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

Amanda D Williams, Olga Y Korolkova, Amos M Sakwe, Timothy M Geiger, Samuel D James, Roberta L Muldoon, Alan J Herline, J Shawn Goodwin, Michael G Izban, Mary K Washington, Duane T Smoot, Billy R Ballard, Maria Gazouli, Amosy E M'Koma, Amanda D Williams, Olga Y Korolkova, Amos M Sakwe, Timothy M Geiger, Samuel D James, Roberta L Muldoon, Alan J Herline, J Shawn Goodwin, Michael G Izban, Mary K Washington, Duane T Smoot, Billy R Ballard, Maria Gazouli, Amosy E M'Koma

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Diagnostic uncertainty and inaccuracy in…
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…
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…
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…
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…
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…
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.

References

    1. Jackson KL, Stocchi L, Duraes L, Rencuzogullari A, Bennett AE, Remzi FH. Long-Term Outcomes in Indeterminate Colitis Patients Undergoing Ileal Pouch-Anal Anastomosis: Function, Quality of Life, and Complications. J Gastrointest Surg 2017;21:56–61. doi:
    1. Carvalho RS, Abadom V, Dilworth HP, Thompson R, Oliva-Hemker M, Cuffari C. Indeterminate colitis: a significant subgroup of pediatric IBD. Inflamm Bowel Dis 2006;12:258–62. doi:
    1. Cantoro L, Di Sabatino A, Papi C, Margagnoni G, Ardizzone S, Giuffrida P, et al. The time course of diagnostic delay in inflammatory bowel disease over the last sixty years: an Italian multicentre study. J Crohn's Colitis 2017. March 18 [Epub ahead of print].
    1. Clark C, Turner J. Diagnostic Modalities for Inflammatory Bowel Disease: Serologic Markers and Endoscopy. Surg Clin North Am 2015;95:1123–41. doi:
    1. Odze RD. A contemporary and critical appraisal of 'indeterminate colitis'. Mod Pathol 2015;28 Suppl 1:S30–46.
    1. M'Koma AE, Seeley EH, Washington MK, Schwartz DA, Muldoon RL, Herline AJ, et al. Proteomic profiling of mucosal and submucosal colonic tissues yields protein signatures that differentiate the inflammatory colitides. Inflamm Bowel Dis 2011;17:875–83. doi:
    1. Seeley EH, Washington MK, Caprioli RM, M'Koma AE. Proteomic patterns of colonic mucosal tissues delineate Crohn's colitis and ulcerative colitis. Proteomics Clin Appl 2013;7:541–9. doi:
    1. M'Koma AE, Blum DL, Norris JL, Koyama T, Billheimer D, Motley S, et al. Detection of pre-neoplastic and neoplastic prostate disease by MALDI profiling of urine. Biochem Biophys Res Commun 2007;353:829–34. doi:
    1. M'Koma AE, Wise PE, Muldoon RL, Schwartz DA, Washington MK, Herline AJ. Evolution of the restorative proctocolectomy and its effects on gastrointestinal hormones. Int J Colorectal Dis 2007;22:1143–63. doi:
    1. Shen B, Remzi FH, Brzezinski A, Lopez R, Bennett AE, Lavery IC, et al. Risk factors for pouch failure in patients with different phenotypes of Crohn's disease of the pouch. Inflamm Bowel Dis 2008;14:942–8. doi:
    1. Shen B, Remzi FH, Lavery IC, Lashner BA, Fazio VW. A proposed classification of ileal pouch disorders and associated complications after restorative proctocolectomy. Clin Gastroenterol Hepatol 2008;6:145–58. doi:
    1. Zaghiyan K, Kaminski JP, Barmparas G, Fleshner P. De novo Crohn's Disease after Ileal Pouch-Anal Anastomosis for Ulcerative Colitis and Inflammatory Bowel Disease Unclassified: Long-Term Follow-Up of a Prospective Inflammatory Bowel Disease Registry. Am Surg 2016;82:977–81.
    1. Shen B. Crohn's disease of the ileal pouch: reality, diagnosis, and management. Inflamm Bowel Dis 2009;15:284–94. doi:
    1. Lee HS, Choe J, Lee HJ, Hwang SW, Park SH, Yang DH, et al. Change in the diagnosis of inflammatory bowel disease: a hospital-based cohort study from Korea. Intest Res 2016;14:258–63. doi:
