Inflammatory Dysregulation and Persistent Epigenetic Modifications of A20 in Necrotizing Enterocolitis

Authors:
Heather Grubbs, Christopher Luschen, Catherine Hunter

Body of Abstract:
Background

The pathogenesis of necrotizing enterocolitis (NEC) centers on dysregulation of inflammation, intestinal barrier function, and cell death. A20 is an inflammatory cascade-suppressing protein which we hypothesize is decreased in active NEC. Additionally, we hypothesize that decreased expression is maintained following recovery due to epigenetic modifications of the TNFAIP3 gene promoter region which encodes for A20.

Methods

Intestinal tissue from neonates with active NEC, following NEC recovery, or without NEC was snap frozen. RNA was extracted for RT-qPCR analysis of TNFAIP3 expression. DNA was also extracted and underwent bisulfite conversion followed by PCR amplification of the TNFAIP3 promoter region and sanger sequencing. Site-specific methylation percentage was then calculated in the TNFAIP3 promoter region. Statistical significance was determined with ANOVA.

Results

Intestinal tissue from patients with no history of NEC had elevated A20 expression compared to active and recovered tissue (p=0.0039 and p=0.0009, respectively). Additionally, there was no difference in expression between expression in active and recovered tissue (p=0.9063). Three methylation sites were identified in the TNFAIP3 promoter region at positions -115, -26, and -5 from gene start with higher percent methylation in active and recovered NEC compared to controls (p=0.0006 and p=0.0005, respectively). No difference was found between active and recovered NEC percent methylation (p=0.9755).

Conclusions

The inflammatory regulator A20 was found to have decreased expression in NEC and recovered NEC tissue compared to control, emphasizing the dysregulated inflammatory response seen in NEC. Additionally, the promoter region of the TNFAIP3 gene was found to have significantly higher percent methylation at three sites in recovered and active NEC tissue compared to control. Therefore, epigenetic modifications seen in both active and recovered NEC tissue may be associated with decreased transcription of A20 and therefore impaired inflammatory regulation in NEC which persists following recovery.