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
3.5.6. Lung homogenate cytokine level analysis
Harvested lungs were homogenized to determined cytokine levels. A total of 7 to 8 lungs samples per infected test and mock-infected control in each genotype group were picked randomly, processed individually and cytokine analysis performed using the Bio-Rad BioPlex system on a 23-Plex panel.
The 23-Plex panel of cytokines can be divided into its characteristic groups based on the investigated cytokines, namely, the proinflammatory and T subsets cytokines, as well as chemokines. Data of cytokines / chemokines deemed having a significant difference upon statistical analysis are presented here. Data is also presented as a fold change of the infected test over the mock-infected control samples.
The absolute amounts are also presented here as reference.
In the panel of proinflammatory cytokines, it was discovered that IL-1α, IL-1β, IL-6, GM-CSF and TNF-α had significant differences in cytokines levels when compared between IRF-4 +/+ and -/-. In IL-1α levels, there was a fold change of 1.4 in the +/+ samples (Test +/+: 37.9pg/ml; Control +/+: 26.9pg/ml) and 1.5 fold change in the -/- samples (Test -/- 48.9pg/ml; Control -/- 32.0pg/ml). Surprisingly, the expression of IL-1α in +/- samples was, however, had a reduction fold change of -1.9 (Test +/- 23.3pg/ml; Control +/- 44.7). There was no significant difference between the +/+ and -/-. In IL-1β levels, a fold change of 22.9 was observed in +/+ samples (Test 15,843.4pg/ml; Control 693pg/ml). There was a significant reduction in the fold change of +/- samples, at a fold change of 7.8 (Test 954.0pg/ml; Control 122pg/ml). A further reduction in fold change was observed in -/- samples, at a fold change of 6.4 (Test 304.0pg/ml; Control 47.8pg/ml). A similar trend was observed in fold change for IL-6 expression levels. Samples of +/+ genotype had a fold change of 286.3 (Test 206.1pg/ml; Control 0.7pg/ml), followed by 76.7 in +/- (Test 118.2pg/ml; Control
156 1.5pg/ml) and 57.2 in -/- (Test 118.2pg/ml; Control 2.1pg/ml). There was an opposite trend observed in GM-CSF and TNF-α. In GM-CSF, +/+ samples had a fold change of 2.8 (Test 131.9pg/ml; Control 47.8pg/ml). Almost at similar levels were +/- at fold change of 2.2 (Test 85.5pg/ml; Control 38.7pg/ml). However there was a highly significant increase in fold change of 7.2 in -/- samples (Test 158.4pg/ml; Control 22.1pg/ml). TNF-α had similar levels for +/+ (Fold change 2.4; Test 48.3pg/ml;
Control 20.2pg/ml) and +/- (Fold change 2.6; Test 48.7pg/ml; Control 19.1pg/ml), but a significant increase at fold change of 7.7 in -/- samples (Test 121.2pg/ml; Control 15.7pg/ml). A closely related cytokine to have proinflammatory characteristics, IL3, had a similar upward fold change trend from +/+ (Fold change 4.4; Test 2.8pg/ml;
Control 0.7pg/ml), +/- (Fold change 5.4; Test 2.5pg/ml; Control 0.5pg/ml) to -/- samples (Fold change 10.6; Test 3.6pg/ml; Control 0.3pg/ml). The presence of the IRF-4 gene in +/+ samples, heterozygousity in +/- samples, or total absence in -/- samples appeared to cause a random non-standard trend in the increase or decrease of fold change in proinflammatory cytokine levels.
Th1 cytokines of IL-2 and IFN-γ had a downward fold change trend in samples of as the presence of IRF-4 diminishes in +/+, +/- and -/- samples. In IL-2, +/+ samples had a fold change of 2.2 (Test 2.9pg/ml; Control 1.3pg/ml), whereas it was found that in +/- samples, a suppression of IL-2 levels occurred, with a reduction fold change of -1.3 (Test 1.4pg/ml; Control 1.9pg/ml) due to the InfAV infection.
