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
8.12 Graphical data of invidual gene expression analysis in lung homogenate
Measured by Quantitative Real-Time PCR
Genotype Mean Comparative Expression Level (Fold Change) 2-
ΔΔCp
Corrected Mean Comparative Expression Level, If MCE < 1, Reduction Fold Change (-
1/MCE)
SD
/ ACTB / ACTB / ACTB
NS1 PR8 H1N1 WT +/+ 65040.7 65040.7 100212.6
HZ +/- 3959.7 3959.7 3978.8
KO -/- 1325840.9 1325840.9 1838114.5
IRF-4 WT +/+ 1.1 1.1 1.3
HZ +/- 0.5 -1.9 -0.9
KO -/- 0.2 -4.4 -0.8
IFN-Alpha WT +/+ 0.3 -3.6 -0.82
HZ +/- 0.7 -1.5 -1.53
KO -/- 0.2 -6.1 -0.43
IFN-Beta WT +/+ 0.1 -14.9 -0.1
HZ +/- 0.1 -8.4 -0.2
KO -/- 0.0 -32.6 -0.2
65040.7 3959.7
1325840.9
0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06 3.0E+06 3.5E+06
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) Mean Comparative Expression Level of NS1PR8 H1N1 InfAV in IRF-4 mice
lung homogenate
***
***
1.1
-1.9
-4.4 -6.0
-5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) IRF-4 Fold Change in IRF-4 mice lung homogenate
**
-3.6
-1.5
-7.0 -6.1 -6.0 -5.0 -4.0 -3.0 -2.0 -1.0 0.0
WT +/+ HZ +/- KO -/-
Mean Comprarative Expression Level
Terminal (Days 7-11 Post Infection) IFN-Alpha Fold Change in IRF-4 mice lung homogenate
-14.9
-8.4
-32.6 -35.0
-30.0 -25.0 -20.0 -15.0 -10.0 -5.0 0.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post-Infection) IFN-Beta Fold Change in IRF-4 mice lung homogenate
Figure 8.7. mRNA expression analysis of Lung homogenate by qRT-PCR (1)
342
Genotype Mean Comparative Expression Level (Fold Change) 2-
ΔΔCp
Corrected Mean Comparative Expression Level, If MCE < 1, Reduction Fold Change (-
1/MCE)
SD
/ ACTB / ACTB / ACTB
IFN-Gamma WT +/+ 89.9 89.9 44.0
HZ +/- 4.3 4.3 3.7
KO -/- 2.4 2.4 2.7
IRF-1 WT +/+ 4.1 4.1 4.0
HZ +/- 2.0 2.0 2.0
KO -/- 0.7 -1.4 -0.9
IRF-3 WT +/+ 0.5 -1.9 -0.4
HZ +/- 0.5 -1.8 -0.4
KO -/- 0.1 -7.6 -0.1
IRF-5 WT +/+ 3.0 3.0 1.3
HZ +/- 2.1 2.1 1.3
KO -/- 0.4 -2.5 -0.4
89.9
4.3 2.4
0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post-Infection) IFN-Gamma Fold Change in IRF-4 mice lung homogenate
***
***
4.1
2.0
-2.0 -1.4 0.0 2.0 4.0 6.0 8.0 10.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) IRF-1 Fold Change in IRF-4 mice lung homogenate
***
**
-1.9 -1.8
-7.6 -9.0
-8.0 -7.0 -6.0 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post-Infection) IRF-3 Fold Change in IRF-4 mice lung homogenate
***
***
3.0
2.1
-2.5 -4.0
-3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) IRF-5 Fold Change in IRF-4 mice lung homogenate
