Observational Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Cases. Sep 6, 2019; 7(17): 2463-2476
Published online Sep 6, 2019. doi: 10.12998/wjcc.v7.i17.2463
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
ORCID number: Francesco Giudici (0000-0002-6879-5685); Letizia Lombardelli (0000-0001-9986-0956); Edda Russo (0000-0003-3141-1091); Tiziana Cavalli (0000-0001-8673-7190); Daniela Zambonin (0000-0001-8892-2445); Federica Logiodice (0000-0002-3426-304X); Ornela Kullolli (0000-0002-7744-4596); Lamberto Giusti (0000-0002-9972-090X); Tatiana Bargellini (0000-0002-5381-8813); Marilena Fazi (0000-0003-1117-7461); Livia Biancone (0000-0002-9056-1957); Stefano Scaringi (0000-0002-6838-8318); Ann Maria Clemente (0000-0003-0386-590X); Eloisa Perissi (0000-0001-9089-9205); Giovanni Delfino (0000-0002-5434-5923); Maria Gabriella Torcia (0000-0003-4740-4646); Ferdinando Ficari (0000-0003-2248-6243); Francesco Tonelli (0000-0002-0527-9851); Marie-Pierre Piccinni (0000-0003-1810-5209); Cecilia Malentacchi (0000-0002-3315-1396).
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

Abstract
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.

Key Words: Crohn’s disease, Ileum, Colon, Messenger ribonucleic acid, Th1/Th17, Microbiota, Inflammation

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.


  • Citation: Giudici F, Lombardelli L, Russo E, Cavalli T, Zambonin D, Logiodice F, Kullolli O, Giusti L, Bargellini T, Fazi M, Biancone L, Scaringi S, Clemente AM, Perissi E, Delfino G, Torcia MG, Ficari F, Tonelli F, Piccinni MP, Malentacchi C. Multiplex gene expression profile in inflamed mucosa of patients with Crohn’s disease ileal localization: A pilot study. World J Clin Cases 2019; 7(17): 2463-2476
  • URL: https://www.wjgnet.com/2307-8960/full/v7/i17/2463.htm
  • DOI: https://dx.doi.org/10.12998/wjcc.v7.i17.2463

INTRODUCTION

The pathogenesis of Crohn’s disease (CD), one of the major inflammatory bowel diseases (IBD) together with ulcerative colitis (UC), has been extensively investigated. It is generally accepted that both genetic and environmental factors contribute to the etiology of the disease. In CD patients, strong associations between genes involved in maintaining intestinal barrier function, epithelial anti- microbial defence, innate immune regulation, reactive oxygen species (ROS) generation, autophagy, and metabolic pathways have been identified[1,2].

Environmental risk factors involved in the progression of the disease include smoking, low-fiber and high-carbohydrate diet, gut microbiota (GM) alteration, and treatments with antibiotics or non-steroidal anti-inflammatory drugs[3].

CD is characterized by a transmural inflammation which can potentially affect any segment of the gastrointestinal tract[4]. However, recent studies reported a selective location in the terminal ileum in 50% of the patients and location in the colonic district in 20% of the patients. Ileum and colon district were involved in the remaining 30% of the patients. A different clinical course and surgical requirement was reported according to disease’s localization but currently the reasons underlying the differences in the clinical course have not been defined. In addition, the immunological pathways involved in colonic inflammation are different from those involved in ileal inflammation[5].

The mutual interplay between GM and the immune system is involved in the pathogenesis and prognosis of intestinal diseases[6] as the GM is a key modulator of intestinal inflammation[7]. In CD patients, a reduced GM diversity and lower bacterial load in inflamed vs non-inflamed tissues was observed[8]. In addition, several evidences report that the small bowel is responsible for the systemic tolerance towards microbes. A recent study revealed that the ileum harbors a distinctive niche of the GM that differs more from the colonic[9]. This different GM composition could be attributed to the activation of distinctive immunological pathways.

In the present study, we used the multiplex gene assay[10,11] to analyze surgical specimens of CD patients with prevalent ileal localization.

