitive, precise and reproducible measurement of specific mRNA sequences. Gene expression data can then be evaluated from real-time quantitative PCR by amplifying cDNA synthesized from mRNA. In order to quantify gene expression, real-time RT-PCR was used. The quantitative endpoint for RT-PCR is the threshold cycle (CT), where this can be defined as the PCR cycle at which the fluorescent signal of the reporter dye crosses an arbitrarily placed threshold. The comparative CT method (2-ΔΔCT method) used here makes the assumption that the PCR has perfect efficiency and the PCR of the target gene is similar to the internal control gene (Livak and Schmittgen 2001, Schmittgen and Livak 2008). Advantage of this method includes the ease of use and the ability to present data as ‘fold change’ in expression.
In order to attempt to decipher the relationship of the IRF-4 gene to the innate and adaptive immune responses involved in influenza viral infections, the mRNA expression of particularly important genes linked to such responses were investigated.
Additionally, the relationship of the IRF-4 gene to other family members of the transcription factor of IRFs was looked at. Genes of interest included IRF-1, -3, -4, -5, -7, -8, IFN-α, -β, -γ, , MyD88 and NFKB2.
164 Mice infected intra-tracheally with 500pfu PR8 H1N1 InfAV were culled when the weight loss achieved 25-30% of its original body weight, approximately Days 7-11 Post Infection, whereas PBS-mock-infected control mice were culled at Days 14 Post Infection. Lungs were harvested and RNA extraction was performed before reverse-transcription reactions were done to synthesize cDNA.
In innate immunity, the gene expression of Type I Interferons (IFN-α and IFN- β) were found to have a non-significant general reduction in fold change [(IFN-α; +/+
fold change -3.6; +/- fold change -1.5; -/- fold change -6.1) (IFN-β; +/+ fold change - 14.9; +/- fold change -8.4; -/- fold change 32.6)]. Expression of MyD88 were found to have undergone significant reduction in fold change in the IRF-4 -/- sample (fold change -2.9) when compared to the upregulated IRF-4 +/+ (fold change 2.3) and IRF- 4 +/- (fold change 1.6) samples.
As for adaptive immunity, expression of NFKβ also showed a similar significant trend where there was a upregulation in the IRF-4 +/+ samples (fold change 1.4) but a reduction in fold change in the IRF-4 +/- (fold change -1.3) and IRF-4 -/- (fold change -3.8) samples. This trend was also common in the Type II Interferon (IFN-γ). The IFN-γ gene expression was affected with the graded absence of the IRF-4 gene during the terminal phase of the InfAV infection, where a lower upregulation of IFN- γ was observed in IRF-4 +/- (fold change 4.3) and IRF-4 -/- (fold change).
Gene expression of IRF-4 during InfAV infection was also examined. In IRF- 4 +/+ there was a fold change of 1.1. However, a significant reduction in fold change existed in IRF-4 -/- (fold change -4.4). As IRF-8 interacted closely with IRF-4, gene expression of IRF-8 was also examined. Fold change followed the trend as well. In IRF-4 +/+, expression of IRF-8 was increased 2.2 times, followed by 1.3 times in
165 IRF-4 +/-. However, there was suppression in IRF-8 gene expression in IRF-4 -/- (fold change -3.3). In fact, gene expression of IRF-1 also followed the same trend where there was a significant suppression in IRF-1 gene expression in IRF-4 -/- (fold change -1.4) as compared to IRF-4 +/+ (fold change 4.1) and IRF-4 +/- (fold change 2.0).
Being direct transducers of virus-mediated signaling, the gene expression of IRF-3, -5 and -7 were examined. It appeared that the total absence of IRF-4 significantly affected the gene expression of all three transcription factors. The graded absence of IRF-4 gene caused a significant reduction in IRF-3. In IRF-4 -/- a reduction in fold change -7.6 was observed compared to the fold changes of IRF-4 +/+ (fold change -1.9) and IRF-4 +/- (fold change -1.8). A similar suppression in gene expression of IRF-5 was also observed in IRF-4 -/- (fold change -2.5). This, compared to the significant increase in IRF-5 gene expression in IRF-4 +/+ (fold change 3.0) and IRF-4 +/- (fold change 2.1). The InfAV infection caused an upregulation of IRF-7 in IRF-4 +/+ (fold change 1.4). However there was a significant suppression in IRF-7 in IRF-4 +/- (fold change -1.3) and IRF-4 -/- (fold change -3.8).
(A)
166 (B)
(C)
167 Figures 3.25 (A), (B) and (C). Cytokine expression in lung homogenates of IRF-4 mice infected with 500pfu PR8 H1N1 InfAV, (harvested at Days 14 Post-Infection or upon weight loss reaching 25-30% of the initial weight of the mouse), by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. (A) and (B) Expression of Interferon Type I and II and important key genes involve in innate and adaptive immune responses in lung homogenates of PR8 H1N1 InfAV-infected IRF-4 mice. (C) Expression of other members of IRF family in lung homogenates of PR8 H1N1 InfAV-infected IRF-4 mice.
Data expressed as ‘the expression of the gene of interest relative to the internal control in InfAV infected-samples compared with the Control mock-infected control’ - Comparative Mean Expression levels [2-∆∆CP], derived from the following steps: (i) average Cp (threshold cycle) calculated for both the gene of interest (GOI) and the housekeeping gene (Beta Actin), (ii) the mean of difference between Cp of GOI and normalizer (housekeeping) gene is determined (∆Cp), (iii) ∆∆Cp is determined from the difference between the Infected and Uninfected samples and (iv) Comparative Mean Expression levels (2-∆∆CP) is then obtained.
Number of mice used: PR8-infected Test IRF-4 mice (n=20/genotype/experimental round) and Control PBS-mock infected IRF-4 mice (n=10/genotype/experimental round), conducted over 2 investigation rounds. Mice found dead from InfAv infection were excluded from study. One way ANOVA was performed to determine statistical significance (p < 0.05).