Chapter 3. RESULTS
3.4 Analysis of major transcriptional changes in Theiler’s murine
After running the EDGE natural cubic spline analysis for the 45101 probes of the Affymetrix mouse genome 430 2.0 chips, 425 probe sets in the deep cLN and six probe sets in the spleen were differentially expressed in TMEV-infected mice over the study period at a false discovery rate of < 5.0% (q-value < 0.05). Log2 mean fold changes of the individual time points of the differentially expressed genes in TMEV-infected versus sham-infected mice were calculated.
Results of the deep cLN showed that differentially expressed probe sets matched to 387 unique official gene symbols (Table S1). 27.4% of the genes (106/387 genes) presented fold changes higher than +/- 1.5 at 14 dpi, while there were no gene transcriptions higher than +/-1.5 fold changes at later time points. In order to detect similarities in the expression patterns of the 387 differentially expressed genes of the lymph node, the log2 mean fold changes from sham-infected and TMEV-infected mice for each time point were analyzed through k-means cluster analyses, and visualized through heat maps. The resulting five statistically significant k-mean clusters (k-means clusters I to V, Figure 3-6) grouped genes with similar expression.
In order to assign a biological meaning to these genes, the functional annotation tool from DAVID was applied to all k-mean clusters, matching to a functional annotation cluster (Table 3-1).
50 Chapter 3 – Results
Figure 3-6. Expression profile of differentially expressed genes in the deep cervical lymph node.
Each row represents one of the 387 genes and each column one of the four experimental days. The log2-transformedfold changes are indicated by a color scale ranging from -2 (relative low expression in TMEV-infected mice) in green to 2 (relative high expression in TMEV-infected mice) in red. The majority of the differentially expressed genes were organized into five k-means cluster (I-V). (A) Represents k-means cluster I, showing gene up-regulation in the acute phase of TME. (B) K-means cluster II, consisted of down-regulated genes during the acute phase. (C) K-means cluster III, presented gene up-regulation at the acute phase. (D) and (E) are k-means cluster IV and V, respectively, presenting down-regulation of genes during the acute phase.
Chapter 3 – Results 51
Table 3-1. Significantly enriched pathways in the deep cervical lymph node.
K-means cluster Pathway FDR (%)
mRNA processing 0.00
Purine nucleoside triphosphate metabolic process 0.15 I
Pyrimidine nucleoside triphosphate metabolic process
0.67
II Protein metabolic process 0.26
Positive regulation of immune response 0.00
B-cell mediated immunity 0.00
Regulation of immune response 0.00
Complement activation 0.03
III
Regulation of B-cell mediated immunity 0.08
IV Not determined* 6.00
V Contractile fiber part 0.62
K-means clusters: number refers to the respective cluster of genes with a similar expression pattern in k-means cluster analysis as displayed in Figure 3-6. Pathway: from each cluster of functionally related gene ontology terms derived by DAVID, one term that subjectively gave the best description of the whole cluster was manually chosen. FDR: false discovery of significant enrichment for the respective gene ontology term. *Not determined: ontology term did not meet the cut off selection criteria of FDR < 1.0%, therefore, a specific ontology term could not be assigned to k-means cluster IV.
At this, k-means cluster I showed 156 genes (Table S2). 100% of them were up-regulated at 14 dpi, while 13.5% were up-regulated at 42 dpi, 10.3% were up-regulated at 98 dpi, and 71.2% of genes were up-regulated at 196 dpi. At 14 dpi, 7.7% of genes presented fold changes higher than +/-1.5. Genes grouped in this cluster were significantly associated (FDR < 1.0%) to gene ontology terms such as
52 Chapter 3 – Results
mRNA processing, purine and pyrimidine nucleoside triphosphate metabolic processes (Table 3-1).
K-means cluster II was composed of 89 genes (Table S3). 98.9% were down-regulated at 14 dpi, while 82.0% and 80.9% of genes showed an up-regulation at 42 and 98 dpi, respectively. A down-regulation of 56.2% of genes was observed at the last time point. The enriched gene ontology term for this cluster was significantly associated (FDR = 0.26%) to protein metabolic processes (Table 3-1).
K-means cluster III included 41 genes. All genes were up-regulated at 14 dpi with fold changes > 1.5 (Table S4), including the ten most up-regulated genes throughout the study period (Table 3-2). All of these genes were down-regulated at 42 dpi, and 95.1% of them were down-regulated at 98 dpi. At 196 dpi, 97.6% of cluster III genes were up-regulated. According to DAVID, the most enriched ontology terms (FDR < 1.0%) for this cluster were positive regulation of immune response, B-cell mediated immunity, regulation of immune response, complement activation and regulation of B-cell mediated immunity (Table 3-1).