MATERIALS AND METHODS
Patients

14 consecutive patients undergoing surgery for ileal CD aged 15 to 57 and hospitalized at the Surgery Unit of Azienda Ospedaliero-Universitaria Careggi, University of Florence, were recruited (Table 1). CD was diagnosed based on both histological and clinical/endoscopic criteria. Table 1 reports the clinical characteristics of the patients.

Table 1 Clinical characteristics of patients with ileal Crohn’s disease.
PatientPt1Pt2Pt3Pt4Pt5Pt6Pt7Pt8Pt9Pt10Pt11Pt12Pt13Pt14
LocalizationL1L1L1L1L1L1L3L1L3L1L3-L4L1L3L1
of CDa
Age of CD5715535525313942162419184630
onset (yr)
Surgery / relapse1st1stRelapse1stRelapseRelapseRelapse1stRelapseRelapseRelapseRelapseRelapse1st
surgerysurgerysurgerysurgerysurgery
Disease behaviorbB2B2, B3B2B3B2, B4B2B3B2B2B2B2B2B3B2
TherapycC, F, I, BF, CCNoF, C, IC, F, II, BF, CC, I, F, BCF, C, BF, C, IC, F, B, IF, C, I
Smoking statusdNoCurNoNo10月10日NoNo20/3010月10日Cur10月20日NoNoNo
20/15.510
GenotypewtR702wthzG881RwtwtwtwthzG881RR702No seqNo seqwtwt

Peripheral blood samples from each patient were collected in EDTA tubes and genomic DNA was extracted using QIA-AMP DNA Blood Maxi Kit (Qiagen GmbH, Hilden, Germany). The main polymorphisms of the gene Card15/Nod2 (R702W, G908R, and 1007fs) were analysed in each sample[12].

mRNA extraction and multiplex Gene Assay

Tissue samples were taken both from the tract affected by CD and from the apparently healthy and disease-free margins (internal controls). The surgical specimens were opened longitudinally.

All samples were stored in RNA later (Qiagen, Germany) before homogenization. Then each sample was weighed and the appropriate lysis solution was added to a final volume of 150 µL containing 50% Lysis Mixture (Thermo-Fisher, MA, United States) and 1 g/L proteinase. The mixture was agitated for 30 min at 65 °C to lyse the cells. The lysate was stored at -80 °C for later use. We used a microarray panel of 24 genes implicated in CD etiopathogenesis[10]. We evaluated the expression of these genes in both non-inflamed and inflamed ileal biopsies. Table 2 indicates the panel of the examined genes, the number of Mendelian Inheritance in Man (MIM) (used as a reference), accession number and their corresponding encoded product and function. To improve the analysis of the results, the selected genes were divided into four groups according to their biological role: (1) Transport across epithelia: ABCB1, SLC40A1, SLC22A4, SLC22A5, HAMP; (2) Immune response: CCR6, IL-17F, IL-17A, MICA, MYD88, STAT3, IL-23R, JAK2, IFNG, NOD2; (3) Antimicrobial activity: HAMP, CAMP, LRRK2.DEFB4; and (4) Physiological activities: STAT3, ESR1, LRRK2, TNFSF15, CARD14, DLG5 BMP2 ATG16L1.