Surprisingly, no IL-2 could be detectable in -/- samples in both the Mock-Infected Control as well as the Infected Test group. It appears there was a total suppression of IL-2 protein level in IRF-4 -/- mice. Another Th1 cytokine investigated in this panel was IFN-γ. It appeared to follow the IL-2 trend, where in the progressive absence of IRF-4 in the mouse model, the expression fold change of IFN- γ caused by the PR8
157 H1N1 InfAV infection decreased as well. Samples of +/+ had a fold change of 45.1 (Test 256.1pg/ml; Control 5.7pg/ml) and followed by +/-, with a fold change of 19.6 (Test 125.0pg/ml; Control 6.4pg/ml). There was a significant lower fold change in -/- samples (Fold change 7.7; Test 41.2pg/ml; Control 5.3pg/ml).
Infection with the PR8 H1N1 InfAV caused Th2 and Th17 cytokines of IL-4, IL-10 and IL-17 to respond significantly with an upward fold change trend in samples from IRF-4 +/+ to -/-. IL-4 in +/+ had a fold change of 1.9 (Test 6.8pg/ml; Control 3.5pg/ml). Samples of +/- had similar levels of fold change, at 1.7 (Test 5.3pg/ml;
Control 3.1pg/ml). However, there was a significant increase in -/- samples, with fold change of 9.4 (Test 6.9pg/ml; Control 0.7pg/ml). Similarly in IL-10 levels, samples from +/+ group had a fold change of 4.9 (Test 396.2pg/ml; Control 81.3pg/ml), following an upward trend in +/- samples, with a fold change of 6.7 (Test 397.7pg/ml;
Control 59.5pg/ml) and in -/- sample, with a fold change of 14.4 (Test 118.6pg/ml;
Control 8.2pg/ml). IL-17 secreted by Th17 cells appeared to have an increase in fold change in the absence of IRF-4, as displayed in the -/- sample. In +/+ and +/- samples, however, there was a slight suppression in IL-17 expression caused by the InfAV infection. The suppression in fold change were found to be -1.3 and -1.4, respectively (+/+; Test 4.8pg/ml; Control 6.2pg/ml) (+/-; Test 4.3pg/ml; Control 6.0pg/ml).
However in the absence of IRF-4 gene, the InfAV infection caused a fold change of 1.5 in -/- samples (Test 10.5pg/ml; Control 6.8pg/ml). From the data, it appeared that IRF-4 functions to suppress IL-17 expression during the InfAV infection. However in the absence of IRF-4, an infection would trigger an increase in IL-17.
In essence in the traditional classification of Th1 and Th2 cytokines investigated, it appears that Th1 cytokines had a lower fold change in -/- samples. The IRF-4 gene appears to cause a suppression of IL-2 and IFN-γ. In Th2 cytokines, it
158 appears that there was an augmentation of the fold change in an infection model caused by the absence of the IRF-4 gene.
For chemokines investigated by this panel, CXCL1 had a downward trend in - /- samples whereas the opposite trend was observed in CCL3 and CCL5. In +/+
samples, CXCL1 (KC) had a fold change of 13.7 (Test 1017.3pg/ml, Ctrl 74.4pg/ml).
A stepwise drop in fold change was observed in +/- samples, with a fold change of 8.6 (Test 367.9pg/ml; Ctrl 42.7pg/ml). Compared to +/+ samples, a significant drop in fold change was also observed in -/- samples, with only a fold change of 3.3 (Test 222.9pg/ml; Ctrl 68.1pg/ml). The absence of the IRF-4 gene appeared to have an effect on CCL3 (MIP-1α) during InfAv infection. In +/+ samples, a fold change of 2.0 was observed (Test 1004.6pg/ml; Ctrl 500.4pg/ml). However in +/- and -/- samples, a single or double knock out of the IRF-4 gene caused a significant increase in fold change of CCL3 during the infection with fold changes at 10.4 and 9.6, respectively [(+/-: Test 3698.6pg/ml; Ctrl 355.8pg/ml) and (-/-: Test 1290.4pg/ml; Ctrl 135.1pg/ml)]. CCL5 (RANTES) was affected positively in term of the fold change by the graduated absence of the IRF-4 gene. In +/+ samples, a fold change of 2.8 was observed (Test 2853.7pg/ml; Ctrl 1014.0pg/ml). However, a significant increase was seen in +/- and -/-, with fold changes of 4.9 and 5.6, respectively [(+/-: Test 6622.4pg/ml; Ctrl 1359.3pg/ml) and (-/-: Test 3395.3pg/ml; Ctrl 709.8pg/ml)].