***
***
Figure 8.8. mRNA expression analysis of Lung homogenate by qRT-PCR (2)
343
Genotype Mean Comparative Expression Level (Fold Change) 2-
ΔΔCp
Corrected Mean Comparative Expression Level, If MCE < 1, Reduction Fold Change (-
1/MCE)
SD
/ ACTB / ACTB / ACTB
IRF-7 WT +/+ 18.4 18.4 14.6
HZ +/- 8.6 8.6 7.5
KO -/- 1.7 1.7 2.1
IRF-8 WT +/+ 2.0 2.0 1.3
HZ +/- 1.3 1.3 0.9
KO -/- 0.3 -3.1 -0.2
MyD88 WT +/+ 2.3 2.3 1.8
HZ +/- 1.6 1.6 1.4
KO -/- 0.3 -2.9 -0.3
NFKb WT +/+ 1.4 1.4 1.5
HZ +/- 0.8 -1.3 -0.5
KO -/- 0.3 -3.8 -0.3
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post-Infection) IRF-7 Fold Change in IRF-4 mice lung homogenate
*** *
***
2.0
1.3
-4.0 -3.1 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) IRF-8 Fold Change in IRF-4 mice lung homogenate
*** ***
2.3
1.6
-4.0 -2.9 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) MyD88 Fold Change in IRF-4 mice lung homogenate
***
1.4
-1.3
-4.0 -3.8 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0
WT +/+ HZ +/- KO -/-
Mean Comparative Expression Level
Terminal (Days 7-11 Post Infection) NFKb Fold Change in IRF-4 mice lung homogenate
***
*
***
Figure 8.9. mRNA expression analysis of Lung homogenate by qRT-PCR (3)
344 Table 8.1. Top 30 differentially expressed genes between IRF-4 +/+ WT vs +/- HZ vs -/- KO. Comparison was made between the PR8 H1N1 InfAV-infected mice of three IRF-4 genotypes (IRF4 +/+ WT, IRF-4 +/- HZ and IRF-4 -/- KO). Significance analysis was performed with 1-way ANOVA with fold change analysis and the genelist is arranged by the p-value.
ProbeID Symbol Definition p FC ([+/- Test] vs
[+/+ Test])
FC ([-/- Test] vs [+/+ Test])
FC ([-/- Test] vs [+/- Test])
3130707 Pdgfb 1.63E-05 -1.2 -1.1 1.1
5810332 Mtap1b Mus musculus microtubule-associated protein 1
B (Mtap1b), mRNA. 4.17E-05 -1.1 -1.4 -1.3
1580519 Anxa3 Mus musculus annexin A3 (Anxa3), mRNA.
4.24E-05 -1.1 1.4 1.5
4570458 Sn 4.30E-05 -1.3 1.5 1.9
6550687
Slc7a8
Mus musculus solute carrier family 7 (cationic amino acid transporter, y+ system), member 8
(Slc7a8), mRNA. 4.59E-05
-1.2 -1.3 -1.1
4290072 BC021614 4.79E-05 1.1 -2.3 -2.6
2350440 Gpr68 Mus musculus G protein-coupled receptor 68
(Gpr68), mRNA. 5.12E-05 1.1 -2.9 -3.3
610324 Cd3d
5.59E-05 1.2 -2.4 -2.9
2760131 2410042D21Rik Mus musculus RIKEN cDNA 2410042D21
gene (2410042D21Rik), mRNA. 5.82E-05 -1.1 1.2 1.3
1580431 Tpbg Mus musculus trophoblast glycoprotein (Tpbg),
mRNA. 6.04E-05 1.0 1.4 1.4
5820392 Cfl1 Mus musculus cofilin 1, non-muscle (Cfl1),
