gene-deficient mouse
3.4.3 Colon gene expression
3.4.3.2 Colon gene expression between mouse genotypes fed the OA diet Approximately twice as many genes were differentially expressed in the colon of 12-
Table 3.3 Numbers of differentially expressed genes in the colon of Il10-/- mice and C57BL/6J mice fed AIN-76A diets, either unmodified, or enriched with oleic acid (OA) or eicosapentaenoic acid (EPA). Samples were obtained at 9 or 12 weeks of age and the effects of the EPA diet compared to the AIN-76A and OA control diets. Genes passing a fold-change (≥ |1.5|) and p-value (≤ 0.005) cut-off were considered differentially expressed. Data represent four biological replicates per group, with the exception of C57BL/6J mice fed the AIN-76A diet where n = 3.
Contrast mRNA transcript levels
1 Uploaded into Ingenuity Pathway Analysis
Increased Decreased Analysis-ready molecules2
Genotype comparison (Il10-/- relative to C57)
9 weeks OA 900 476 938
EPA 1251 1269 820 (FC = 1.8 and P = 0.001) 12 weeks AIN-76A 1455 1163 932 (FC = 1.8 and P = 0.001) OA 1558 1339 816 (FC = 2 and P = 0.001) EPA 1127 975 975 (FC = 1.5 and P = 0.001)
Diet comparison (EPA relative to OA)
9 weeks C57 22 2 13
Il10-/- 9 17 26
12 weeks C57 113 57 133
Il10-/- 14 102 95
(1) FC ≥ |1.5| and p-value ≤ 0.005; genes may be represented with more than one probe on the array; (2) Reducing genes to 1000 genes for pathway analysis by applying more stringent FC and p-value as recommended in the IPA handbook.
(REG3G), indoleamine 2,3-dioxygenase 1 (IDO1), S100 calcium binding proteins A8 and A9 (S100A8 and S100A9), and chemokine (C-X-C motif) ligands 6, 9, and 10 (CXCL6,
CXCL9 and CXCL10). The expression of these genes was increased in Il10-/- mice
compared to C57BL/6J mice, with FC ranging from 3.7 to 23.2, irrespective of age (Table
3.4). Generally, the colon gene expression profiles of Il10-/- mice fed the OA diet at 9 and
12 weeks of age were similar (Pearson correlation coefficient = 0.98).
The ten highest p-value-ranked biological functions associated with the
differentially expressed genes were similar between 9-week-old Il10-/- mice and 12-week-
old Il10-/- mice fed the OA diet (vs. C57BL/6J mice) and mostly related to the cellular
immune system (Table 3.5). These were for example “Activation” and “Quantity” of blood cells and “Activation”, “Migration”, “Movement”, and “Quantity” of leukocytes, with the functions predicted to be increased in activity (activation z-score ≥ 2). At 9 weeks of age, the predictions for these 10 functions were based on 367 unique genes. Merging the genes into a biological interaction network (not shown due to the large number of genes) identified six central transcription regulators that affected the expression of many
genes in the network. These were the interferon regulatory factor 1 and 8 (IRF1 and
IRF8), TNF, Spi-1 proto-oncogene (SPI1), PPARA, and spleen tyrosine kinase (SYK). The mRNA expression levels of these genes, except for PPARA, were increased by
approximately 1.5 to 3.4-fold in Il10-/- mice compared to C57BL/6J mice, both fed the
OA diet, indicating important roles in the regulation of the immune response in the colon
of Il10-/- mice at 9 weeks of age.
At 12 weeks of age, the predictions for these ten functions were based on 376 unique genes. Merging the genes into a biological interaction network (not shown due to the large number of genes) identified five central transcription regulators that affected the expression of many genes in the network. These were IRF1, GATA binding protein 3
(GATA3), PPARA, FBJ murine osteosarcoma viral oncogene homolog (FOS), and
lymphocyte-specific protein tyrosine kinase (LCK). The mRNA levels of these genes,
except for PPARA, were increased by approximately 2.5-fold in Il10-/- mice compared to
C57BL/6J mice, both fed the OA diet, indicating important roles in the regulation of the
Table 3.4 Genes with the highest fold-changes (FC) between the colon from Il10-/- mice and C57BL/6J mice fed the oleic acid (OA) diet (9 and 12 weeks of age). Ten genes with the highest positive FC and ten genes with the highest negative FC between 9-week-old Il10-/- mice vs. C57BL/6J mice and between 12-week-old Il10-/- mice vs. C57BL/6J mice are shown. Data represent four biological replicates per group.