    1. Nakamura K, Sakuragi N, Takakuwa A, Ayabe T. Paneth cell alpha-defensins and enteric microbiota in health and disease. Biosci Microbiota Food Health 2016;35:57–67. doi:
    1. Durand A, Donahue B, Peignon G, Letourneur F, Cagnard N, Slomianny C, et al. Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1). Proc Natl Acad Sci U S A 2012;109:8965–70. doi:
    1. Li X, LeBlanc J, Elashoff D, McHardy I, Tong M, Roth B, et al. Microgeographic Proteomic Networks of the Human Colonic Mucosa and Their Association With Inflammatory Bowel Disease. Cell Mol Gastroenterol Hepatol 2016;2:567–83. doi:
    1. VanDussen KL, Liu TC, Li D, Towfic F, Modiano N, Winter R, et al. Genetic variants synthesize to produce paneth cell phenotypes that define subtypes of Crohn's disease. Gastroenterology 2014;146:200–9. doi:
    1. Lawrance IC, Fiocchi C, Chakravarti S. Ulcerative colitis and Crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes. Hum Mol Genet 2001;10:445–56.
    1. Puri KS, Suresh KR, Gogtay NJ, Thatte UM. Declaration of Helsinki, 2008: implications for stakeholders in research. J Postgrad Med 2009;55:131–4. doi:
    1. Targan SR, Karp LC. Inflammatory bowel disease diagnosis, evaluation and classification: state-of-the art approach. Curr Opin Gastroenterol 2007;23:390–4. doi:
    1. James SD, Wise PE, Zuluaga-Toro T, Schwartz DA, Washington MK, Shi C. Identification of pathologic features associated with "ulcerative colitis-like" Crohn's disease. World J Gastroenterol 2014;20:13139–45. doi:
    1. Jong VL, Novianti PW, Roes KC, Eijkemans MJ. Selecting a classification function for class prediction with gene expression data. Bioinformatics 2016;32:1814–22. doi:
    1. Myers JN, Schaffer MW, Korolkova OY, Williams AD, Gangula PR, M'Koma AE. Implications of the Colonic Deposition of Free Hemoglobin-alpha Chain: A Previously Unknown Tissue By-product in Inflammatory Bowel Disease. Inflamm Bowel Dis 2014;20:1530–47. doi:
    1. Gmyr V, Bonner C, Lukowiak B, Pawlowski V, Dellaleau N, Belaich S, et al. Automated digital image analysis of islet cell mass using Nikon's inverted eclipse Ti microscope and software to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers. Cell Transplant 2015;24:1–9. doi:
    1. Tulay P, Naja RP, Cascales-Roman O, Doshi A, Serhal P, SenGupta SB. Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts. J Assist Reprod Genet 2015;32:1757–64. doi:
    1. Farin HF, Karthaus WR, Kujala P, Rakhshandehroo M, Schwank G, Vries RG, et al. Paneth cell extrusion and release of antimicrobial products is directly controlled by immune cell-derived IFN-gamma. J Exp Med 2014;211:1393–405. doi:
    1. Ballard BR, M'Koma AE. Gastrointestinal endoscopy biopsy derived proteomic patterns predict indeterminate colitis into ulcerative colitis and Crohn's colitis. World J Gastrointest Endos 2015;7:670–4.
    1. Zaharie R, Tantau A, Zaharie F, Tantau M, Gheorghe L, Gheorghe C, et al. Diagnostic Delay in Romanian Patients with Inflammatory Bowel Disease: Risk Factors and Impact on the Disease Course and Need for Surgery. J Crohn's Colitis 2016;10:306–14.
    1. M'Koma AE. Diagnosis of inflammatory bowel disease: Potential role of molecular biometrics. World J Gastrointest Surg 2014;6:208–19. doi:
    1. Matsui T. [Diagnosis of inflammatory bowel diseases. 1. Diagnosis, disease type and severity classification]. Nihon Naika Gakkai Zasshi 2009;98:31–6.
    1. Feakins RM. Ulcerative colitis or Crohn's disease? Pitfalls and problems. Histopathology 2014;64:317–35. doi:
    1. Gu J, Stocchi L, Kiran RP, Shen B, Remzi FH. Do clinical characteristics of de novo pouch Crohn's disease after restorative proctocolectomy affect ileal pouch retention? Dis Colon Rectum 2014;57:76–82. doi:
