Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment

Abstract

Background: Antimicrobial peptides (AMPs) protect the host intestinal mucosa against microorganisms. Abnormal expression of defensins was shown in inflammatory bowel disease (IBD), but it is not clear whether this is a primary defect. We investigated the impact of anti-inflammatory therapy with infliximab on the mucosal gene expression of AMPs in IBD.

Methodology/principal findings: Mucosal gene expression of 81 AMPs was assessed in 61 IBD patients before and 4-6 weeks after their first infliximab infusion and in 12 control patients, using Affymetrix arrays. Quantitative real-time reverse-transcription PCR and immunohistochemistry were used to confirm microarray data. The dysregulation of many AMPs in colonic IBD in comparison with control colons was widely restored by infliximab therapy, and only DEFB1 expression remained significantly decreased after therapy in the colonic mucosa of IBD responders to infliximab. In ileal Crohn's disease (CD), expression of two neuropeptides with antimicrobial activity, PYY and CHGB, was significantly decreased before therapy compared to control ileums, and ileal PYY expression remained significantly decreased after therapy in CD responders. Expression of the downregulated AMPs before and after treatment (DEFB1 and PYY) correlated with villin 1 expression, a gut epithelial cell marker, indicating that the decrease is a consequence of epithelial damage.

Conclusions/significance: Our study shows that the dysregulation of AMPs in IBD mucosa is the consequence of inflammation, but may be responsible for perpetuation of inflammation due to ineffective clearance of microorganisms.

Figure 1. Individual microarray log2 expression values for selected AMP genes in intestinal mucosa of IBD patients before and after infliximab treatment and controls.

(A) DEFB1 (probe set 210397_at), (B) LEAP2 (probe set 1552362_a_at), (C) PYY (probe set 207080_s_at), (D) DEFA5 (probe set 207529_at), (E) DEFA6 (probe set 207814_at) and (F) DEFB4 (probe set 207356_at). Lines between 2 points represent the change in expression before and after treatment for one patient. R: responders.

Figure 2. Scatterplot representing correlation between colonic microarray log2 expression values of DEFB1 and VIL1.

R: responders; T: treatment.

Figure 3. Immunohistochemical detection of DEFB1 protein in intestinal mucosa.

Intense immunostaining of epithelial cells at the mucosal surface in normal colon (A) and ileum (B). Severely reduced DEFB1 expression in UC (C) and CDi (D) before infliximab treatment. Partial restoration of epithelial cell mass with DEFB1 staining in UC (E) and CDi (F) in responders to infliximab therapy (original magnification (OM): ×50).

Figure 4. Immunohistochemical detection of DEFA5 protein in intestinal mucosa.

No immunostaining can be seen in normal colon (A), while Paneth cells and follicle centers in Peyer's patches are immunoreactive in normal ileum (B). Mucosal defects with inflammatory cells staining for DEFA5 in untreated UC (C) and Paneth cell loss with diminished DEFA5 staining in active CDi (D). Rare DEFA5-positive metaplastic Paneth cells in colonic mucosa of UC responders after infliximab (E). No difference in DEFA5 immunoreactivity when comparing CDi responders with untreated CDi (F). (OM (A): ×100; OM (B–F): ×50).