Gene name (Gene ID) Il10
-/- vs. C57BL/6J
9 weeks of age 12 weeks of age
Genes with decreased expression
Cytochrome P450, family 4, subfamily B, polypeptide 1
(CYP4B1)
-10.2 -16.6
Sulfotransferase family, cytosolic, 1C, member 2 (SULT1C2) -5.6 -13.6
Galactose-3-O-sulfotransferase 2 (GAL3ST2) -6.8 -12.5
Adiponectin, C1Q and collagen domain containing (ADIPOQ) n.s. -11.9
Carbonic anhydrase III, muscle specific (CA3) n.s. -11.9
Cytochrome P450, family 2, subfamily C, polypeptide 18
(CYP2C18)
n.s. -11.0
Complement factor D (adipsin) (CFD) n.s. -8.8
Chromogranin B (secretogranin 1) (CHGB) -2.9 -7.6
Chromogranin A (parathyroid secretory protein 1) (CHGA) -3.1 -7.0
Resistin (RETN) n.s. -6.8
Colipase, pancreatic (CLPS) -4.4 -5.6
Transient receptor potential cation channel, subfamily M, member 6 (TRPM6)
-3.6 -5.1
Protease, serine, 23 (PRSS23) -4.2 -4.6
Neurexophilin and PC-esterase domain family, member 2
(NXPE2)
-5.3 -3.6
Metallothionein 1 (Mt1) -4.5 n.s.
Metallothionein 2 (Mt2) -5.3 n.s.
Phosphatidylinositol-4-phosphate 5-kinase, type I, alpha
(PIP5K1A)
-4.9 n.s.
Genes with increased expression
Indoleamine 2,3-dioxygenase 1 (IDO1) 6.7 23.2
S100 calcium binding protein A9 (S100A9) 5.6 22.2
Chemokine (C-X-C motif) ligand 9 (CXCL9) 11.5 19.5
Chemokine (C-X-C motif) ligand 10 (CXCL10) 8.1 13.8
Chemokine (C-X-C motif) ligand 6 (CXCL6) 4.0 13.0
Granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) (GZMA)
8.2 12.9
Regenerating islet-derived 3 gamma (REG3G) 14.6 12.8
Table 3.5 Most significantly affected biological functions in the colon of Il10-/- mice compared to C57BL/6J mice fed the oleic acid (OA) diet (9 and 12 weeks of age). Functions with an activation z-score
< |2| were excluded and biological functions limited to show the ten highest p-value-ranked functions. “# genes” indicates the number of genes associated with the biological function and individual genes may be represented in more than one function. Data represent four biological replicates per group.