K-means cluster IV grouped 73 genes in total (Table S5). All genes grouped within this cluster were down-regulated at 14 dpi, and 33.8% of them were presented with fold changes > 1.5. There was no enriched functional annotation cluster for this cluster from DAVID (Table 3-1), since the highest ranked ontology term did not meet the cut off selection criteria of FDR < 1.0% (FDR = 6.0%). Therefore, a specific ontology term could not be assigned to cluster IV.
From the 28 genes reported in k-means cluster V (Table S6), 32.1% belong to the ten most down-regulated genes at 14 dpi with a fold change of < -1.5 (Table 3-3).
Up-regulated genes in this cluster were observed at 42 and 98 dpi (89.3% and 92.9%, respectively). At the last time point (196 dpi), 64.3% of the genes were down-regulated. The ontology term significantly related (FDR = 0.62%) to this cluster was contractile fiber part pathway.
Chapter 3 – Results 53
Table 3-2. Official gene symbols, gene names and fold changes for the ten most up-regulated genes in the deep cervical lymph node at different time points.
Time
period Probe set ID Gene symbol Gene name Fold
change 1450792_at Tyrobp TYRO protein tyrosine kinase binding
protein
3.59
1460242_at CD55 Decay accelerating factor 1 3.43
1424007_at Gdf10 Growth differentiation factor 10 3.26
1449164_at CD68 CD68 molecule 3.11
1436996_x_at Lyz1 Lysozyme 1 2.92
1418340_at Fcer1g Fc receptor, IgE, high affinity I, gamma polypeptide
2.6
1419599_s_at Ms4a6d Membrane-spanning 4-domains, subfamily A, member 6D
2.58
1449401_at C1qc Complement component 1, q
subcomponent, c chain
2.56
1417063_at C1qb Complement component 1, q
subcomponent, beta polypeptide
2.5 14 dpi
1424754_at Ms4a7 Membrane-spanning 4-domains,
subfamily A, member 7
2.46
1439143_at A930018M24Rik Riken cDNA A930018M24 gene 1.43 1435358_at Cuedc1 Coupling of ubiquitin conjugation to
endoplasmic reticulum degradation domain containing 1
1.04
1459878_a_at A430107O13Rik Riken cDNA A430107O13 gene 1.02
1427104_at Zfp612 Zinc finger protein 612 0.99
1420136_a_at Ogfrl1 Opioid growth factor receptor-like 1 0.74 1433661_at Nlrx1 Nod-like receptor family member X1 0.72
1442715_at Tsga8 Testis specific gene A8 0.72
1439106_at Zfp462 Zinc finger protein 462 0.65
1436326_at Rora Retinoic acid receptors related orphan receptor alpha
0.64 42 dpi
1429463_at Prkaa2 Protein kinase, AMP-activated, alpha 2 catalytic subunit
0.62
54 Chapter 3 – Results
1450888_at Napb N-ethylmaleimide sensitive fusion protein attachment protein beta
0.71
1427638_at Zbtb16 Zinc finger and BTB domain containing 16
0.71
1424007_at Gdf10 Growth differentiation factor 10 0.68 1435344_at 1110029I05Rik Riken cDNA 1110029I05 gene 0.67
1427104_at Zfp612 Zinc finger protein 612 0.66
1443315_at Dmd Dystrophin, muscular dystrophy 0.65
1420136_a_at Ogfrl1 Opioid growth factor receptor-like 1 0.63
1442715_at Tsga8 Testis specific gene A8 0.62
98 dpi
1439143_at A930018M24Rik Riken cDNA A930018M24 gene 0.62 1450792_at Tyrobp TYRO protein tyrosine kinase binding
protein
1.05
1418340_at Fcer1g Fc receptor, IgE, high affinity I, gamma polypeptide
0.86
1444546_at BC027057 cDNA sequence BC027057 0.76
1449164_at CD68 CD68 molecule 0.75
1448416_at Mgp Matrix GLA protein 0.65
1436996_x_at lyz1 Lysozyme 1 0.6
1451882_a_at Fgf8 Fibroblast growth factor 8 0.59
1448183_a_at Hif1a Hypoxia inducible factor 1, alpha subunit
1432304_a_at 9030624J02Rik Riken cDNA 2610029J22 gene 0.55 Probe set ID: represents the unique probe set identifier of the Affymetrix mouse genome 430 2.0 array. Fold change: calculated as the ratio of the inverse-transformed arithmetic means of the log2
transformed expression values of TMEV-infected versus sham-infected mice. Down regulations are shown as negative reciprocal values. dpi: days post infection.
Chapter 3 – Results 55
Table 3-3. Official gene symbols, gene names and fold changes for the ten most down-regulated genes in the deep cervical lymph node at different time points.
