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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., Competing Interests: The authors declare conflicts of interests to disclose, A.E. M’Koma has received Honoraria fees for Educational Presentation from Lipscomb University Health Sciences. Further, he is an inventor of two Patents: (i) Assay methods for diagnosing and treating inflammatory bowel disease with human alpha-defensin 5 (US16/571,034, 2020) and (ii) A.E. M’Koma and A.M. Sakwe - Targeted DEFA5 antibody and assay methods for diagnosing and treating inflammatory bowel disease (US62/522,652, 2020). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: We analyzed inflamed mucosal/submucosal layers of ulcerative colitis (UC = 63) and Crohn's colitis (CC = 50), and unexpectedly, we unveiled a pool of free hemoglobin alpha (Hb-α) chain. Patients with colitides have increased reactive oxidative stress (ROS), DNA oxidation products, free iron in mucosa, in preneoplastic, and in colitis-cancers and increased risks of developing colorectal cancer. All inflammatory bowel disease-related colorectal cancer lesions are found in segments with colitis. Linking this information, we investigated whether free Hb-α is key transformational stepping that increases colitis-related colorectal cancer vulnerability., Methods: UC/CC samples were profiled using matrix-assisted laser desorption/ionization mass spectrometry; protein identification was made by liquid chromatography. Diverticulitis was used as control (Ctrl). The presence of Hb(n) (n = α, β, or hemin)/Hb was validated by Western blotting and immunohistochemistry. We tested for DNA damage (DNAD) by exposing normal colonic epithelial cell line, NCM460, to 10 μM and 100 μM of Hb(n)/Hb, individually for 2, 6, and 12 hours. Quantification of Hb-α staining was done by Nikon Elements Advance Research Analysis software. ROS was measured by the production of 8-OHdG. DNAD was assessed by Comet assay. Colonic tissue homogenate antioxidants Nrf2-, CAT-, SOD-, and GPx-expressions were analyzed densitometrically/normalized by β-actin., Results: Immunohistochemistry of CC/UC mucosal/submucosal compartments stained strongly positive for Hb-α and significantly higher versus Ctrl. NCM460 exposed to Hb(n)/Hb exhibited steadily increasing ROS and subsequent DNAD. DNAD was higher in 10 μM than 100 μM in Hb-β/hemin the first 2 hours then plateaued followed by DNAD repair. This may be likely due to apoptosis in the later concentration. Nrf2 enzyme activities among UC, CC, and ulcerative colitis-associated colon cancer (UCAC) were observed impaired in all inflammatory bowel disease subjects. Decreased levels of Nrf2 among patients with UC versus patients with CC with active disease were insignificant as well as versus Ctrls but significantly lower in UCAC versus Ctrl. SOD was decreased in UC and UCAC and GPx in CC but statistically not significant. Comparing CC versus UC, SOD was significantly lower in CC (P < 0.05). CAT was observed increased among patients with CC/UC/UCAC and GPx in UC and UCAC versus Ctrl, respectively, and significantly increased in CC versus Ctrl (P < 0.01)., Conclusions: In the colitides, mucosal/submucosal tissue microenvironments demonstrated pool of free Hb-α chain. In vitro exposure of NCM460 cells to Hb(n)/Hb induced ROS and DNAD. Toxic effect of free Hb-α, in colonic epithelial cells, is therefore through production of ROS formation modulated by impairment of antioxidant effects. Targeting reduction-oxidation-sensitive pathways and transcription factors may offer options for inflammatory bowel disease-management and colitis-related cancer prevention.