Table 2 Panel of the 24 genes investigated.
SymbolComplete nameGroupAccession numbermimGene product function (s)Ref.
HPRT1Hypoxanthine phosphoribosyltransferase 1Low expression housekeeping geneM26434308000It plays a central role in the generation of purine nucleotides, chosen as a low expression housekeeping gene[39]
ACTBActin betaprovidedHigh expression housekeeping geneM28424102630Is involved in the cell motility, structure, and integrity[40]
SLC40A1Solute carrier family 40 (iron-regulated transporter), member11AF215636604653Exports iron from duodenal epithelial cells[41]
ABCB1ATP-binding cassette, sub-family B (MDR/TAP), member11M14758171050Transports various molecules across extra- and intra-cellular membranes. It belongs to a protein sub-family involved in multidrug resistance[42]
SLC22A5Solute carrier family 22 (organic cation/carnitine transporter), member 51AF057164603377Transports several small organic cations in the liver, kidney, intestine. It is involved in elimination of drugs and environmental toxins[43]
SLC22A4Solute carrier family 22 (organic cation/ergothioneine transporter), member 41AB007448604190Polyspecific transporter of organic cations in the liver, kidney, intestine, and involved in the elimination of these molecules.[44]
CCR6Chemokine (C-C motif) receptor62U68030601835Induces B-lineage maturation and antigen-driven B-cell differentiation[45]
IL17AInterleukin 17A2U32659603149Produced by Th17-type CD4+ cells. Regulates the activities of NF-kB and mitogen-activated protein kinases[26]
IL17FInterleukin 17F2AF384857606496Produced by Th17-type CD4+ cells. Stimulates the production of other cytokines, including IL6,IL8.It also inhibits angiogenesis by endothelial[46]
cells.
STAT3Signal transducer and activator of transcription 3 (acute-phase response factor)2-4BC014482102582Activates transcription of cell growth and apoptosis’ genes asresponses to inflammation[22,47]
MICAMHC class I polypeptide-related sequence A2L14848600169Acts as a stress-induced antigen broadly recognized by intestinal intra-epithelial gamma delta T cells.[48,49]
MYD88Myeloid differentiation primary response gene (88)2U84408602170Acts as an essential signal transducer in the interleukin-1 and Toll-like receptor signaling[50]
pathways
IL23RInterleukin 23 receptor2AF461422607562Expressed on Th17 cells. Involvedin the IL23A signaling pathways with the receptor molecule IL12RB1/IL12Rbeta1.[20]
JAK2Janus kinase 223717147796Is involved in cytokine receptor signaling pathways and is required for responses to gamma interferon[51]
IFNGInterferon, gamma23458147570It encodes a cytokine with antiviral,immunoregulatory and anti-tumor properties and activates macrophages[52]
CAMPCathelicidin antimicrobial peptide3BC055089600474It is an antimicrobial protein (defensin)[53]
CARD15Nucleotide-binding oligomerization domain containing 22AF178930605956Induces immune response to intracellular bacterial by recognizing the muramyl dipeptide (MDP)[54]
DEFB4Defensin, beta 4A3AJ314835602215Acts as an antibiotic peptide locally regulated by inflammation.[55,56]
HAMPHepcidin antimicrobialpeptide1/2/3AF309489606464It is involved in iron transport, antimicrobial, defence and inflammatory responses[57,58]
LRRK2Leucine-rich repeat kinase 23/4AK026776609007It is involved in autophagy and implicated in clearance of intracellular bacteria.[59,60]
TNFSF15Tumor necrosis facto (ligand) superfamily, member 154AF039390604052Induces apoptosis in endothelial cells[61]
CARD14Caspase recruitment domain family, member 144AF322642607211Regulates the molecular scaffolding process and activates NF-kappa B[62,63]
ATG16L1ATG16 autophagy related 16-like 14AK000897610767Induces autophagy processes involved in degradation of cell organelles[64]
ESR1Estrogen receptor 14X03635133430Involved in the metabolic pathway of the hormones and in several diseases including osteoporosis[65]
BMP2Bone morphogenetic protein 24650112261Induces bone and cartilage formation[66]
DLG5Discs, large homolog 54U61843604090It encodes for scaffolding molecules involved in cell-cell contact and in the maintenance of epithelial cell integrity. Its products are also involved in the transmission of extracellular signals[67]

The messenger ribonucleic acid (mRNA) expression for CCR6, IL-17A, IL-17F, BMP2, TNFSF15, ABCB1, IL-23R, DEFB4, CARD14, STAT3, SLC40A1, JAK2, SLC22A5, ACTB, ATG16L1, CAMP, DLG5, ESR1,CARD15, MICA, MYD88, SLC22A4, IFN-γ, LRRK2, HAMP, ACTB (high expression housekeeping gene), HPTR1 (low expression housekeeping gene) was measured using the QuantiGene® Plex assay (Thermo-Fisher, MA, United States).

