Manuscript Doi : 10.1093/ecco-jcc/jjv056
Journal of Crohn's and Colitis Advance Access published March 28, 2015
Changes in epithelial barrier function in response to parasitic infection: implications for IBD pathogenesis Joan Antoni Fernández-Blancoa, Javier Estéveza, Terez Shea-Donohueb, Vicente
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Martíneza,c,d and Patri Vergaraa,c,d a
Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de
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Barcelona, Barcelona, Spain. bUniversity of Maryland School of Medicine, Department
of Medicine, Division of Gastroenterology & Hepatology and Mucosal Biology Research Center, Baltimore, MD, USA. cInstituto de Neurociencias; Universitat Autònoma de Barcelona, Barcelona, Spain.
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Centro de Investigación Biomédica en Red de
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Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid,
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Spain.
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Running title: Mast cells and tight junctions in gut barrier dysfunction
Keywords: Epithelial permeability; Mast cell proteinases; Post-infectious; Tight junction; Trichinella spiralis.
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Address for correspondence
Vicente Martínez
Unit of Physiology – Veterinary School Department of Cell Biology, Physiology and Immunology Universitat Autònoma de Barcelona 08193 – Bellaterra, Barcelona Spain Phone: +34 93 581 3834 FAX: +34 93 581 2006 e-mail: [email protected]
Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
Abbreviations: Cq: Quantification cycle FD4: 4KD Fluorescein Isothiocyanate-Labeled Dextran G: Conductance
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GLP-2: Glucagon-like peptide 2 IBD: Inflammatory bowel disease
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IBS: Irritable bowel syndrome IL-6: Interleukin 6 JAM-1: Junctional adhesion molecule 1
MC: Mast cell
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MLCK: Myosin light chain kinase
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LCM: Laser capture microdissection
PAR: Proteinase-activated receptor
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rMCP: Rat mast cell proteinase TJ: Tight junction
VCU: Villus-crypt unit
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ZO: Zonula occludens
Abstract Background and Aims: Mast cells (MCs) are implicated in epithelial barrier alterations that characterize inflammatory and functional bowel disorders. In this study, we
proteins kinetics in a rat model of postinfectious gut dysfunction.
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describe mast cell proteinases (chymases and tryptases) and tight junction (TJ)
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Methods: Jejunal tissues of control and Trichinella spiralis-infected rats were used. Inflammation-related changes in MCs and the expression of TJ-related proteins were evaluated by immunostaining and RT-qPCR. Epithelial barrier function was assessed in vitro (Ussing chambers) and in vivo.
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Results: After infection, intestinal inflammation was associated with a generalized overexpression of MC chymases, peaking between days 6 and 14. Thereafter, a
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mucosal MC hyperplasia and a late increase in connective tissue MC counts were observed. From day 2 post-infection, TJ proteins occludin and claudin-3 expression was
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down-regulated while the pore-forming protein claudin-2 was overexpressed. The expression of proglucagon, precursor of the barrier-enhancing factor glucagon-like peptide-2, was reduced. These changes were associated with an increase in epithelial
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permeability, both in vitro and in vivo.
Conclusions: Proteinases expression and location of mucosal and connective tissue MCs indicate a time-related pattern in the maturation of intestinal MCs following T. spiralis infection. Altered expression of TJ-related proteins is consistent with a loss of epithelial tightness, and provides a molecular mechanism for the enhanced epithelial
permeability observed in inflammatory conditions of the gut.