    1. Wagner-Bartak NA, Levine MS, Rubesin SE, Laufer I, Rombeau JL, Lichtenstein GR. Crohn's disease in the ileal pouch after total colectomy for ulcerative colitis: findings on pouch enemas in six patients. AJR Am J Roentgenol 2005;184:1843–7. doi:
    1. Price AB. Overlap in the spectrum of non-specific inflammatory bowel disease—'colitis indeterminate'. J Clin Pathol 1978;31:567–77.
    1. Langner C, Aust D, Ensari A, Villanacci V, Becheanu G, Miehlke S, et al. Histology of microscopic colitis-review with a practical approach for pathologists. Histopathology 2015;66:613–26. doi:
    1. Langner C, Magro F, Driessen A, Ensari A, Mantzaris GJ, Villanacci V, et al. The histopathological approach to inflammatory bowel disease: a practice guide. Virchows Arch 2014;464:511–27. doi:
    1. Abdelaal K, Jaffray B. Variables associated with loss of ileoanal pouches constructed in childhood. J Pediatr Surg 2017;52:281–5. doi:
    1. Robbins L, Zaghiyan K, Melmed G, et al. Outcomes with Anti-Tumour Necrosis Factor-Alpha Therapy and Serology in Patients with Denovo Crohn's Disease After Ileal Pouch Anal Anastomosis. J Crohn's Colitis 2017;11:77–83.
    1. Das P, Smith JJ, Tekkis PP, Heriot AG, Antropoli M, John Nicholls R. Quality of life after indefinite diversion/pouch excision in ileal pouch failure patients. Colorectal Dis 2007;9:718–24. doi:
    1. Lepisto A, Luukkonen P, Jarvinen HJ. Cumulative failure rate of ileal pouch-anal anastomosis and quality of life after failure. Dis Colon Rectum 2002;45:1289–94. doi:
    1. Magro F, Gionchetti P, Eliakim R, Ardizzone S, Armuzzi A, Barreiro-de Acosta M, et al. Third European Evidence-Based Consensus on Diagnosis and Management of Ulcerative Colitis. Part 1: Definitions, diagnosis, extra-intestinal manifestations, pregnancy, cancer surveillance, surgery, and ileo-anal pouch disorders. J Crohn's Colitis 2017;11:649–670
    1. Keighley MR. The final diagnosis in pouch patients for presumed ulcerative colitis may change to Crohn's disease: patients should be warned of the consequences. Acta Chir Iugosl 2000;47:27–31.
    1. Turina M, Remzi FH. The J-pouch for patients with Crohn's disease and indeterminate colitis: (when) is it an option? J Gastrointest Surg 2014;18:1343–4. doi:
    1. Le Q, Melmed G, Dubinsky M, McGovern D, Vasiliauskas EA, Murrell Z, et al. Surgical outcome of ileal pouch-anal anastomosis when used intentionally for well-defined Crohn's disease. Inflamm Bowel Dis 2013;19:30–6. doi:
    1. Li Y, Wu B, Shen B. Diagnosis and differential diagnosis of Crohn's disease of the ileal pouch. Curr Gastroenterol Rep 2012;14:406–13. doi:
    1. Shen B, Patel S, Lian L. Natural history of Crohn's disease in patients who underwent intentional restorative proctocolectomy with ileal pouch-anal anastomosis. Aliment Pharmacol Ther 2010;31:745–53. doi:
    1. Courth LF, Ostaff MJ, Mailander-Sanchez D, Malek NP, Stange EF, Wehkamp J. Crohn's disease-derived monocytes fail to induce Paneth cell defensins. Proc Natl Acad Sci U S A 2015;112:14000–5. doi:
    1. Tan G, Zeng B, Zhi FC. Regulation of human enteric alpha-defensins by NOD2 in the Paneth cell lineage. Eur J Cell Biol 2015;94:60–6. doi:
    1. Wang C, Shen M, Gohain N, Tolbert WD, Chen F, Zhang N, et al. Design of a potent antibiotic peptide based on the active region of human defensin 5. J Med Chem 2015;58:3083–93. doi:
    1. Hayashi R, Tsuchiya K, Fukushima K, Horita N, Hibiya S, Kitagaki K, et al. Reduced Human alpha-defensin 6 in Noninflamed Jejunal Tissue of Patients with Crohn's Disease. Inflamm Bowel Dis 2016;22:1119–28. doi:
    1. Wagner JA, Williams SA, Webster CJ. Biomarkers and surrogate end points for fit-for-purpose development and regulatory evaluation of new drugs. Clin Pharmacol Ther 2007;81:104–7. doi:
    1. Vermeire S, Van Assche G, Rutgeerts P. Laboratory markers in IBD: useful, magic, or unnecessary toys? Gut 2006;55:426–31. doi:

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