A panel of oligonucleotide capture probes was covalently linked to carboxylated fluorescently encoded beads (Luminex, Bio-Rad, MA, United States). Each probe has a unique sequence of 15 bases. Each sample lysate diluted at 1:1 and 1:2 was mixed with the pooled capture beads in a round-bottom assay well and hybridized for 16 h at 54 °C (final volume in each well was 100 µL). The assay mixture was moved to a MultiScreen® Filter Plate (Millipore, Billerica, MA, United States) and unbound material was filter-washed from the wells by rinsing 3 times with wash buffer. The plate was hybridized with 100 µL/well of bDNA amplifier in Amplifier Diluent (Panomics, CA, United States) at 54 °C for 1 h. After the plate was filter- washed twice with wash buffer and incubated at 50 °C for 1 h with 100 µL/well of 5’-dT(Biotin)-conjugated label probe (Panomics, CA, United States) diluted in Label Probe Diluent (Panomics, CA, United States). After 2 washes, streptavidin-conjugated R-phycoerythrin diluted in SA-PE diluent (20 mmol/L Tris-HCl, 400 mmol/L lithium chloride, 1 mL/L Tween 20, 1 mL/L bovine serum albumin, and 5 mL/L Micr-O-protect) was added and the plate was shaken and incubated at room temperature for 30 min. We washed the beads to remove unbound SA-PE and then evaluated them with Bio-Plex® 200 system (Bio-Rad, MA, United States). The SA-PE fluorescence measured from each bead was proportional to the number of mRNA transcripts captured by the beads. Expression of target-specific RNA molecules was calculated as the mean values from triplicate cultures and normalized against Actin gene (high expression housekeeping gene).

Polymorphism analysis

A standard non-enzymatic method, using the QIA-AMP® DNA Blood Maxi Kit (Qiagen GmbH, Hilden, Germany) was used to extract Genomic DNA from peripheral blood leucocytes of all CD patients and healthy controls. In addition, DNA samples from 70 healthy Caucasian subjects (140 alleles) were analysed as controls. Three exon of the CARD15/NOD2 gene (Exon 4, Exon 8, Exon 11), were amplified by PCR using pairs of primers derived from the published sequence of the gene (available upon request). Each exon is associated with the three main single-nucleotide polymorphisms (SNPs) (R702W-C2104T; G908R-G2722C; 1007fs-3020insC). These three main variants, associated with susceptibility to CD, represented 32%, 18%, and 31%, respectively, of the total CD mutations[13-15].

The BigDye® Terminator Cycle Sequencing kit (Applied Biosystems, CA, United States) was used to perform direct sequencing of PCR amplified products (SNPs rs87950, rs127951, and rs137955) of the CARD15/NOD2 gene. The samples were analysed in an ABI Prism® 310 genetic analyzer (Applied Biosystems, CA, United States). The of the sequences were confirmed with the analysis of newly-amplified fragments and the sequencing of both DNA strands.

Statistical analysis

SSPS software vers. 10 (SPSS Inc., IL, United States) was used to perform the statistical analysis. All comparisons of genes mRNA expression in tissues (non-inflamed and inflamed areas) were performed by non-parametric assay (Mann-Whitney test, Wilcoxon test). Data are reported as mean and ranges unless otherwise stated. A P-value < 0.05 was accepted as statistically significant. Furthermore, to better characterize the different clinical CD phenotypes, we compared the results regarding the CARD15, CCR6, interferon gamma, and IL-17A genes to colonic CD patients previously examined for these same genes.

Histological analysis

Once removed, tissue samples were rinsed in 0.1 M, pH 7.0 cacodylate buffer, the same used in prefixation and further steps of histological preparation. Samples were then placed in Karnovsky (1965)[16], aldehyde solution, and after 3 h prefixation (4°C), underwent prolonged washing in the buffer. Surgery specimens were reduced into approximately 20 mm3 fragments that were post-fixed (1 h 30 min, 4°C) with 1% OsO4 in cacodylate. These specimens were washed in the buffer, dehydrated in graded ethanol series, soaked in propylene oxide, and embedded in Epon 812. Flat blocks were obtained after polymerization, which were reduced into semi-thin sections (1.5 µm thick), using an 8800 ULTROTOME III LKB equipped with glass knives. Semi-thin sections were stained with borax buffered 1% toluidine blue, and observed with a LEITZ DMRB, in order to collect LM digital images (JPG) for structural analysis. Subsequent ultrastructural observations were carried out on ultrathin sections, obtained with an ULTROTOME NOVA LKB, using a DIATOME diamond knife. Ultrathin sections with gold yellow to silver gray interference colour were selected and collected on uncoated 200-300 mesh copper grids to be electron-dense stained with a hydroalcoholic saturated solution (25 mg/mL) of uranyl acetate, followed by alkaline lead citrate (2 mg/mL). These sections where finally observed (80 KV) with a PHILIPS 201 TEM (BIO, UNIFI), and analogic images were collected, which were later acquired and stored as digital TIFF files using a DIMAGE SCAN DUAL (MINOLTA).

