Neutral endopeptidase (EC 3.4.24.11) downregulates the onset of intestinal inflammation in the nematode infected mouse

Abstract

Background and aims: Substance P (SP) release from sensory nerves induces neurogenic inflammation. Neutral endopeptidase (NEP) degrades SP, thereby limiting its proinflammatory effects. Intestinal inflammation following Trichinella spiralis infection markedly downregulates NEP, resulting in diminished SP degradation, with unknown functional consequences. We hypothesised that diminished expression of NEP would exacerbate T spiralis induced enteritis.

Methods: NEP knockout (NEP-/-) and wild-type (NEP+/+) mice were infected with T spiralis and studied at 6, 12, 24, and 48 hours post infection (PI). Tissue inflammation was quantified by computerised cell counting and myeloperoxidase activity (MPO). The leucocyte adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), and SP were assessed by immunohistochemistry.

Results: Before infection, the lack of NEP was not associated with changes in mucosal cellularity or MPO activity. Twelve hours PI, NEP-/- mice showed a 2.5-fold increase in MPO activity at a time when values in NEP+/+ mice were still within normal limits. MPO activity and cellularity peaked at 24 hours PI. This was accompanied by increased staining for both ICAM-1 and SP in NEP-/- mice. Infusion of rhNEP to NEP-/- mice significantly reduced MPO activity 24 hours PI.

Conclusions: These findings demonstrate that NEP downregulates the early onset of nematode intestinal inflammation and that increased bioavailability of SP and overexpression of ICAM-1 in NEP-/- mice likely play a role in the earlier onset of intestinal inflammation.

Figure 1

Degradation of substance P (SP) and bradykinin (BK) by intestinal membranes from wild-type (NEP+/+) and knockout (NEP−/−) neutral endopeptidase mice. (A) High pressure liquid chromatograms of the degradation products after 120 minute incubation period with or without thiorphan. (B) Time course of degradation of SP and BK with or without thiorphan, presented as per cent degradation compared with controls in which membranes were omitted. In NEP+/+ mice, SP and BK were rapidly degraded and the NEP inhibitor thiorphan reduced degradation of SP by 43% and BK by 70% (determined after 120 minute incubation period of peptides with membranes). Membranes from NEP−/− mice degraded SP 43% more slowly and BK 33% more slowly than membranes from NEP+/+ mice (determined after 120 minute incubation period of peptides with membranes). The results in (A) are representative chromatograms from 4–6 analyses. The results in (B) are the mean of n = 2 or 3 experiments, each in duplicate. OD, optical density.

Figure 2

Extravasation of Evans blue in the jejunum. (A) Basal, substance P (SP), and bradykinin (BK) stimulated extravasation of Evans blue in wild-type (NEP+/+) and knockout (NEP−/−) neutral endopeptidase mice (mean (SEM) (n)). *p<0.05 compared with basal (ANOVA, Bonferroni t test). (B) Effects of thiorphan (Thio) and phosphoramidon (Phos) on basal extravasation of Evans blue in NEP+/+ mice, and effects of human recombinant NEP (rhNEP), SR140333, and HOE140 on basal extravasation in NEP−/− mice (mean (SEM) (n)). *p<0.05 compared with basal for wild-type mice; †p<0.05 compared with basal for NEP knockout mice (ANOVA, Bonferroni t test).

Figure 3

Quantification of lamina propria and submucosal cells at the indicated time points after T spiralis infection in wild-type (NEP+/+) and knockout (NEP−/−) neutral endopeptidase mice. Mucosal cellularity in NEP−/− mice significantly increased over non-infected NEP−/− mice 24 and 48 hours post infection. No changes in total cellularity were identified at the same time points post infection in NEP+/+ mice. Values are means (SEM). *Significant difference versus NEP−/− non- infected mice (p<0.05); significant difference versus NEP+/+ infected mice (48 hours) (p<0.05).

Figure 4

Myeloperoxidase (MPO) activity from the jejunum at the indicated time points after T spiralis infection in wild-type (NEP+/+) and knockout (NEP−/−) neutral endopeptidase mice. After infection, MPO activity increased significantly earlier (12 hours) and with a higher magnitude in NEP−/− mice in comparison with NEP+/+ mice. Values are means (SEM). **Significant difference versus non-infected mice (p<0.01); ††significant difference versus NEP+/+ studied at the same time post infection (p<0.01).

Figure 5

Myeloperoxidase (MPO) activity from the jejunum of non-infected and T spiralis infected (+24 hours) knockout (NEP−/−) neutral endopeptidase mice continuously treated with recombinant human NEP (rhNRP) or with non-relevant peptide as a control. rhNEP significantly reduced the increase in MPO activity after infection. Values are means (SEM). *Significant difference versus NEP−/− non-infected mice (p<0.05); †significant difference versus NEP−/− infected mice treated with non-relevant peptide.

Figure 6

Representative photomicrographs of jejunum showing substance P (SP) immunoreactivity in neuronal cell bodies and nerve processes before and after T spiralis infection. Under basal conditions, SP immunoreactive neural pattern and staining intensity was comparable in wild-type (NEP+/+) (A) and knockout (NEP−/−) (B) neutral endopeptidase mice. T spiralis infection (24 hours) evoked an increase in SP staining intensity in NEP+/+ mice (C). However, the increase in both SP density and staining intensity after infection in NEP−/− mice (D) was markedly enhanced. Calibration bar 25 µm.

Figure 7

Representative photomicrographs showing intercellular adhesion molecule 1 (ICAM-1) immunoreactive pattern throughout the jejunal wall before and after T spiralis infection. Under basal conditions, ICAM-1 was weakly expressed in the submucosal and mucosal layer in wild-type (NEP+/+) neutral endopeptidase mice (A), this pattern showed a detectable increase in knockout (NEP−/−) mice (B). Following T spiralis infection (24 hours), no evident increase in ICAM-1 immunoreactivity was appreciated in NEP+/+ infected mice (C). However, NEP−/− infected (24 hours) mice showed markedly enhanced ICAM-1 immunoreactivity both at the submucosal and mucosal levels (D). Calibration bar 25 μm.