1. Introduction Mast cells (MCs) participate in the pathophysiology of inflammatory and functional gut disorders. Indeed, an increased number of intestinal MCs, together with a persistent state of activation, have been described for both inflammatory bowel disease (IBD)
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and irritable bowel syndrome (IBS).1-3 MC mediators can modulate a variety of
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secretomotor and sensory functions within the gut, including epithelial barrier function. There is evidence that MCs participate in the enhancement of intestinal permeability associated with IBD and IBS.4
Regardless of the species studied, mature MCs are classified taking into account
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their tissue location (mucosa vs. connective tissue) and their enzymatic characteristics, particularly their content of serine proteinases (chymases and tryptases). Accordingly,
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in rodents, MCs are usually classified as mucosal MCs or connective tissue MCs. In the intestine, mucosal MCs are preferentially located in the epithelium and the lamina
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propria, whereas connective tissue MCs are situated in the submucosa and the serosa layers. In the rat, mucosal MCs express predominantly chymases; in particular, rat mast cell proteinase (rMCP)-2, -3, -4 and the three members of the rMCP-8 subfamily
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(rMCP-8, -9 and -10). In contrast, rat connective tissue MCs express two chymases, rMCP-1 and -5, and two tryptases, rMCP-6 and -7.5,6 Since each proteinase cleaves different target substrates, the profile of serine
proteinases expressed by MCs conditions the potential biological effects to be observed in states of cell activation-degranulation.5,6 In the gut, one of the main effects of MC proteinases is to modulate epithelial permeability.7-11 MC proteinases are able to directly alter the expression and location of epithelial tight junction (TJ) proteins.8,10 At the same time, they also regulate TJs indirectly, by their effects over scaffolding
proteins, like the zonula occludens (ZO) proteins, and the cytoskeleton. 8,12 Overall, MC activation and the associated proteinases release disrupt the epithelial barrier leading to an increase in epithelial permeability. Within the gut, increased epithelial permeability might act as a defensive
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mechanism, favoring the movement of fluids to the lumen and facilitating intestinal
motility, as a way to eliminate potentially harmful luminal contents.10,13 However, in
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parallel, epithelial barrier disruption may also facilitate passage of luminal antigens across the mucosal barrier, activating the immune system and generating an inflammatory-like state.4 Therefore, MC proteinases-mediated effects on the epithelial
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barrier may contribute to the underlying pathophysiological alterations observed in inflammatory and functional bowel disorders.
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The main goal of the present study was to characterize the changes in MC populations and epithelial barrier function in a rat model of postinfectious gut
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dysfunction. Trichinella spiralis infection in the rat is an accepted model of inflammatory and functional gastrointestinal diseases characterized by the presence of jejunal MC hyperplasia and epithelial barrier dysfunctions.14,15 During acute
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inflammation and in the recovery postinfectious phase (days 2 to 30 post-infection), we assessed the kinetics of MC hyperplasia. In this regard, we emphasized the study of the appearance of phenotypically differentiated MC populations and time-related changes in MC serine proteinases gene expression. In addition, we assessed infectionassociated epithelial barrier alterations, both in vitro and in vivo, and changes in the expression of TJ-related proteins, as a possible underlying mechanism. Finally, we evaluated the expression of proglucagon, as precursor of the barrier-enhancing factor glucagon-like peptide 2 (GLP-2).16
2. Materials and Methods 2.1 Animals Adult male OFA Sprague-Dawley rats (7-8 weeks old, 250-275 g; Charles River Laboratories, Lyon, France) were used. Rats were housed under conventional
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conditions in a light (12h/12h light-dark cycle) and temperature controlled (20-22°C)
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room, with access to tap water and laboratory rat chow ad libitum. Animals were kept
in groups of two to three per cage. All experimental procedures were approved by the Ethics Committee of the Universitat Autònoma de Barcelona and the Generalitat de
2.2 Trichinella spiralis Infection
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Catalunya (protocols 5352 and 5564).
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Muscle-stage larvae of T. spiralis were obtained from infected CD1 mice as previously described.17-19 Rats were infected at 8-9 weeks of age by administration of 7.500 T.