RESULTS
Expression of CD susceptibility genes in the inflamed ileum tissue

The simultaneous expression of 24 genes involved in the pathogenesis of CD was studied in surgical specimens from 14 CD patients with ileal localization of disease. The expression of genes in inflamed ileal mucosa was compared to that of non-inflamed ileal sites collected from the same patient. We observed a significant increase in mRNA levels of twelve genes compared to internal control (Figure 1).

Figure 1
Figure 1 Quantitative evaluation of gene expression using multiplex gene assay in surgical ileum specimens of CD patients. The abscissa shows the genes evaluated in inflamed and not inflamed tissue; the axis of the ordinates shows the value of expression of the gene normalized to the housekeeping actin (gene/β actin ratio). The first seven genes are more closely related to immunity. P value is reported only when statistically significant (P < 0.05).

Figure 1 shows that genes related to innate immune response (NOD 2, ATG16L1, DEFB4), and to adaptive immune response (CCR6, IL17A, IL17F, IL23R, IFN-γ) were significantly increased in inflamed mucosa of CD patients compared with non-inflamed sites. Moreover, the levels of mRNA for genes involved in physiological functions of epithelial cells, such as JAK2, TNFSF15, and SLC22A4 were higher in inflamed mucosa compared to non-inflamed mucosa and the differences in expression reached statistical significance.

Detection of CARD15 polymorphism

DNA samples obtained from peripheral blood were sequenced to investigate the presence of polymorphisms of CARD15/NOD2 gene. The results of this analysis showed that four patients (28.5%) included in this study are carriers of at least one of the polymorphisms investigated, suggesting that genetic factors might contribute to the dysregulated expression of CARD15/NOD2 gene[17-19].

Morphological analysis

In order to observe the morphology of inflamed tissue samples, light microscopy (LM) and TEM micrographs were obtained. Both gut wall and Lieberkühn crypts retain usual features in both lining epithelium and lamina propria. Epithelial cells consist of constitutive enterocytes along with goblet mucocytes, whereas the underlying connective tissue contains large amounts of cells with wide morphological variety (Figure 2). Goblet cells are involved in impressive secretory processes, releasing a moderately opaque product into cryptal and gut lumen through gaps between enterocytes apices (Figure 3). As a consistent pattern, the lamina propria contains granulocytes and plasma cells.

Figure 2
Figure 2 Representative Light microscope (LM) of the lamina propria between Lieberkühn crypts in inflamed ileal tissue of CD patient number 7b. Notice muciparous goblet cells in cryptal epithelium and large amount and variety of immune cells (arrows) in the connective tissue. Semithin (1 µm thick) section, toluidine blue staining; cc = goblet cell, ec = enterocyte. Scale bar (4 cm) = 70 µm.
Figure 3
Figure 3 Representative TEM micrograph of Lieberkühn crypt wall and lumen that contains mucous product released (large arrows) by goblet cells (CC) in inflamed ileal tissue CD patient number 7b. Small arrows indicate transport processes involving apical and lateral surfaces of enterocytes. Scale bar (1 cm) = 1 µm.
DISCUSSION

Among the numerous genes that have been studied so far with respect to CD, strong and replicated associations have been identified with NOD2, IL23R, and ATG16L1 genes[20]. Environmental factors like smoking, low-fiber and, high-carbohydrate diet, altered GM, and medications such as non-steroidal anti-inflammatory drugs interact with genetic background and induce abnormal inflammation and dysregulation of the immune response. Clinical symptomatology relates to such dysregulation.

