Linking bacterial enterotoxins and alpha defensin 5 expansion in the Crohn's colitis: A new insight into the etiopathogenetic and differentiation triggers driving colonic inflammatory bowel disease

Abstract

Evidence link bacterial enterotoxins to apparent crypt-cell like cells (CCLCs), and Alpha Defensin 5 (DEFA5) expansion in the colonic mucosa of Crohn's colitis disease (CC) patients. These areas of ectopic ileal metaplasia, positive for Paneth cell (PC) markers are consistent with diagnosis of CC. Retrospectively, we: 1. Identified 21 patients with indeterminate colitis (IC) between 2000-2007 and were reevaluation their final clinical diagnosis in 2014 after a followed-up for mean 8.7±3.7 (range, 4-14) years. Their initial biopsies were analyzed by DEFA5 bioassay. 2. Differentiated ulcer-associated cell lineage (UACL) analysis by immunohistochemistry (IHC) of the CC patients, stained for Mucin 6 (MUC6) and DEFA5. 3. Treated human immortalized colonic epithelial cells (NCM460) and colonoids with pure DEFA5 on the secretion of signatures after 24hr. The control colonoids were not treated. 4. Treated colonoids with/without enterotoxins for 14 days and the spent medium were collected and determined by quantitative expression of DEFA5, CCLCs and other biologic signatures. The experiments were repeated twice. Three statistical methods were used: (i) Univariate analysis; (ii) LASSO; and (iii) Elastic net. DEFA5 bioassay discriminated CC and ulcerative colitis (UC) in a cohort of IC patients with accuracy. A fit logistic model with group CC and UC as the outcome and the DEFA5 as independent variable differentiator with a positive predictive value of 96 percent. IHC staining of CC for MUC6 and DEFA5 stained in different locations indicating that DEFA5 is not co-expressed in UACL and is therefore NOT the genesis of CC, rather a secretagogue for specific signature(s) that underlie the distinct crypt pathobiology of CC. Notably, we observed expansion of signatures after DEFA5 treatment on NCM460 and colonoids cells expressed at different times, intervals, and intensity. These factors are key stem cell niche regulators leading to DEFA5 secreting CCLCs differentiation 'the colonic ectopy ileal metaplasia formation' conspicuously of pathogenic importance in CC.

Fig 1. Representative H & E staining of the colon.

A, Normal—normal crypt; B, UC–sporadic to no CCLCs even in severe disease activity; and C, CC–abundant CCLCs in the crypt base (arrow) seen even in mild disease activity.

Fig 2. Immunohistochemical detection of CCLCs marker (positive for Paneth cell) DEFA5 and UACL marker MUC6 in the intestinal mucosa of colonic CC.

Seral adjacent tissue sections were stained with MUC6 (left column) and DEFA5 (right column) on diseased colon from Crohn’s patients. Full tissue section (row A) and enlarged regions associated with MUC6 (box B) or DEFA5 (box C) demonstrate that DEFA5 expression within the apparent CCLCs is not localized within the UACL region of the diseased colon.

Fig 3. Multivariable logistic regression model for probability of being CC.

A, twenty-one (#21) IC patients were followed for 8.7±3.7 (range, 4–14) years. Although most IC resolved to UC or CC, but the mean diagnostic delay was 7 (range, 4–14) years. 28.5% of the patients could still not be diagnosed into authentic UC or CC. The mean area fraction of DEFA5 count (%) by NEARAS IHC staining agrees with final diagnostic outcome of every IC patient samples tested. B, Clinical data of these 21 IC patients who correlate with 3A data could have been diagnosed using DEFA5 immunoreactivity during the first endoscopy biopsy to avoid diagnosis delay. C, Bioinformatic data depicting a fit logistic model with group CC vs. UC (in 3A) as the outcome and DEFA5 as independent variable discriminator. The R2 of the model fit is 1 and area under the ROC curve is 1. The area under the ROC curve was not plotted since it will look similar as the orange curve in ROC plot. Three statistical methods were used: (1) Univariate analysis: Mann Whitney U test between two groups for each peak, adjusting p-values by FDR; (2) LASSO; and (3) Elastic net. As the scale of all measures were small, we multiplied all values by 10,000.

Fig 4. Treatment of colonoids with bacterial enterotoxins.

A–B, Equal numbers of colonoids were treated with/without enterotoxins (LPS, LTA, SAE) following a time course of up to 14 days. At each time point, we collected both the colonoids and the spent medium and determined the expression and/or secretion of DEFA5 by ELISA and IHC staining for lysozymes. We also seeded the colonoids at low density in triplicate, allowed to attach overnight, and then treated one set with enterotoxins and the other set without. We allowed the colonoids to form colonies for 14 days. Colonies were stained with anti-DEFA5 to determine whether the treatment led to differentiation into apparent CCLCs. The expression and/or secretion of DEFA5 and proinflammatory cytokines, etc. was determined by ELISA. C, Lysozyme assay: The assay was performed using lysozyme ELISA kit firm abcam (catalog no. ab108880 as per manufacturer’s instructions.

Fig 5. Treatment of immortalized colonic epithelial cells and colonoids with DEFA5.

We graphically present the data on the signatures for immortalized colonic epithelial cells and colonoids separately. We did not perform statistical tests on these data. Immortalized colonic epithelial cells (NCM 460) were received by a Material Transfer Agreement from INCELL Corporation (San Antonio, Texas, USA) [8]. NCM 460 cells (1x10⁶ cells) were plated in a 100 mm culture dish prior to starting the experiment. At the end of the treatment period, the cell culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. Abcam Cytokine Antibody Array (catalog no. ab133998) was used for the simultaneous detection of multiple cytokines following the manufacturer’s protocol. Colonoids were purchased from Cellesce Limited (Medicentre, Cardiff, UK). Organoid culture was performed as per instructions provided by the manufacturer (Cellesce). Two thousand colonoids per well were plated in a 12-well plate. After 2 days of growth in complete medium, the colonoids were treated with purified Human DEFA5 (5 μg/ml) for 6hr. and 24hr. The control colonoids were not treated. At the end of the treatment period, the colonoids culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. The experiments were repeated twice. At each time point, we collected both the colonoids and the spent medium and determined the expression and/or secretion of DEFA5 by ELISA and IHC staining for lysozymes. We also seeded the colonoids at low density in triplicate, allowed to attach overnight, and then treated one set with enterotoxins and the other set without and allowed the colonoids to form colonies for up to 14 days. Cytokines, chemokines, and growth factor measurements (signatures) are depicted: A, 24hr measurement; B, (24hr measurement) minus (6hr measurement) and C, (24hr measurement) minus (Control 24hr measurement) respectively.