Paneth cell proteins DEFA6 and GUCA2A as tissue markers in necrotizing enterocolitis

Abstract

Previous studies suggest that Paneth cells are involved in NEC development. Defensin alpha 6 (DEFA6) and guanylate cyclase activator 2A (GUCA2A) are selective protein markers of Paneth cells. The objective was to explore DEFA6 and GUCA2A expression in intestinal tissue samples from newborn infants with and without NEC. Tissue samples from histologically intact intestine were analyzed from 70 infants: 43 underwent bowel resection due to NEC and 27 controls were operated due to conditions such as intestinal atresia, dysmotility, aganglionosis, pseudo-obstruction or volvulus. Each tissue sample was immunohistochemically stained for DEFA6 and GUCA2A. Semi-automated digital image analysis was performed to determine protein expression. Clinical data and protein expressions were compared between the groups. DEFA6 expression was lower in the NEC group (p = 0.006). Low DEFA6 correlated with risk of developing NEC in a logistic regression analysis, independently of gestational age and birth weight (OR 0.843 [CI 0.732-0.971]; p = 0.018). GUCA2A expression did not differ between the two groups.

Conclusion: Lower expression of DEFA6 together with intact GUCA2A expression indicates that NEC patients have well-defined Paneth cells but diminished defensin activity. Our results suggest that DEFA6 could be used as a biomarker for NEC.

What is known: • Previous studies of defensin activity in NEC have been inconsistent, showing that defensin levels may be increased or diminished in NEC. GUCA2A has to our knowledge never been studied in NEC.

What is new: • This study benchmarks two specific Paneth cell markers (DEFA6 and GUCA2A) and their activity in individuals with and without NEC. • The key finding is that the NEC group had a lower DEFA6 expression compared to the Controls, while the expression of GUCA2A did not differ between the groups.

Keywords: Biomarker; Necrotizing enterocolitis; Paneth cell.

Fig. 1

a–e Image processing to detect DAB staining. a Digital image of the immunohistochemically stained tissue sample in RGB color. b Selected region of interest (ROI), stretching 500 µm above the basal lamina. c Color selection with the IHC Toolbox plugin in ImageJ, representing DAB staining. d RGB image converted into an 8-bit image; lowest pixel intensity represents the highest DEFA6 expression and vice versa. e Pixel intensity is inverted, so the highest pixel intensity corresponds to the highest DEFA6 expression

Fig. 2

Equations used to define protein expression in each tissue sample. a The region of interest area (ROI) was divided by the height of mucosa (500 µm). This gives the “length” of mucosa in the selected ROI in the tissue sample. b The DAB intensity is divided with the “length” of mucosa, found in a; the result is protein expression/μm mucosa

Fig. 3

a–f Clinical data. Statistical difference when comparing the NEC group and Controls is marked with *. a Birth weight in grams. b Z-score for birth weight. c Birth weight in percentiles. d Gestational age in weeks. e Postnatal age (PNA) in days. f Postmenstrual age (PMA) in weeks

Fig. 4

a–dRepresentative tissue samples. a DEFA6 staining in an individual with NEC. b DEFA6 staining in a control. c GUCA2A staining in an individual with NEC. d GUCA2A staining in a contro

Fig. 5

a–h Protein expression per μm mucosa in NEC group (NEC), Controls, and Adults. a Expression of DEFA6 in NEC group, Controls, and Adults. b–d Scatter plot of DEFA6 correlated to gestational age (b), postnatal age (PNA) at surgery (c), and postmenstrual age (PMA) at surgery (d). * marks significant difference between NEC and Controls. NEC group is marked in orange and Controls in blue. e Expression of GUCA2A in NEC group, Controls, and Adult. f–h Scatter plot of GUCA2A correlated to gestational age (f), PNA at surgery (g), and PMA at surgery (h). NEC group is marked in orange and Controls in blue