CCL4 (MIP-1β) had a general increase in fold change in all three genotypes as a result of the InfAV infection. (+/+: fold change 4.1; Test 482.6pg/ml; Ctrl 116.7pg/ml) (+/-: fold change 8.1; Test 633.5; Ctrl 78.1pg/ml) (-/-: fold change 5.1;
Test 328.2pg/ml; 64.8pg/ml). However, the graduated absence of the IRF-4 gene appeared to make no significant difference to the fold changes.
159 Surprisingly, the cytokines responsible for airway inflammation IL-9 and IL- 13 appeared to be not affected by the graduated absence of the IRF-4 at the terminal phase of the InfAV infection. [IL9: (+/+: fold change 1.5; Test 33721.6pg/ml; Ctrl 22185.0pg/ml) (+/-: fold change -1.5; Test 4047.9pg/ml; Ctrl) (-/-: fold change -1.1;
Test 184.2pg/ml; Ctrl 206.0pg/ml)] [IL-13: (+/+: fold change 1.0; Test 9805.4pg/ml;
Ctrl 9461.2pg/ml) (+/-: fold change -1.3; Test 1460.4pg/ml; Ctrl 1961.8pg/ml) (-/-:
fold change 1.5; Test 92.4pg/ml; Ctrl 60.9pg/ml)].
Other cytokines examined found to be not affected by the graded absence of IRF-4 during the terminal phase of InfAV infection included IL-5 and G-CSF. [IL-5:
(+/+: fold change 15.1; Test 70.0pg/ml; Ctrl 4.6pg/ml) (-/-: fold change 21.0; Test 73.5pg/ml; Ctrl 3.5pg/ml)] [G-CSF: (+/+: fold change 101.2; Test 1334.1pg/ml; Ctrl 13.2pg/ml) (+/-: fold change 81.7; Test 903.3pg/ml; Ctrl 11.1pg/ml) (-/-: fold change 113.5; Test 2092.7pg/ml; Ctrl 18.4pg/ml)].
(A)
160 (B)
(C)
161 (D)
(E)
162 (F)
(G)
Figures 3.24 (A), (B), (C), (D), (E), (F) (G) and (H). Cytokine expression in lung homogenates of IRF-4 mice infected with 500 PFU of PR8 H1N1 InfAV, (harvested at Days 14 Post-Infection or upon weight loss reaching 25-30% of the initial weight of the mouse), as determined by Bio-Plex PROTM Cytokine Assay. (A), (B) and (C) Fold change in Lung homogenate pro-inflammatory cytokine (IL-1α, Il-1β, GM-CSF, TNF-α, IL-6 and IL-3) expression of IRF-4 mice infected with PR8 H1N1 InfAV. (D) and (E) Fold change in Lung homogenate Th1, Th2 and Th17-related cytokine (IL-2, IL-4, IL-10, IL-12p70, IL-17 and IFN-γ) expression of IRF-4 mice infected with PR8 H1N1 InfAV. (F) Fold change of IL-3 in IRF-4 mice lung homogenate. (G) Fold
163 Change in Lung homogenate chemokine (CXCL1, CCL3, CCL4 and CCL5) expression of IRF-4 mice infected with PR8 H1N1 InfAV.
Data expressed as Average Fold Change (conducted over 2 investigation rounds), derived from the ratio of absolute amounts of cytokine in PR8-infected Test IRF-4 mice (n=20/experimental round) over Control PBS-mock infected IRF-4 mice (n=10/experimental round) of same genotype. Mice found dead were excluded from study. One way ANOVA was performed to determine statistical significance (p <
0.05).