The clinical course of CD is conditioned by several parameters such as disease location, extra-intestinal manifestation, and age at onset[21]. Strictures and fistulas are more frequent in patients with ileal disease, whereas Crohn's colitis remains uncomplicated for many years. On the whole, almost 80% of patients with CD require intestinal surgery, with a permanent stoma required by almost 10%. The presence of selected mutations in the NOD2 gene (see, e.g., 605956.0001-605956.0003) (IBD1; 266600) has been associated with susceptibility to ileum-localized CD[22]; patients homozygous for the 1007fs mutation had an early disease onset with long-segment ileal stenoses and entero-enteral fistulas; they frequently needed surgical intervention and had a high risk of recurrence[23,24]. Beside NOD2 gene, huge genome-wide linkage-analyses and meta-analyses have described several CD susceptibility regions including IBD5 locus, DLG5, and autophagy- related 16-like 1 (ATG16L1) gene, JAK2, STAT3 interleukin-23 receptor (IL23R), SLC22A4 and SLC22A5 TNFSF15[14].

In this paper, we evaluated the expression of 24 genes that were associated to CD susceptibility[10]. mRNA was extracted from gut specimens obtained from patients with CD ileal localization of CD, undergoing surgery. We used a multiplex gene assay which directly quantifies the mRNA amounts without need of reverse transcription and gives a detailed picture of the inflammation process for each patient[11]. The same technique was used to quantify gene expression in colonic mucosa from surgical specimens or endoscopic bioptic fragments obtained by CD patients with predominant colonic (L2) location[10].

The analysis revealed a clear activation of immune-adaptive Th17 response in association with a Th1 response in inflamed mucosa of patients undergoing surgery suggesting a dysregulated and very aggressive immune-inflammatory response.

Here gene expression analysis of inflamed ileal mucosa revealed an increased expression of genes involved in adaptive immune response compared to non-inflamed tissue. In particular, we found a significant increase of IL17A and IL17F, IL 23R and CCR6 gene expression suggesting an activation of a Th17 adaptive response[25,26] similar to that found in gut mucosa of patients with colonic localization. According with this hypothesis, three additional genes involved in Th17 differentiation as JAK2, STAT3 and TNFSF15[27,28] were found to be overexpressed in inflamed ileal mucosa of CD patients compared to non- inflamed sites. Furthermore, as we expected, the expression of the antimicrobial peptides as defensin (DEFB4)[29] and Hepcidin 6 (HAMP)[30] were significantly increased in inflamed mucosa of CD patients compared with non-inflamed sites, suggesting the overwhelming stimulation of epithelial cells by commensal GM. Indeed, while the human β-defensin (HBD) 1 is constitutively expressed, other genes, like HBD2 (gene name DEFB4), show pathogen and/or inflammation dependent upregulation[31] while also being inducible by probiotic bacteria[32]. Conversely, HAMP transcription mediates the effects of host defence and inflammation. Shanmugam et al. provided persuasive evidence in support of an important role for the GM composition in influencing hepcidin expression during intestinal inflammation in mouse models of colitis[33].

As the position of the pathogenic tissue may condition not only the clinical course of the disease but also the probability to require surgery, we also compared with the same methodology (Quantigene 2.0) the expression of selected genes (IFN-γ, CCR6, IL17A, NOD2) involved in immune responses in inflamed mucosa with predominant ileal location with the one previously studied[10] in inflamed mucosa with colonic location. mRNA expression for IFN-γ γ and for the chemokine CCR6 appeared significantly higher in ileal site compared to colonic site (ileal CD = 2.7 ± 1.5; colonic CD = 0.2 ± 0.06; P = 0.01). The mRNA for IL-17 and NOD2 appeared to be expressed at higher levels in ileal site compared to colonic site, even if the difference is not statistically significant (P ≥ 0.05). The significant differences in the expression levels of IFN-γ gene (higher expression in specimens from patients with ileal localization compared to patients with colonic localization) may suggest an increased damage of the ileal mucosa due to the simultaneous presence of Th1 and Th17 effector cells and/or the shift of Th17 cells to Th1 effectors functionally more aggressive than Th17 unshifted cells[34,35].