mRNA. 6.26E-05 1.1 -1.7 -1.8
540138 1500012F01Rik Mus musculus RIKEN cDNA 1500012F01
gene (1500012F01Rik), mRNA. 7.21E-05 1.1 1.5 1.4
240435 Rnf30 Mus musculus tripartite motif-containing 54
(Trim54), mRNA. 1.14E-04 -1.0 -1.6 -1.6
4850382 Sfrs3 Mus musculus splicing factor, arginine/serine-
rich 3 (SRp20) (Sfrs3), mRNA. 1.16E-04 -1.0 1.5 1.6
10411 Sytl1 Mus musculus synaptotagmin-like 1 (Sytl1),
mRNA. 1.24E-04 1.1 -2.0 -2.1
3310274 1700007G11Rik Mus musculus RIKEN cDNA 1700007G11
gene (1700007G11Rik), mRNA. 1.38E-04 -1.0 1.6 1.6
20201 Akap7 Mus musculus A kinase (PRKA) anchor
protein 7 (Akap7), mRNA. 1.47E-04 1.0 1.3 1.3
3310026 Clec3b Mus musculus C-type lectin domain family 3,
member b (Clec3b), mRNA. 1.51E-04 -1.1 -1.7 -1.6
1690079 Cd8b
1.53E-04 1.1 -4.4 -5.0
430670 Foxa1
1.65E-04 -1.1 2.0 2.2
1070307 Selpl Mus musculus selectin, platelet (p-selectin)
ligand (Selplg), mRNA. 1.86E-04 1.0 -1.5 -1.5
670204 Ian3 1.89E-04 1.2 -1.7 -2.0
2230341 A930008G19Rik 2.02E-04 -1.1 -1.6 -1.5
2690292
Mapk9 Mus musculus mitogen activated protein kinase 9 (Mapk9), transcript variant 1, mRNA.
2.05E-04
-1.1 1.1 1.1
6520725 Gpr43 Mus musculus free fatty acid receptor 2
(Ffar2), mRNA. 2.23E-04 1.1 2.1 2.0
5220725 A730042J05Rik
2.55E-04 1.2 -1.6 -1.9
2450239 Icos Mus musculus inducible T-cell co-stimulator
(Icos), mRNA. 2.76E-04 -1.1 -5.7 -5.0
4050609 Vti1a 2.88E-04 1.1 -1.5 -1.6
630678 Mta3 Mus musculus metastasis associated 3 (Mta3),
mRNA. 2.90E-04 1.1 -1.4 -1.5
990438 Cotl1 Mus musculus coactosin-like 1 (Dictyostelium)
(Cotl1), mRNA. 3.02E-04 -1.0 -1.8 -1.7
2100196 Fnbp1 Mus musculus formin binding protein 1 (Fnbp1),
transcript variant 1, mRNA. 3.05E-04 -1.1 -1.5 -1.4
345 Table 8.2. Fold Change of significant and non-signifcant differentially expressed genes between IRF-4 +/+ WT vs IRF-4 +/- HZ vs IRF-4 -/- KO, concurrently investigated for Cytokine Analysis by BioPlex and mRNA expression by qRT-PCR, arranged alphabetically by gene name.
ProbeID Symbol Definition p FC ([+/- Test] vs [+/+
Test])
FC ([-/- Test] vs [+/+
Test])
FC ([-/- Test] vs [+/- Test]) 1190241 Ccl1
Mus musculus chemokine (C-C motif) ligand 1 (Ccl1),
mRNA. 0.04464356 -1.1 -1.3 -1.2
110112 Ccl2 0.3512405 -1.3 1.5 1.9
2760047 Ccl3 0.9349415 1.0 1.2 1.1
6940184 Ccl4 0.03324678 -1.4 -2.0 -1.4
7150500 Ccl4 0.6306312 -1.3 -1.5 -1.1
1690768 Ccl5
Mus musculus chemokine (C-C motif) ligand 5 (Ccl5),
mRNA. 0.5489506 -1.1 1.0 1.2
2480296 Ccl8
Mus musculus chemokine (C-C motif) ligand 8 (Ccl8),
mRNA. 0.61776745 1.2 -1.0 -1.2
3610082 Cxcl1