Time period
Probe set ID Gene symbol Gene name Fold
change
1442715_at Tsga8 Testis specific gene A8 -3.76
1440635_at Palld Palladin, cytoskeletal associated protein -3.66
1442710_at Pdlim5 PDZ and LIM domain 5 -2.96
1459289_at C530030K21Rik Riken cDNA c530030k21 gene -2.79
1443315_at Dmd Dystrophin, muscular dystrophy -2.76
1439143_at A930018M24Rik Hypothetical protein 9330167e06 -2.66
1443299_at Pdlim3 PDZ and LIM domain 3 -2.63
1420136_a_at Ogfrl1 Opioid growth factor receptor-like 1 -2.6 1427638_at Zbtb16 Zinc finger and BTB domain containing 16 -2.51 14 dpi
1457338_at Ppp1r12b Protein phosphatase 1, regulatory (inhibitor) subunit 12B
-2.43
1456280_at Clspn Claspin homolog (Xenopus laevis) -1.21
1450792_at Tyrobp TYRO protein tyrosine kinase binding protein
-1.05
1418281_at Rad51 RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)
-1.04
1460242_at CD55 Decay accelerating factor 1 -1.03
1419599_s_at Ms4a6d Membrane-spanning 4-domains, subfamily A, member 6D
-0.94
1421731_a_at Fen1 Flap structure specific endonuclease 1 -0.89 1417161_at Cdk2ap2 Cyclin-dependent kinase 2 associated
protein 2
-0.85
1419872_at Csf1r Colony stimulating factor 1 receptor -0.82 1417947_at Pcna Proliferating cell nuclear antigen -0.81 42 dpi
1426133_a_at Mitd1 Microtubule interacting and transport, domain containing 1
-0.77
56 Chapter 3 – Results 1426808_at Lgals3 Lectin, galactose binding, soluble 3 -1.5 1418340_at Fcer1g Fc receptor, IgE, high affinity I, gamma
polypeptide
-1.21
1419599_s_at Ms4a6d Membrane-spanning 4-domains, subfamily A, member 6D
-1.14
1450792_at Tyrobp TYRO protein tyrosine kinase binding protein -1.14
1444546_at BC027057 cDNA SEQUENCE BC027057 -1.12
1424754_at Ms4a7 Membrane-spanning 4-domains, subfamily A, member 7
-1.08
1448183_a_at Hif1a Hypoxia inducible factor 1, alpha subunit -1.03 1417063_at C1qb Complement component 1, q
subcomponent, beta polypeptide
-1.01
1417876_at Fcgr1 Fc receptor, IgG, high affinity I -0.93
98 dpi
1439129_at Dock5 Dedicator of cytokinesis 5 -0.85
1458309_at Dip2c Disco-interacting protein 2 homolog C (Drosophila)
-0.75
1446921_at --- --- -0.75
1442473_at Etl4 Enhancer trap locus 4 -0.72
1446982_at --- --- -0.72
1446656_at Angpt1 Angiopoietin 1 -0.61
1458057_at Cald1 Caldesmon 1 -0.61
1432686_at 4833406M21Rik Riken cDNA 4833406M21 gene -0.57 1441114_at 9330156P08Rik Riken cDNA 9330156P08 gene -0.55 1449114_at Stk3 Serine/threonine kinase 3 (Ste20, yeast
homolog)
transformed expression values of TMEV-infected versus sham-infected mice. Down regulations are shown as negative reciprocal values. --- : indicates a missing value, in this case unknown gene symbol and name. dpi: days post infection.
Chapter 3 – Results 57
In the spleen, differentially expressed probe sets matched to six unique official gene symbols related to cellular processes, such as cell division and metabolism (Table 3-4). However, genes could not be grouped using the k-mean clustering analysis.
Table 3-4. Official gene symbols, gene names and fold changes for all six gene probes sets differentially expressed in the spleen at different time points.
Fold change
Probe set
ID Gene symbol Gene name 14 dpi 42 dpi 98 dpi 196 dpi
1433231_at 4933426I03Rik Riken cDNA 4933426I03 gene 2.22 0.21 1.67 -3.08 1425912_at Cep164 Centrosomal protein 164 0.23 -4.20 0.78 -1.57 1446169_at Dgcr8 Digeorge syndrome critical region
gene 8
-0.39 -1.46 2.80 0.35
1423435_at Gpc3 Glypican 3 0.88 0.22 -3.21 0.54
1431039_at Otud4 Otu domain containing 4 -0.08 -2.75 0.29 2.98 1423439_at Pck1 Phosphoenolpyruvate carboxykinase
1, cytosolic
-0.44 -3.50 -0.10 0.59
Probe set ID: represents the unique probe set identifier of the Affymetrix mouse genome 430 2.0 array. Fold change: calculated as the ratio of the inverse-transformed arithmetic means of the log2
transformed expression values of TMEV-infected versus sham-infected mice. Down regulations are shown as negative reciprocal values. dpi: days post infection.
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