Role of Inflammation and the NF-κB Signaling Pathway in Hirschsprung's Disease

Abstract

Hirschsprung's disease (HSCR, incidence 1/5000 live births) is caused by the failure of neural crest-derived precursors to migrate, survive, proliferate, or differentiate during the embryonic development of the Enteric Nervous System (ENS), which could be disrupted by many factors, including inflammatory processes. The NF-κB family controls several biological processes, including inflammation, neurogenesis, and cell migration. With the aim of studying the potential role of NF-κB in HSCR, we have analyzed the expression of the NF-κB main subunits and other NF-κB-related genes by RT-qPCR in HSCR tissue samples (sub-divided into ganglionic and aganglionic segments). We found decreased gene expression of the NF-κB main subunit RELA but also of NFKBIA, TNFA, TFGBR2, and ERBB3 in the pathologic distal aganglionic segments compared to the proximal ganglionic segments. Moreover, we could also confirm the lower protein expression of RelA/p65 in the aganglionic distal segments by immunofluorescence staining. Further, we show that the expression of RelA/p65 protein in the proximal segments concurs with lymphocyte infiltration in the bowel tissue, indicating a pro-inflammatory activation of p65 in the proximal ganglionic HSCR tissue in the patients analyzed. All in all, our findings suggest that the modulation of NF-κB signaling in the neuro-enteric system does obviously contribute to the pathological effects of HSCR.

Keywords: Enteric Nervous System (ENS); Hirschsprung’s disease (HSCR); NF-κB pathway; inflammation; neuronal migration.

Figure 1

Comparison of gene expression by RT-qPCR analysis in the HSCR samples (distal segments relative to the proximal segments). All values were normalized to the average Ct values of the internal reference gene GAPDH. (a) Expression of genes encoding the neuronal markers (TUBB3 and PGP9.5), genes related to neuronal development (RET and GDNF), and genes encoding TGFB2, TGFBR2, ERBB2, and ERBB3. (b) Expression of genes encoding NF-κB subunits RELA and NF-KB1, and NF-κB-related genes, NFKBIA: Inhibitor of NF-κB (F.N. Test p = 0.0004), TNFA (F.N Test p = 0.0006), and TRAF6. (c) Fold change comparison of 2^−ΔΔCt from distal to proximal samples (Distal/Proximal).

Figure 1

Comparison of gene expression by RT-qPCR analysis in the HSCR samples (distal segments relative to the proximal segments). All values were normalized to the average Ct values of the internal reference gene GAPDH. (a) Expression of genes encoding the neuronal markers (TUBB3 and PGP9.5), genes related to neuronal development (RET and GDNF), and genes encoding TGFB2, TGFBR2, ERBB2, and ERBB3. (b) Expression of genes encoding NF-κB subunits RELA and NF-KB1, and NF-κB-related genes, NFKBIA: Inhibitor of NF-κB (F.N. Test p = 0.0004), TNFA (F.N Test p = 0.0006), and TRAF6. (c) Fold change comparison of 2^−ΔΔCt from distal to proximal samples (Distal/Proximal).

Figure 2

Immunohistochemistry of RelA/p65 and NFκB1/p50 in the proximal (mucosa, submucosa, and muscle layer) and distal (muscle layer) colon segments of a Hirschsprung patient. RelA/p65 and NFκB1/p50 were stained in red, neurons were labeled with TubβIII in green. A non-HSCR sample (muscle layer) was included as staining control. White bars: scale bar (25 μm).

Figure 3

Immunohistochemistry of RelA/p65 (a) and NFκB1/p50 (b) in the muscle layers of the proximal and the distal colon segments of a Hirschsprung patient compared to a non-HSCR tissue. RelA/p65 and NFκB1/p50 were stained in red, neurons were stained with TubβIII in green. Images obtained with confocal laser scanning microscope (Leica TCS SP8), objective magnification ×40 (25 μm), resolution (XY): 1024 × 1024. White bars: scale bar (25 μm).

Figure 3

Immunohistochemistry of RelA/p65 (a) and NFκB1/p50 (b) in the muscle layers of the proximal and the distal colon segments of a Hirschsprung patient compared to a non-HSCR tissue. RelA/p65 and NFκB1/p50 were stained in red, neurons were stained with TubβIII in green. Images obtained with confocal laser scanning microscope (Leica TCS SP8), objective magnification ×40 (25 μm), resolution (XY): 1024 × 1024. White bars: scale bar (25 μm).

Figure 4

Quantification of RelA/p65 protein expression in distal samples HSCR compared to proximal HSCR samples. * Statistical significance cut-off (p < 0.003).

Figure 5

Immunohistochemistry of RelA/p65 (red) and LCA (leukocyte common antigen) (purple) in the distal (mucosa and muscle layer) colon segments (mucosa and muscle layer) of a Hirschsprung patient. RelA/p65, Mouse mAb (1:500) and LCA, Mouse mAbs (1:100) in fast red. Images obtained with Inverted fluorescence microscope (KEYENCE, Corporation of America, Itasca, IL, USA), Objective magnification ×20 (50 μm) and ×60 (12.5 μm). Images were taken from the same area of tissue. The “Negative control” was only stained with fast red with no primary antibody.