Functions annotation P-value # genes Predicted activation
state (activation z-score) Category
9 weeks of age
Blood cells Activation 1.73E-33 134 Increased (+4.9) 2, 5 Quantity 1.42E-38 182 Increased (+2.6) 2 3 Leukocytes Activation 1.86E-33 128 Increased (+4.8) 2, 5, 6, 7
Migration 4.43E-37 159 Increased (+4.6) 6, 13 Movement 4.67E-33 139 Increased (+4.4) 2, 6, 13 Quantity 2.61E-41 173 Increased (+2.7) 2, 3 Lymphocytes Quantity 5.51E-34 136 Increased (+3.6) 2, 3 Mononuclear leukocytes Quantity 8.61E-34 139 Increased (+3.7) 2, 3 Glucose metabolism disorder 6.00E-44 205 Increased (+3.4) 12 Infection of mammalia 1.27E-38 96 Decreased (-5.3) 14
12 weeks of age
Inflammatory response 9.17E-42 142 Increased (+4.0) 7
Cells Activation 1.31E-41 162 Increased (+4.1) 5
Blood cells Activation 1.43E-41 137 Increased (+4.7) 2, 5 Quantity 2.33E-47 183 Increased (+3.2) 2, 3 Leukocytes Activation 9.06E-43 133 Increased (+4.7) 2, 5, 6, 7
Migration 3.93E-53 172 Increased (+5.0) 6, 13 Movement 9.74E-47 150 Increased (+4.8) 6, 13 Quantity 2.66E-51 176 Increased (+3.1) 2, 3 Glucose metabolism disorder 1.20E-54 207 Increased (+3.9) 12 Infection of mammalia 7.77E-48 101 Decreased (-5.2) 14
Categories: 1 Cellular Function and Maintenance; 2 Hematological System Development and Function; 3 Tissue Morphology; 4 Cellular Growth and Proliferation; 5 Cell-To-Cell Signalling and Interaction; 6 Immune Cell Trafficking; 7 Inflammatory Response; 8 Cell-mediated Immune Response; 9 Cellular Development; 10 Hematopoiesis; 11 Lymphoid Tissue Structure and Development; 12 Metabolic Disease; 13 Cellular Movement; 14 Infectious Disease; 15 Tissue Development; 16 Cell Morphology; 17 Cell Death and Survival; 18 Cellular Compromise; 19 Lipid Metabolism; 20 Small Molecules Biochemistry; 21 Organismal Survival; 22 Molecular Transport; 23 Organismal Development; 24 Cellular Assembly and Organisation; 25 Humoral Immune Response; 26 Protein Synthesis; 27 Immunological Disease; 28 Cardiovascular Disease.
GSEA supported the IPA results, showing that irrespective of age, the colon
transcriptomic profile for Il10-/- mice fed the OA diet was associated with immune-related
KEGG pathway gene sets compared to C57BL/6J mice fed the same diet (P ≤ 0.01 (Table 3.6)). There was a good concordance of affected KEGG pathway gene sets between 9-
week-old Il10-/- mice and 12-week-old Il10-/- mice, with 43 sets in common (Table 3.6).
For example, within the biological process Organismal systems (function Immune system), the KEGG pathways Antigen processing and presentation and signalling pathways against bacteria and other pathogens such as Intestinal immune network for IgA production, Cytosolic DNA-sensing pathway and Natural killer cell mediated
cytotoxicity, were increased in expression in the colon of Il10-/- mice (all P ≤ 0.01 vs.
C57BL/6J mice both fed the OA diet (Table 3.6)).
GSEA indicated decreased PPAR signalling in the colon of Il10-/- mice fed the
OA diet (vs. C57BL/6J mice fed the same diet), but differences were dependent on age of mice (Table 3.6). At 9 weeks of age, PPARA mRNA expression levels were decreased 2.4-fold and at 12 weeks of age, a 4.3-fold decrease was detected (P ≤ 0.005). As a gene set, the KEGG pathway PPAR signalling did not pass the significance criteria for
differential expression in 9-week-old Il10-/- mice (P = 0.05, with 25% genes decreased).
In 12-week-old Il10-/- mice, almost twice as many genes were affected in the PPAR
signalling pathway (approximately 47% decreased (P = 0.003)), indicating a loss of
functionality of PPAR signalling associated with the development of colitis in Il10-/- mice
(Table 3.6).
Large increases in mRNA expression levels of the gene IDO1 which catalyses the metabolism of tryptophan to kynurenine (Figure 3.6) has previously been observed in
the large intestine of Il10-/- mice [6, 342] and was also shown in the current experiment,
increased (14%) in expression in Il10-/- mice compared to C57BL/6J mice both fed the
OA diet (P ≤ 0.01 (Table 3.6)).
Within the biological processes Organismal systems and Metabolism, the expression of KEGG pathways associated with the digestion, absorption and metabolism
of several nutrients was reduced in Il10-/- mice fed the OA diet compared to C57BL/6J
mice fed the same diet (Table 3.6). These included for example, amino acids, carbohydrates, lipids, and cofactors and vitamins. This reduction in metabolism-related processes may indicate loss of functionality of biochemical pathways in the colon of