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spiralis larvae suspended in 1 mL of saline by oral gavage and studies were performed on days 2, 6, 14 and 30 post-infection. Age- and time-matched rats dosed orally with 1 mL of saline were used as controls. During this time, animals were regularly monitored
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for clinical signs and body weight changes. Normal course of the infection was confirmed by a significant decrease of body weight after T. spiralis infection compared
with controls, with a peak reduction on days 8-10 and a subsequent increase over time, as previously described by us.14,15,19
2.3 Tissue Sampling At the time of the experiments, animals were euthanatized by decapitation, except otherwise stated. A laparotomy was performed and jejunal samples (beginning 10 cm
distal to the ligament of Treitz) were excised. Immediately, the intestines were flushed and placed in ice cold oxygenated Krebs buffer [(in mM): 115.48 NaCl, 21.90 NaHCO3; 4.61 KCl; 1.14 NaH2PO4; 2.50 CaCl2; 1.16 MgSO4 (pH: 7.3-7.4)] containing 10mM glucose. Jejunal segments were used to perform in vitro epithelial barrier function
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studies or preserved for morphological or molecular biology studies (see below). In
some cases, the mucosa and submucosa ( 2cm) were scraped off with blunt-edged
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glass slides obtaining the mucosa-submucosa and underlying smooth muscle-serosa samples separately. For laser capture microdissection (LCM) studies, jejunal samples were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe B.V.,
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Zoeterwoude, The Netherlands) in a cryomold (Peel-A-Way disposable embedding molds S-22; Polysciences Inc., Warrington, PA., USA). The mucosa-submucosa and
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smooth muscle-serosa samples and the cryoblocks were frozen in liquid nitrogen and stored at −80°C until analysis. Samples for histological and immunostaining studies
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were obtained and fixed in 4% paraformaldehyde in phosphate buffer for 24 h. Thereafter, fixed samples were processed routinely for paraffin embedding and 5 µm
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sections were obtained for H&E staining or immunohistochemistry.
2.4 Histopathological Studies H&E-stained slides were evaluated in a blinded fashion by two independent investigators. A histological score based on the epithelial structure (0-3), presence of edema (0-3), presence of ulcerations (0-3), presence of inflammatory infiltrate (0-3), relative density of goblet cells (0-3) and relative thickness of the intestinal wall (0-3) was assigned to each animal (maximal score of 18).
2.5 Epithelial Barrier Function Measurements 2.5.1 In vitro assessment of epithelial barrier function (Ussing chambers)
Jejunal
segments stripped of the outer muscle layers and myenteric plexus were mounted in Ussing chambers, following procedures previously described.14,15 Tissues were bathed
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bilaterally with 5 mL of 37°C oxygenated Krebs buffer. The basolateral buffer contained
10 mM glucose, osmotically balanced with 10 mM mannitol in the apical buffer. A zero
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potential difference was maintained injecting the required short-circuit current in each
moment. A voltage step of 1mV was applied every 5 min and the change in shortcircuit current was used to calculate tissue conductance (G) by Ohm’s law, as a
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measure of intestinal permeability. Tissues were allowed to stabilize for 15-25 min before baseline values were recorded. Data were digitized with an analog-to-digital
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converter and measurements were recorded and analyzed with Acqknowledge computer software. G was normalized for the exposed surface area (0.67 cm2).
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Paracellular permeability was further assessed by measuring mucosal to basolateral flux of fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4kD (FD4; Sigma Aldrich, St Louis, MO, USA). After stabilization,
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FD4 was added to the mucosal reservoir to a final concentration of 2.5x10-4 M. Basolateral samples (250 μL; replaced by 250 µL of appropriate buffer solution) were taken for subsequent fluorescence measurement (Infinite F200; Tecan, Crailsheim, Germany), with an excitation wavelength of 485 nm and an emission wavelength of 535 nm, against a standard curve. Readings are expressed as a percentage (%) of the total amount of FD4 added to the mucosal reservoir. Hydroelectrolytic transport capacity of the tissues was tested at the end of the permeability experiments by assessing responses to CCh (10 -4 M) added to the
basolateral side. Tissues with abnormal baseline values of electrophysiological parameters or with a lack of response to CCh were considered damaged and were excluded. Overall, these represented less than 5% of the preparations tested.
Non-infected controls and T.
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2.5.2 In vivo assessment of epithelial barrier function
spiralis-infected animals (at days 14 and 30 post-infection) were dosed orally with FD4
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(10 mg/rat, 0.2 mL) and returned to their home cages. Thirty minutes later, each animal was deeply anesthetized with isoflurane (Isoflo®, Esteve Veterinaria, Barcelona, Spain) and a blood sample was obtained by intracardiac puncture. Samples were
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maintained in ice in a dark box until processing. Serum was obtained by centrifugation (3000xg, 10 min, 4°C) and the concentration of FD4 (µg/mL) determined by
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fluorimetry, as described above for in vitro experiments.