Furthermore, according to a worse clinical course of patient with ileal localization of CD compared with patient with colonic localization[36], the increased expression of IL17 and NOD2 in mucosal fragments from patients with ileal CD compared to patients with colonic CD is in agreement with the NOD2 –dependent regulation of immunity in mouse intestinal tract[37]. We suppose that the above differences between the two gut tracts (ileal and colonic) may be due to the Paneth cells at the bottom of the crypts of Lieberkühn in the small intestine, which produce antimicrobial peptides and hinder commensal GM and pathogenic bacteria to penetrate gut mucosa. Initially described as innate immune cells producing antimicrobial products, Paneth cells have recently been suggested to constitute a cardinal component of the intestinal stem cell niche. In fact, Paneth cells contribute to controlling the luminal flora as well as repairing the intestinal barrier following an insult. Genomic alterations that impede the Paneth cell compartment functionality can potentially increase the propensity to develop CD[38].

As a consistent trait, cryptal globlet cells produce large amounts of mucus that performs the double role of barrier and holder of antimicrobial products. The microscopic anatomy analysis aims to provide some details that illustrate phenotypic features: the large cell variety in the lamina propria includes immune lines that represent a further defense tool. Although these morphological traits are not directly related to specific gene outputs, they illustrate the tissue responses to key gene deregulation.

As a pilot study, our study presents a low number of subjects investigated which may have influenced the statistical power of the results. To confirm these results, studies with a larger number of patients are needed. In addition, gene expression was evaluated with Multiplex Gene Assay only. This method directly quantifies the mRNA amounts without need of reverse transcription and gives a detailed picture of the metabolic processes for each patient but it should be validated by comparisons with additional techniques to evaluate gene expression.

One of the main purposes of our research is therefore to identify new molecules involved in metabolic pathways that could potentially represent new biological drugs to identify the appropriate therapy in relation to the clinical phenotype of the CD patient.

ARTICLE HIGHLIGHTS
Research background

The interplay of environmental, genetic and microbial elements influences the etiopathogenesis of Crohn’s disease (CD). Differences in the clinical course of CD have recently been reported in patients with ileal or colonic localization of the inflammatory process.

Research motivation

Aim of this study was to define biochemical and histological differences in intestinal biopsies from patients with ileal or colonic localization of Crohn disease in order to identify new assays which can be useful for planning individual therapeutic strategies

Research objectives

Main objective of the current research was to investigate the expression of genes involved in immune-inflammatory pathways in gut mucosa from patients with ileal or colonic localization of CD and to correlate the results of gene expression with those obtained through a classical morphological analysis of surgical biopsies.

Research methods

A Multiplex Gene Assay was used to assess the simultaneous expression of 24 genes related to immune-inflammatory process and to CD pathogenesis. Structural and ultrastructural features of gut samples were also evaluated through Light microscopy (LM) and Transmission Electron Microscopy (TEM) techniques.

Research results

We observed a strong activation of genes involved in TH-1- and TH-17 immune response in patients with ileal localization of CD compared to patients with colonic localization. In addition, the expression of genes for antimicrobial peptides as DEFB4 and HAMP was found highly stimulated in ileal mucosa from CD patients suggesting a possible interference with microbial commensals at this site.

Research conclusions

Our results indicate that patients with ileal localization of CD have a stronger activation of TH-1 and TH-17 immune-inflammatory responses compared with patients with colonic localization of the disease thus defining a clear subclinical phenotype of CD.

Research perspectives

These results may suggest that therapeutic strategies with biological drugs in CD patients can be differentiated depending on the location of the disease

ACKNOWLEDGEMENTS

We would like to thank Dr. Michele Tanturli for the statistical support, Dr. Giulia Ricciardi for additional manuscript revision, and all the patients who participated to this study,

Footnotes

Manuscript source: Invited manuscript

Specialty type: Medicine, Research and Experimental

Country of origin: Italy

Peer-review report classification

Grade A (Excellent): 0

Grade B (Very good): B, B

Grade C (Good): C, C

Grade D (Fair): 0

Grade E (Poor): 0

P-Reviewer: El-Hussuna A, Mattar MC, Christodoulou DK, Day AS S-Editor: Dou Y L-Editor: AE-Editor: Zhou BX

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