Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

Abstract

Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.

Fig 1. Diagnostic uncertainty and inaccuracy in IBD clinical setting.

A, Indeterminant colitis. Twenty-one IC patients were followed for approximately ten years. At the end of the 10 year period, 28.5% of the patients could still not be delineated into a precise diagnosis of either UC or CC. B, Crohn’s as Ulcerative colitis. Sixty-seven UC RPC operated patients were followed for re-evaluated after a mean follow-up of 9.4 (range, 8–13) years for changing course of diagnosis. Thirty percent of these patients required a change of diagnosis to de novo Crohn’s disease.

Fig 2. Aberrantly expressed of HD5 in IBD.

A, Transcript levels of HD5 in moderate UC and CC. qRT-PCR confirms an increase in HD5 levels in moderate CC compared to moderate UC (p<0.05). Each point represents one patient sample. B, Representative HD5 Western Blot. HD5 levels (top) are higher in moderate and severe CC levels compared to all other disease states. β-actin loading control is shown on bottom. C, Graphical representation (densitometry) of HD5 Levels. Band intensities were measured for all samples and graphed as a ratio to β-actin loading control. Each point represents an individual sample. Moderate and severe CC levels of HD5 are both significantly higher than all other disease states (p<0.0001). D-G Representative IHC images of HD5 staining. D, Diverticulosis (DVL) no primary antibody control. E, Diverticulitis (DV). F, UC. G, CC (positive, arrow). H, HD5 staining counts. Each point represents one patient sample.

Fig 3. It is possible to use HD5 to determine patient candidacy for IPAA.

A. Representative results from a RPC-operated patient that did not change the diagnosis after surgery and was molecularly tested using HD5 IHC. B, Representative results from a UC RPC and IPAA operated patients that did change the diagnosis from UC to de novo Crohn’s was molecularly tested using HD5 IHC. C. NL-Ileum, control. D., Quantification of NEARAS HD5 IHC staining spot counts for UC RPC and IPAA-operated patients who did not have their original diagnosis changed versus those who did change from UC to de novo Crohn's (Fig 2A vs. 2B). (Ctrl 1 —staining control, UC—Ulcerative Colitis, CC—Crohn’s Colitis, DV—Diverticulitis, DVL—Diverticulosis).

Fig 4. H&E staining on parallel sections the typical morphological appearance of Paneth cell (PCs) including the presence of dense apical eosinophilic granules.

Upper panel: A, Diverticulitis (DV, no PCs), B, Diverticulosis (DVL, no PCs), C, Normal (NL-Colon, Control, no PCs). Middle panel: D, UC (found prodromal PC in one patient, arrow). E, CC, demonstrate abundance of PCs allover colonic basal crypts (arrows). F, Normal (NL-Ileum, Control), with abundance of PCs. Lower panel: IHC detection of Paneth cell markers α-defensin 5 (DEFA5) and lysozyme (LYZ) in the colon. G, NL-Colon, H, CC, and I, NL-Ileum, Control.

Fig 5. Double stain of PCs, lyzosomes and HD5.

Double staining analyses from de novo Crohn’s (Fig. 5A and D), and normal ileum/control (Fig. 5G) are presented. Image deconvolutions are displayed vertically to evaluate lysozyme-specific permanent red (Fig. 5B, E and H) and HD5α-specific DAB (Fig. 5C, F and I). The normal colon image (Fig. 5J), which lacks PCs, was not further processed.

Fig 6. Assessment of HD5 and Paneth cells in inflamed and normal, adjacent tissue.

HD5 staining of CC inflamed and normal, adjacent tissue shows expression of HD5 in all patient samples examined (Fig. 6A), compared to inflamed and adjacent, normal tissue of UC patients (Fig. 6B). H&E stains for Paneth Cells (Fig. 6C and D), were negative for PCs in all tissues.