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2.6 Immunohistochemistry for rat mast cell proteinases 2 (rMCP-2) and 6 (rMCP-6) and mast cells counts
Immunodetection of rMCP-2 and rMCP-6 was carried out on jejunal sections following
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standard immunohistochemical procedures using the monoclonal antibodies MS-RM4 (1:500; Moredun Animal Health, Edinburgh, UK) and sc-32473 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively, as previously described.14,15 Specificity of the staining was confirmed by omission of the primary antibody. Stained mucosal MCs (rMCP-2 positive) were counted in at least 20 well-oriented
villus-crypt units (VCU) per animal, at X400 magnification, and cell counts expressed as mucosal MCs/VCU. The total number of stained connective tissue MCs (rMCP-6 positive) in the submucosa, external smooth muscle and serosa areas was determined
in two complete tissue sections of the jejunum for each animal (X600). Connective tissue MC counting was normalized for the surface area of submucosa, external smooth muscle and serosa layers, as evaluated in digital images. Cell counting was performed using an Axioskop 40 microscope (Carl Zeiss, Jena, Germany; equipped with
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a digital camera, Zeiss AxioCam MRm; image analysis software: Zeiss Axiovision
on coded slides to avoid observer’s bias.
2.7 Immunohistochemistry for claudin 2
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Release 4.8.1). Counting and analysis of all data were performed in a blinded manner
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For claudin 2 immunohistochemistry, tissue sections were rehydrated, and microwave antigen retrieval was performed (10 mM Tris Base, 1 mM EDTA solution, 10 mM, pH 9;
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2 cycles of 5 min, 800 W). Thereafter, samples were incubated for 40 min in H 2O2 (5%), for inhibiting endogenous peroxidases, followed by a 30 min incubation with horse
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serum. Subsequently, slides were incubated (overnight, 4°C) with mouse monoclonal anti-claudin 2 antibody (1:2000; ref.: 32-5600. Invitrogen, Camarillo, CA, USA). Finally, sections were incubated (overnight, 5°C) with biotinylated horse anti-mouse IgG
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(1:200; Santa Cruz Biotechnology). The avidin/peroxidase kit (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) was used for detection, and the antigenantibody complexes were revealed using 3,3’-diaminobenzidine (SK-4100 DAB; Vector Laboratories). Sections were counterstained with hematoxylin. Specificity of the staining was confirmed by omission of the primary antibody. Coded slides were observed with an Axioskop 40 microscope. Claudin 2 immunoreactivity was quantified applying a semiquantitative score (0: no immunoreactivity; 1: some immunoreactivity restricted to the epithelial crypts; 2:
intense immunoreactivity restricted to the majority/all epithelial crypts; 3: intense immunoreactivity within the epithelial crypts and also extending to some of the villi; 4: intense immunoreactivity within the epithelial crypts and extending along the majority of the villi). For quantification, 15 randomly selected fields covering the whole
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thickness of the jejunal mucosa, from at least two tissue sections for animal, were
scored by two independent observers. A final score (0-4) for each animal was
performed on coded slides to avoid any bias.
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2.8 Laser capture microdissection (LCM)
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calculated as the mean of the scores assigned by each observer. All procedures were
LCM was performed as previously described20, with minor modifications. Briefly, 4 µm
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sections were obtained from jejunal cryoblocks using a cryostat and placed in plain uncoated pre-cleaned slides (Corning Inc., Corning, NY, USA) maintained in dry ice, and
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stored at -80°C until needed. Cryosectioned tissues were stained with H&E and dehydrated. Thereafter, LCM was performed with an ArcturusXT microdissection system (Arcturus Engineering Inc., Mountain View, CA, USA). Cells were captured from
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the lamina propria, the submucosa and the external smooth muscle layers, transferred to CapSure Macro LCM Caps (LCM 0211; Arcturus Engineering Inc.) and processed
immediately (see below).
