Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients

Abstract

Background & aims: The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC).

Method: Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis.

Results: Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants.

Conclusion: Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS.

Fig 1. Nerve activation evoked by mucosal biopsy supernatants.

(A, left panel) Mucosal biopsy supernatants from patients with irritable bowel syndrome (14 IBS) and patients with ulcerative colitis in remission (12 UC), but not from healthy controls (7 HC), caused nerve activation as indicated by a significantly increased neuroindex (product of spike frequency and % of responding neurons) (Dunn´s method). (A, right panel) the broad spectrum serine protease inhibitor FUT-175 reduced the neuroindex evoked by UC supernatants (Wilcoxon signed rank test for paired data). (B) Spike discharge in human submucous neurons after pressure application of supernatants (duration indicated by the bars below the traces) from IBS (upper panel) and UC patients (lower panel) before and during incubation with the PAR1 receptor antagonist SCH79797 (10μM). While SCH79797 blocked spike discharge in response to IBS supernatants, it had no effect on spike discharge after application of UC supernatants (each symbol represent one patient sample). (C) Quantification of the neuronal activity evoked by biopsy supernatants. The PAR1 receptor antagonist SCH79797 significantly reduced the neuroindex evoked by IBS supernatants from 14 patients (upper panel) but had no influence on the neuronal activation evoked by UC supernatants from 12 patients (lower panel) (each symbol represent one supernatant; Wilcoxon signed rank test for paired data). Numbers in parentheses indicate number of tissues/ganglia/neurons studied.

Fig 2. Proteome analysis of mucosal biopsy supernatants.

Proteome analysis revealed significantly different protein levels and pattern in biopsy supernatants from IBS, HC or UC. (A) 204 proteins exhibit different levels between the three groups (p < 0.05, Benjamini-Hochberg adjusted). Hierarchical clustering of z-transformed protein levels reveals striking differences between biopsy supernatants. (B) Principal component analysis of all 22 samples using the 204 proteins corroborates this finding. (C) The subset of the 8 proteins which were significantly upregulated in the IBS patients vs. healthy controls were used to construct predictive models using linear discriminant analysis (left panel). All combinations consisting of 2 to 5 proteins (p1-p5) were examined. The prediction accuracy of each is shown as a dot in the figure. The combination of the three proteases cathepsin L1 (CTSL1), proteasome subunit alpha type 4 (PSMA4), and elastase 3A (ELA3A) resulted in the highest value of prediction accuracy of 98%, marked with an arrow. Single ROC curves plotted for the three protein predictors CTSL1, PSMA4, and ELA3A are shown in the right panel. The area under the curve (AUC), as a measure of the discriminatory value of individual or combinatorial proteins, showed the highest value for the protein combination. Note, for none of the individual proteins the specificity to discriminate between the HC and the IBS groups was lower than 85% at a given sensitivity of 75%.

Fig 3. Role of elastase in the activation of submucous neurons.

(A) Traces show the synergistic effect of adding 100nM elastase to a mediator mix containing 1μM histamine and 1μM serotonin. The right panel displays the analysis of several such experiments. Elastase alone had no effect. The mix of histamine and serotonin induced a nerve activation indicated by an increased Ca-neuroindex (product of maximum [Ca²⁺]_i signal x % responding neurons, calculated for each ganglion). Addition of elastase significantly enhanced the [Ca²⁺]_i neuroindex (Mann-Whitney Rank Sum Test, tissue/ganglion/neuron). (B) Inhibitory effect of 15nM SERPIN_BL from Bifidobacterium longum on IBS supernatant evoked spike discharge in human submucous neurons (left panel). The neuroindex (product of spike frequency and % of responding neurons) evoked by IBS supernatants from 10 patients was significantly reduced (Wilcoxon signed Rank Test for paired data; each symbol represents one patient sample; right panel). Bars below the traces indicate the duration of application. Numbers in parentheses indicate number of tissues/ganglia/neurons studied.