Gene expression profiling in non-human primate jejunum, ileum and colon after total-body irradiation: a comparative study of segment-specific molecular and cellular responses

Abstract

Background: Although extensive studies have investigated radiation-induced injuries in particular gastrointestinal (GI) segments, a systematic comparison among the different segments on the basis of mode, magnitude and mechanism has not been reported. Here, a comparative study of segment-specific molecular and cellular responses was performed on jejunum, ileum and colon obtained at three time points (4, 7 and 12 days after irradiation) from non-human primate (Rhesus macaque) models exposed to 6.7 Gy or 7.4 Gy total body irradiation (TBI).

Results: Pathway analysis on the gene expression profiles identified radiation-induced time-, dose- and segment-dependent activation of tumor necrosis factor α (TNFα) cascade, tight junction, apoptosis, cell cycle control/DNA damage repair and coagulation system signaling. Activation of these signaling pathways suggests that colon sustained the severest mucosal barrier disruption and inflammation, and jejunum the greatest DNA damage, apoptosis and endothelial dysfunction. These more pronounced alterations correlate with the high incidence of macroscopic pathologies that are observed in the colon after TBI. Compared to colon and jejunum, ileum was resistant to radiation injury. In addition to the identification a marked increase of TNFα cascade, this study also identified radiation induced strikingly up-regulated tight junction gene CLDN2 (196-fold after 7.4-Gy TBI), matrix degradation genes such as MMP7 (increased 11- and 41-fold after 6.7-Gy and 7.4-Gy TBI), and anoikis mediated gene EDA2R that mediate mucosal shedding and barrier disruption.

Conclusions: This is the first systematic comparative study of the molecular and cellular responses to radiation injury in jejunum, ileum and colon. The strongest activation of TNFα cascades and the striking up-regulation of its down-stream matrix-dissociated genes suggest that TNFα modulation could be a target for mitigating radiation-induced mucosal barrier disruption.

Fig. 1

Validation of selected genes in colon by real-time RT-qPCR (a and b). Expression of the selected genes at the different time points after TBI was plotted by microarray normalized signals (grey bars). The relative expression of the same selected genes was normalized by GAPDH gene at the different time points by real-time RT-qPCR (black bars). The opposite direction of HBB and HBA2 demonstrates their down-regulation after radiation (panel a). Statistics analysis was performed to estimate the consistency between RT-qPCR results and the microarray data (panel b)

Fig. 2

TBI at 6.7 Gy induced significant activation of TNFα cascades in colon tissue at day 4 (z-score, 6.7; P-value = 3.85e-23). An absolute z-score of ≥ 2 is considered significant

Fig. 3

Comparative analysis of the dynamic activation of upstream regulators in jejunum, ileum and colon after 6.7 Gy and 7.4 Gy TBI. An absolute z-score of ≥ 2 is considered significant. An upstream regulator was considered increased (activated) if the z-score was ≥ 2 (orange), and decreased (inhibited) if the z-score was ≤ −2 (blue)

Fig. 4

Comparative analysis demonstrates that the tight junction signaling pathway is only activated in colon on day 4 after 7.4 Gy TBI (panel a). Pathways with a –log value (P-value) above the threshold (dashed line) were significantly activated. Altered transcripts involved in tight junction signaling in jejunum, ileum and colon at the different time points after 6.7 Gy and 7.4 Gy TBI (panel b). The font color of A1 to A6 represents segment of jejunum (black), ileum (red) and colon (blue). A1 to A6 represent the time point of days 4, 7 and 12 after 6.7 Gy and 7.4 Gy TBI

Fig. 5

The various immune response signaling pathways activated in jejunum on day 4 after 6.7 Gy TBI were significantly suppressed by day 7. Stacked bar charts demonstrate IPA-generated activated pathways after irradiation. The width of a bar indicates the percentage of transcripts that changed in the particular pathway (red bar: up-regulated; green bar: down-regulated). Pathways with a P-value (yellow square) above the threshold (dashed line) were significantly regulated (panel a). A comparative analysis of granulocyte and agranulocyte adhesion signaling and diapedesis in jejunum, ileum and colon on days 4, 7 and 12 after 6.7 Gy and 7.4 Gy TBI revealed pathways that were significantly activated above the threshold –Log value (P-value) (dashed line) (panel b)

Fig. 6

TBI induced significant activation of the AhR-mediated apoptosis signaling pathway in jejunum, ileum and colon at the early time point of 4 days after TBI. Pathways with a –log value (P-value) above the threshold (dashed line) were significantly activated (panel a). Genes involved in AhR signaling altered at the different time points after 6.7 Gy and 7.4 Gy TBI (panel b)

Fig. 7

Activation of cell cycle control and DNA damage repair signaling pathway in jejunum, ileum and colon after TBI. Pathways with a –log value (P-value) above the threshold (dashed line) were significantly activated (panel a). Genes involved in G2/M DNA damage checkpoint regulation altered at the different time points after 6.7 Gy and 7.4 Gy TBI (panel b)

Fig. 8

TBI induced significant activation of the coagulation system in jejunum, but not in ileum or colon. Pathways with a –log value (P-value) above the threshold (dashed line) were significantly regulated (panel a). Genes involved in the coagulation system altered at the different time points after 6.7 Gy and 7.4 Gy TBI (panel b)

Fig. 9

Up-regulation of EDA2R and matrix-dissociated transcripts in jejunum, ileum and colon after 6.7 Gy and 7.4 Gy TBI

Fig. 10

Immunohistochemistry in colon tissue sections for TNFα protein. In unirradiated colon, TNFα protein is weakly distributed across the section; however, a streak of some visible signals can be seen beneath the intact mucosal epithelial lining (panels a and b, solid white arrow). At day 4 after 7.4-Gy TBI, TNFα staining in the interstitial space from serosa to mucosa and submucosa of the colon exhibits significantly increased TNFα staining. Note the strong streak of TNFα staining in the mucosal region close to the epithelial barrier, forming an interface between swollen/disrupted cells and normal-shaped cells (panels c and d, open arrows)