A Specific Calprotectin Neo-epitope [CPa9-HNE] in Serum from Inflammatory Bowel Disease Patients Is Associated with Neutrophil Activity and Endoscopic Severity

Abstract

Background and aims: Endoscopy and the use of faecal calprotectin [faecal CP] are among the least-favoured methods for assessing disease activity by inflammatory bowel disease [IBD] patients; the handling/processing of faecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin enzyme-linked immunosorbent assay [ELISA], CPa9-HNE, with the aim of quantifying neutrophil activity and neutrophil extracellular trap [NET]-osis and proposing a non-invasive method for monitoring disease activity in IBD patients.

Methods: In vitro cleavage was performed by mixing calprotectin [S100A9/S100A8] with human neutrophil elastase [HNE], and a novel HNE-derived calprotectin neo-epitope [CPa9-HNE] was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis [UC, n = 43], Crohn's disease [CD, n = 93], and healthy subjects [HS, n = 23]. For comparison, faecal CP and MRP8/14 biomarkers were also measured.

Results: CPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were 4-fold higher in CD [p <0.0001] and UC [p <0.0001] patients than in HS. CPa9-HNE correlated well with the Simple Endoscopic Score [SES]-CD score [r = 0.61, p <0.0001], MES [r = 0.46, p = 0.0141], and the full Mayo score [r = 0.52, p = 0.0013]. CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: area under the curve [AUC] = 0.82 [p = 0.0003], UC: AUC = 0.87 [p = 0.0004]). The performance of CPa9-HNE was equipotent or slightly better than that of faecal CP.

Conclusions: Serum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.

Keywords: Calprotectin; IBD; biomarkers; inflammation; neutrophil elastase; neutrophil extracellular traps [NETs]; neutrophil granulocyte.

Figure 1.

Neutrophil granulocytes release both calprotectin [CP] and human neutrophil elastase [HNE]. A] Inactive neutrophils express CP and HNE, where CP is passively released. B] Upon activation, the neutrophils will initiate the process of neutrophil extracellular trap [NET] formation, which releases CP and HNE in huge amounts into the extracellular space, where HNE will begin to degrade the surrounding tissue and proteins, including CP. C] CP is cleaved by HNE, and small HNE-derived CP neo-epitope protein fragments [CPa9-HNE] are released into the blood and intestinal lumen and can be quantified by ELISA using protein fingerprint technology. D] Simplified depiction of how HNE cleaves CP and how specific monoclonal antibodies recognise only the specific HNE-derived CP neo-epitope fragment. ELISA, enzyme-linked immunosorbent assay.

Figure 2.

Overview of the calprotectin sequence of both A] S100A9 and B] S100A8 dimers. The calprotectin fragments generated by human neutrophil elastase [HNE]-mediated cleavage and selected for ELISA development are depicted by an arrow down [↓], where the neo-epitope selected for ELISA development is depicted by the color grey. Relative peptide abundance of selected HNE-derived calprotectin neo-epitope cleavage fragments; antibody specificity test and biological evaluation of the CPa9-HNE ELISA. C] The relative peptide abundance for the CPa9-HNE sequence was tested using mass spectrometry in samples containing HNE-cleaved calprotectin, full-length calprotectin, or HNE. Furthermore, D] antibody specificity was tested against the selection peptide, elongated peptide, truncated peptide, non-sense peptide, and finally E] biological evaluation of the CPa9-HNE assay was tested in samples containing HNE + calprotectin, calprotectin [Control 1], and HNE [Control 2]. Error bars represent the standard error of the mean [SEM]. ELISA, enzyme-linked immunosorbent assay.

Figure 3.

Biomarker levels of CPa9-HNE in Crohn’s disease [CD: n = 54] and ulcerative colitis [UC: n = 43] vs. healthy subjects [HS: n = 23]. A-C] CPa9-HNE biomarker and D-F] MRP8/14 biomarker were quantified in serum from ulcerative colitis and Crohn’s disease and compared with those of the healthy subjects. Data are depicted as interquartile range [IQR] with 10–90 percentile. Asterisks [*] depict significant differences between HS, CD, and UC patients, calculated using one-way ANOVA, **p <0.01. ***p <0.001, ****p <0.0001. ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance.

Figure 4.

Biomarker levels of CPa9-HNE, faecal CP, and MRP8/14 serum calprotectin stratified according to endoscopic disease severity based on the A-C] SES-CD for Crohn’s disease [n = 54], D-F], full Mayo score for ulcerative colitis [n = 43], and G-I] MES score for ulcerative colitis [n = 30]. Data are depicted as interquartile range [IQR] with 10–90 percentile. Statistical differences were calculated using one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. CP, calptotectin; ANOVA, analysis of variance; SES-CD, Simple Endoscopic Score for Crohn’s Disease; MES, Mayo Endoscopic Score.

Figure 5.

Ex vivo cultures of neutrophil granulocytes demonstrating CPa9-HNE biomarker level fold-change compared with inactive neutrophils, reflecting true neutrophil activity. CPa9-HNE biomarker levels after A] 4 h of cultivation with neutrophil agonists B] and 24 h of cultivation; MRP8/14 biomarker measurements after C] 4 h of cultivation with neutrophil agonists D] and 24 h of cultivation. Definition: negative control [only tissue], S. aureus [tissue + Staphylococcus aureus], inactive neutrophils [tissue + neutrophils], neutrophils + S. aureus [tissue + neutrophils + Staphylococcus aureus], neutrophils], neutrophils + PMA [tissue + neutrophils + phorbol myristate acetate], neutrophils + Cal iono [tissue + neutrophils + calcium ionophore]. Error bars represent the standard error of the mean [SEM].