Trinitrobenzene sulfonic acid-induced intestinal injury in neonatal mice activates transcriptional networks similar to those seen in human necrotizing enterocolitis

Abstract

Background: We have shown previously that enteral administration of 2, 4, 6-trinitrobenzene sulfonic acid in 10-d-old C57BL/6 pups produces an acute necrotizing enterocolitis with histopathological and inflammatory changes similar to human necrotizing enterocolitis (NEC). To determine whether murine neonatal 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-mediated intestinal injury could be used as a NEC model, we compared gene expression profiles of TNBS-mediated intestinal injury and NEC.

Methods: Whole-genome microarray analysis was performed on proximal colon from control and TNBS-treated pups (n = 8/group). For comparison, we downloaded human microarray data of NEC (n = 5) and surgical control (n = 4) from a public database. Data were analyzed using the software programs Partek Genomics Suite and Ingenuity Pathway Analysis.

Results: We detected extensive changes in gene expression in murine TNBS-mediated intestinal injury and human NEC. Using fold-change cut-offs of ±1.5, we identified 4,440 differentially-expressed genes (DEGs) in murine TNBS-mediated injury and 1,377 in NEC. Murine TNBS-mediated injury and NEC produced similar changes in expression of orthologous genes (r = 0.611, P < 0.001), and also activated nearly-identical biological processes and pathways. Lipopolysaccharide was top predicted upstream regulator in both the murine and human datasets.

Conclusion: Murine neonatal TNBS-mediated enterocolitis and human NEC activate nearly-identical biological processes, signaling pathways, and transcriptional networks.

Figure 1. Microarray profiles of TNBS-mediated murine neonatal intestinal injury and human NEC

(a) Principal component analysis (PCA) of microarray data from control (green) and TNBS-treated mice (red) showed distinct clustering of the two groups. N=8 pups/group. The graph is a scatter plot of the values of 3 principal components based on the correlation matrix of the total normalized array intensity data; each point represents one animal. Ellipsoids represent 95% confidence intervals of the clusters. X-, Y-, and Z-axis correspond to principal component 1 (PC1), PC2, and PC3; (b) PCA of datasets from uninflamed human neonatal intestine (green) and NEC (red) show that these two groups also aggregated in separate clusters. N=5 patients with NEC, 4 controls with uninflamed intestine.

Figure 2. Differentially-expressed genes (DEGs) in TNBS-mediated intestinal injury and human NEC

(a) DEGs in murine TNBS-mediated intestinal injury vs. control. Top: Scatterplot shows DEGs upregulated in intestinal injury (red) and control (green). Each gene is denoted by an X, the size of which is inversely proportional to the p-value. X- and Y-axis show signal intensities on a log₂ scale. Data were analyzed by ANOVA. Grey marks indicates genes that were filtered out; Bottom: Hierarchical clustering of DEGs in TNBS-mediated injury vs. control highlights the distinct gene expression profiles of the two groups. Dendrogram above the heat map depicts hierarchical clustering of the samples (controls shown as green, injury samples red). Cluster distance is based on the average distance between all the pairs of objects in the two clusters. Dendrograms for DEGs are not depicted. In the heat maps, red shows increased expression, blue indicates decreased expression, whereas grey shows no change. Expression value intensities are illustrated by color with a range of −3 to +3 on a log scale; (b) DEGs in human NEC and uninflamed neonatal intestine. Top: Scatterplot shows genes upregulated in NEC (red) and uninflamed intestine (green); Bottom: Hierarchical clustering of DEGs shows distinct profiles of human NEC and uninflamed intestine. Settings similar to panel A.

Figure 3. Murine TNBS-mediated intestinal injury and human NEC show similar changes in gene expression

Scatter-plot shows changes in the expression of orthologous genes in the human and murine intestine in NEC and TNBS-mediated intestinal injury, respectively. X-axis shows fold changes in human genes during NEC on a log₂ scale, whereas the Y-axis shows changes in their murine orthologues during TNBS-mediated intestinal injury. Most data points were clustered in the top right (upregulated in both human NEC and murine intestinal injury) or bottom left quadrants (downregulated in both datasets) of the XY plane, indicating that the expression of these orthologous genes changed similarly in murine TNBS-mediated intestinal injury and human NEC.

Figure 4. Top biological processes in murine TNBS-mediated intestinal injury and human NEC

(a) Bar diagrams show the top biological processes activated in TNBS-mediated intestinal injury (left) and human NEC (right). The predicted GO categories were ranked by enrichment score [−log (p-value)]. Single-organism processes included various cellular, metabolic, developmental, and reproductive processes. Multi-organism processes included response to bacteria or other organisms; (b) Bar diagrams show immune system processes activated in TNBS-mediated injury (left) and human NEC (right); (c) Bar diagrams show biological processes leading to cell death in TNBS-mediated injury (left) and human NEC (right).

Figure 5. Top inflammatory pathways in murine TNBS-mediated injury and human NEC

Heat maps show the expression of DEGs involved in 3 top inflammatory pathways: TNF-activated signaling, hematopoietic cell signaling, and cytokine-cytokine receptor interaction in (a) murine TNBS-mediated injury and (b) human NEC. In the heat maps, expression values are shown on a log scale with a range of −3 to +3; red boxes show upregulation, blue show downregulation, and grey indicate no change. Dendrogram above the heat map depicts hierarchical clustering of the samples (controls shown as green, injury samples red). DEGs common to both murine and human datasets are highlighted in orange. Per convention, murine gene symbols are written with the first letter in upper case and the rest in lower case. Human gene symbols are capitalized.

Figure 6. Comparison analysis of murine TNBS-mediated injury and human NEC show a high degree of congruity in the top-ranked canonical pathways, predicted upstream regulators, and disease processes

(a) Top canonical pathways in murine TNBS-mediated injury and human NEC were depicted in a heat map that was clustered hierarchically on two axes (pathways and functions vs. condition); enrichment scores [−log (p-value); range 0 to 16.9] were depicted in grayscale; (b) Heat map shows the top predicted upstream regulators (connected to DEGs through direct or indirect relationships) in murine and human datasets; (c) Heat map shows top disease processes in TNBS-mediated intestinal injury and human NEC.

Figure 7. LPS is predicted to activate similar transcriptional networks in both murine TNBS-mediated injury and human NEC

LPS-activated gene networks in (a) murine TNBS-mediated injury and (b) human NEC. Direct relationships were shown as solid arrows, whereas broken arrows show indirect relationships. Inset: prediction legend. IL = interleukin; TNF = tumor necrosis factor; IFNG = interferon-γ; NFKBIA = nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA)/inhibitor of kappa B-alpha (IκBα); NFKB1 = nuclear factor-kappa B-1; STAT = signal transducer and activator of transcription; JUN = Jun proto-oncogene, AP-1 transcription factor subunit; C/EBP-beta; TP53 = tumor protein 53; NR3C1 = nuclear receptor subfamily 3, group C, member 1/glucocorticoid receptor.