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©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
Multiplex gene expression profile in inflamed mucosa of patients with Crohn’s disease ileal localization: A pilot study
Francesco Giudici, Letizia Lombardelli, Edda Russo, Tiziana Cavalli, Daniela Zambonin, Federica Logiodice, Ornela Kullolli, Lamberto Giusti, Tatiana Bargellini, Marilena Fazi, Livia Biancone, Stefano Scaringi, Ann Maria Clemente, Eloisa Perissi, Giovanni Delfino, Maria G Torcia, Ferdinando Ficari, Francesco Tonelli, Marie-Pierre Piccinni, Cecilia Malentacchi
Francesco Giudici, Letizia Lombardelli, Edda Russo, Daniela Zambonin, Federica Logiodice, Ornela Kullolli, Ann Maria Clemente, Eloisa Perissi, Maria G Torcia, Marie- Pierre Piccinni, Department of Experimental and Clinical Medicine, University of Firenze, Firenze 50134, Italy
Tiziana Cavalli, Dipartimento Chirurgico Ortopedico, Ospedale Carlo Poma di Mantova, Firenze 50134, Italy
Lamberto Giusti, Giovanni Delfino, Cecilia Malentacchi, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Firenze, Firenze 50134, Italy
Tatiana Bargellini, Marilena Fazi, Stefano Scaringi, Ferdinando Ficari, Francesco Tonelli, Surgical Unit, Department of Surgery and Translational Medicine, University of Firenze, Firenze 50134, Italy
Livia Biancone, Department of Internal Medicine, University of Roma Tor Vergata, Roma 00133, Italy
Author contributions: Giudici F and Lombardelli L equally contributed to this work. Malentacchi C and Piccinni MP conceived the study. Giudici F, Cavalli T, Zambonin D and Bargellini T collected the samples. Lombardelli L, Logiodice F, Kullolli O, Russo E and Giusti L performed the experiments. Lombardelli L and Clemente AM performed the statistical analysis. Torcia MG and Piccinni MP partecipated in the writing of the study protocol and in the revision of the manuscript for important intellectual content with Ficari F, Tonelli F, Biancone L, Fazi M, Scaringi S and Delfino G. Perissi E and Russo E edited the manuscript. Malentacchi C, Giudici F, Russo E, Piccinni MP wrote the manuscript. Malentacchi C and Russo E provided funding acquisition. All authors read, commented, and approved the final manuscript.
Institutional review board statement: The study was reviewed and approved by EC of AOUC of Florence on May 2nd, 2011, No. 2011/0018055 rif.95/10, authorization Gen Dir 17/572011 No. 2011/0018055.
Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: The authors declare that they have no competing interests.
Data sharing statement: No additional data are available.
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Edda Russo, PhD, Academic Research, Postdoctoral Fellow, Biologist, Department of Experimental and Clinical Medicine, University of Firenze, Viale Pieraccini 6, Firenze 50134, Italy.
edda.russo@unifi.it
Supported by MIUR-Ministry of Education, University and Research, No. 2008X8NRH4_003; Fondazione Cassa di Risparmio di Firenze, No. 2008.1581, 2009.1301.
Telephone: +39-55-2758330 Fax: +39-55-7947449
Received: March 20, 2019
Peer-review started: March 20, 2019
First decision: May 9, 2019
Revised: July 12, 2019
Accepted: July 27, 2019
Article in press: July 27, 2019
Published online: September 6, 2019
Processing time: 170 Days and 17.3 Hours
BACKGROUND
Crohn’s disease (CD) is a complex disorder resulting from the interaction of genetic, environmental, and microbial factors. The pathogenic process may potentially affect any segment of the gastrointestinal tract, but a selective location in the terminal ileum was reported in 50% of patients.
AIM
To characterize clinical sub-phenotypes (colonic and/or ileal) within the same disease, in order to identify new therapeutic targets.
METHODS
14 consecutive patients undergoing surgery for ileal CD were recruited for this study. Peripheral blood samples from each patient were collected and the main polymorphisms of the gene Card15/Nod2 (R702W, G908R, and 1007fs) were analyzed in each sample. In addition, tissue samples were taken from both the tract affected by CD and from the apparently healthy and disease-free margins (internal controls). We used a multiplex gene assay in specimens obtained from patients with ileal localization of CD to evaluate the simultaneous expression of 24 genes involved in the pathogenesis of the disease. We also processed surgery gut samples with routine light microscopy (LM) and transmission electron microscopy (TEM) techniques to evaluate their structural and ultrastructural features.
RESULTS
We found a significant increase of Th17 (IL17A and IL17F, IL 23R and CCR6) and Th1 (IFN-γ) gene expression in inflamed mucosa compared to non-inflamed sites of 14 CD patients. DEFB4 and HAMP, two genes coding for antimicrobial peptides, were also strongly activated in inflamed ileal mucosa, suggesting the overwhelming stimulation of epithelial cells by commensal microbiota. IFN-γ and CCR6 were more expressed in inflamed mucosa of CD patients with ileal localization compared with patients with colonic localization suggesting a more aggressive inflammation process in this site. Morphological analysis of the epithelial lining of Lieberkün crypts disclosed enhanced release activity from goblet mucocytes, whereas the lamina propria contained numerous cells pertaining to various lines.
CONCLUSION
We observed that the expression of ileal genes related to Th1 and Th17 activity is strongly activated as well as the expression of genes involved in microbiota regulation.
Core tip: Multiplex Gene Assay in specimens obtained from patients with ileal localization of Crohn’s disease (CD) allowed the simultaneous analysis of messenger ribonucleic acid levels for 24 genes, known to be involved in the inflammation processes of CD pathogenesis. The result showed that the expression of genes related to Th1 and Th17 immune response is strongly activated as well as the expression of genes deputized to interact with the commensal microbiota, such as DEFB4 and HAMP, which code for antimicrobial peptides.