Mus musculus chemokine (C-X-C motif) ligand 1 (Cxcl1),
mRNA. 0.01450484 -1.5 1.8 2.6
3140209 Cxcl10
Mus musculus chemokine (C-X-C motif) ligand 10 (Cxcl10),
mRNA. 0.17187747 -1.2 2.8 3.3
7550112 Cxcl12 0.00694924 -1.2 1.6 1.9
1030025 Cxcl12 0.009921 1.0 1.7 1.7
20088 Cxcl12
Mus musculus chemokine (C-X-C motif) ligand 12 (Cxcl12),
transcript variant 3, mRNA. 0.11567923 -1.4 -1.3 1.1
7550112 Cxcl12 0.00694924 -1.2 1.6 1.9
3060040 Cxcl13 0.00224982 -1.2 3.6 4.3
3060040 Cxcl13 0.00224982 -1.2 3.6 4.3
150746 Cxcl14 0.7883063 -1.0 -1.1 -1.0
3520349 Cxcl14 0.8685516 1.1 1.0 -1.1
520403 Cxcl15 0.13632338 1.3 3.6 2.8
630274 Cxcl16 0.0301476 -1.2 -1.3 -1.0
5390639 Cxcl2
Mus musculus chemokine (C-X-C motif) ligand 2 (Cxcl2),
mRNA. 0.02922661 1.7 1.9 1.2
4040524 Cxcl4
Mus musculus chemokine (C-X-C motif) ligand 4 (Cxcl4),
mRNA. 0.1228403 1.0 1.4 1.4
10598 Cxcl9 0.3372854 -1.5 -1.1 1.4
4780072 Ifna1 Mus musculus interferon alpha 1 (Ifna1), mRNA. 0.02945433 1.1 -1.4 -1.5
6280592 Ifnb1 Mus musculus interferon beta 1, fibroblast (Ifnb1), mRNA. 0.00609144 -1.2 2.1 2.5
6280477 Ifng Mus musculus interferon gamma (Ifng), mRNA. 0.03848248 -1.2 -1.8 -1.5
2850092 Ifng Mus musculus interferon gamma (Ifng), mRNA. 0.22179013 -1.0 -1.7 -1.6
3420139 Il1 0.00759154 1.0 1.4 1.4
1260632 Il10 Mus musculus interleukin 10 (Il10), mRNA. 0.00768979 -1.1 -2.2 -2.0
2070468 Il12a 0.14338186 1.2 -1.1 -1.3
5270719 Il13 Mus musculus interleukin 13 (Il13), mRNA. 0.2135997 -1.0 -1.3 -1.3
3420754 Il1b 0.00457027 -1.3 3.3 4.3
6020224 Il4i1 Mus musculus interleukin 4 induced 1 (Il4i1), mRNA. 0.36216855 1.2 1.3 1.2
3400288 Irf1 Mus musculus interferon regulatory factor 1 (Irf1), mRNA. 0.02809242 -1.5 -1.2 1.2
6660634 Irf1 Mus musculus interferon regulatory factor 1 (Irf1), mRNA. 0.14142697 -1.3 -1.4 -1.1
3360138 Irf1 Mus musculus interferon regulatory factor 1 (Irf1), mRNA. 0.3168505 -1.3 -1.1 1.2
1190088 Irf1 Mus musculus interferon regulatory factor 1 (Irf1), mRNA. 0.35852087 -1.1 1.0 1.1
4250291 Irf3 0.13625193 1.1 1.2 1.2
4280193 Irf3 0.14759001 1.0 1.1 1.1
1010397 Irf4 Mus musculus interferon regulatory factor 4 (Irf4), mRNA. 0.08393885 1.6 1.1 -1.5
3140646 Irf5 Mus musculus interferon regulatory factor 5 (Irf5), mRNA. 0.0900343 -1.2 -1.1 1.1
6590653 Irf7 0.05245507 -1.4 1.2 1.7
4810286 Myd88
Mus musculus myeloid differentiation primary response
gene 88 (Myd88), mRNA. 0.21271646 -1.3 -1.0 1.2
2070014 Nfkb2 0.9164811 1.0 1.1 1.0
4050626 Tnf Mus musculus tumor necrosis factor (Tnf), mRNA. 0.13050207 -1.4 -1.0 1.4