Temporal genome expression profile analysis during t-cell-mediated colitis: identification of novel targets and pathways

Abstract

Background: T cells critically regulate inflammatory bowel disease (IBD), with T-cell-dependent experimental colitis models gaining favor in identifying potential pathogenic mechanisms; yet limited understanding of specific pathogenic molecules or pathways still exists.

Methods: In this study we sought to identify changes in whole genome expression profiles using the CD4CD45Rbhi T-cell transfer colitis model compared to genome expression differences from Crohn's disease (CD) tissue specimens. Colon tissue was used for histopathological and genome expression profiling analysis at 0, 2, 4, or 6 weeks after adoptive T-cell transfer.

Results: We identified 1775 genes that were significantly altered during disease progression, with 361 being progressively downregulated and 341 progressively upregulated. Gene expression changes were validated by quantitative real-time polymerase chain reaction (qRT-PCR), confirming genome expression analysis data. Differentially expressed genes were clearly related to inflammation/immune responses but also strongly associated with metabolic, chemokine signaling, Jak-STAT signaling, and angiogenesis pathways. Ingenuity network analysis revealed 25 unique network associations that were associated with functions such as antigen presentation, cell morphology, cell-to-cell signaling and interaction, as well as nervous system development and function. Moreover, many of these genes and pathways were similarly identified in CD specimens.

Conclusions: These findings reveal novel, complex, and dynamic changes in gene expression that may provide useful targets for future therapeutic approaches.

Figure 1. T cell transfer colitis-mediated tissue histopathology over time

A–D: T cell transfer-mediated histological changes illustrated by hematoxylin and eosin staining of tissue harvested at weeks 0, 2, 4 and 6, respectively. Histopathological changes progressively increase over time including neutrophil infiltrate, abnormal crypt architecture, goblet cell loss, and degree of inflammation infiltrate in the lamina propria. Magnification is 200×. Scale bars, 50µm.

Figure 2. T cell transfer colitis model progressively increases multiple disease parameters over time

The tissue histopathology score, colon length, colon weight, and vascular density were measured at weeks 0, 2, 4 and 6. A: colon weight at weeks 0, 2, 4 and 6. B: colon length at weeks 0, 2, 4 and 6. C–F: colon weight to length ration, gross colon score, tissue pathology and vascular density at discrete time points. *P<0.05 vs. week 0 (n=4) by 1-way ANOVA with Bonferroni posttest; n=4 animals per time cohort at weeks 2, 4 and 6.

Figure 3. Genesifter analysis of T cell mediated colitis array data

A: Genesifter program heat map analysis, 1,775 genes were identified from the 39,000 transcripts that significantly changed over the experimental time course at weeks 0, 2, 4 and 6. B: Principal component analysis (PCA) of the gene expression pattern using eigenvalues of covariance. Component 1 (Comp 1) represents the actual change in the gene expression profile, while component 2 (Comp2) illustrates the trend of change in gene expression. X- and y- Axis values are arbitrary units for eigenvalues and gene expression represented by dots. C: sample cluster analysis show that gene expression profiles can be divided into three groups, week 0 and week 2 in one group, which diverge from week 4 (second group) and week 6 (third group).

Figure 4. Different gene expression profile classes during the progression of T cell mediated colitis

1,775 genes for which expression is significantly changed during the time course were classified into 8 types (groups A–H) according to temporal fluctuations in gene expression levels over time. Expression profile classes are ordered from most to least abundant percentage within the total cohort of 1,775 genes.

Figure 5. Quantitative real-time PCR (qRT-PCR) validation of T cell transfer colitis microarray data

qRT-PCR validation was performed for numerous genes identified by microarray analysis. A: microarray vs. qRT-PCR expression heat map for 18 individual genes. B: correlation in gene expression between microarray (x-axis) and qRT-PCR (y-axis) changes in gene expression with r2=0.9072, P<0.0001 at the 95% confidential level, n=72. C, D, E, F: representative validation between microarray and qRT-PCR gene expression for Ifitm6, Mmp3, Ecscr and Wars respectively. *P<0.01 vs week 0 (n=4), at weeks 2, 4 and 6 (n=4).

Figure 6. Ingenuity network 1 of gene expression changes from T cell mediated colitis

Of 1,775 differentially expressed genes, a total of 25 statistically significant networks were identified. Network 1 has a score of 40 and involved 31 genes contained within that network. Major biological functions of network 1 are behavior, nervous system development and function, and gene expression. NFkB complex is centrally located within this network, showing a high degree of relationships with other molecules. Red means the expression value is greater than week 0, green indicates decreased expression compared to week 0. Symbol legend on right indicates the nature of the protein product and the type of relationship.

Figure 7. Ingenuity network 2 of gene expression changes from T cell mediated colitis

Ingenuity network 2 has a score of 38 with 30 genes involved. Major biological functions of network 2 are antigen presentation, cell morphology, cell-to-cell signaling, and interaction. IFNG is centrally located within this network, indicating a high degree of relationships between this molecule and others. Red means the expression value is greater than week 0, green indicates decreased expression compared to week 0. Symbol legend on right indicates the nature of the protein product and the type of relationship.