2.9 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Total RNA extractions from jejunal mucosa-submucosa and smooth muscle-serosa samples were performed using TRI reagent and the Ribopure RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA). RNA was quantified by Nanodrop (Nanodrop
Technologies, Rockland, DE, USA) and 1 µg of RNA was reverse-transcribed in a 20 µL reaction volume for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For LCM samples, RNA was extracted and reverse-transcribed following
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previously described methods20,21 , with minor modifications. Briefly, thermoplastic films containing microdissected cells were incubated with RNA denaturing buffer
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(Stratagene, La Jolla, CA, USA) and β-mercaptoethanol. Then the phenol-chloroformbased method was used for the extraction and RNA was precipitated with sodium acetate aided by glycogen and ice-cold isopropanol. Thereafter, RNA was reverse-
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transcribed with the SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY, USA) using dNTP (0.5 mM each) and random primers (10ng/μl; Hexanucleotide Mix,
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10x conc.; Roche, Penzberg, Germany). Complementary DNA obtained was then preamplified with the TaqMan® preAmp Master Mix Kit and validated TaqMan®
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probes (Applied Biosystems; Table 1), following manufacturers' instructions. The temperature profile was 25°C for 10 min, 42°C for 50 min and 70°C for 15 min for reverse transcription and 95°C for 10 min; 14 cycles of 95°C for 15 s, 60°C for 4 min for
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cDNA preamplification.
Validated TaqMan® gene expressions assays with hydrolysis probes for rat mast
cell proteinases, epithelial barrier function-related proteins, interleukin-6 (IL-6) and reference genes were used (Applied Biosystems) (Table 1). PCR reaction mixtures were transferred to clear 384-well reaction plates (HSP-3801; Bio-Rad, Barcelona, Spain; 20 µL/well), sealed by adhesives and incubated on a CFX384 Touch real-time PCR
detection system (Bio-Rad). Amplification conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 15 s, 60°C for 1min. Fluorescence signals measured during
amplification were processed after amplification. Bio-Rad CFX Manager 2.1 software was used to obtain the quantification cycle (Cq) for each sample. Each sample was run in triplicate and data were analyzed by the comparative Cq method (2 -∆∆Cq), as previously described.22 Rps18 and Actb were tested as reference genes. Actb
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expression levels were used for normalizing the mRNA levels of the target gens
because of their constancy across the different experimental groups. Controls of
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analytical specificity included omission of reverse transcriptase, to exclude contamination with genomic DNA, and no-template controls, omitting the cDNA.
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2.10 Statistical analysis
All data are expressed as mean ± SEM. A robust analysis (one iteration) was used to
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obtain mean ± SEM for RT-qPCR data. Comparisons between multiple groups were performed using a one-way ANOVA, a two-way ANOVA or a Kruskal-Wallis ANOVA, as
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appropriate; followed, when necessary, by a Newman-Keuls or a Dunns multiple comparisons test. In all cases, results were considered statistically significant when
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prMCP-9>rMCP-2>rMCP-6. A similar pattern was detected in smooth muscle-serosa samples (Fig. 5); however, in this case, expression of rMCP-6 was up-regulated, showing a progressive time-
related increase, with the highest expression levels detected by day 30 post-infection (Fig. 5). Overall, relative expression changes were: rMCP-1>rMCP-4>rMCP-6~rMCP8>rMCP-9>rMCP-5~rMCP-10>rMCP-2. Gene expression analysis in LCM samples obtained from the lamina propria, the
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submucosa or the smooth muscle, in which areas containing MCs were selected, were, in general, in consonance with results from whole tissue samples. LCM samples
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confirmed and up-regulation of rMCP-1 in lamina propria and submucosa and of rMCP2 in submucosa (where mucosal MCs are preferentially found) (Fig. 6). Similarly, LCM samples from the muscle layers confirmed the up-regulation on rMCP-6 observed in
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smooth muscle-serosa samples. Moreover, although rMCP-6 was not detected in control conditions, this proteinase was expressed respectively in 2 and 4 (out of 5) of
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(Fig. 6).
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the lamina propria and the submucosa samples obtained from T. spiralis-infected rats
3.4 Effects of T. spiralis infection on epithelial barrier function in vitro and in vivo At days 14 and 30 post-infection, an increase in epithelial conductance was observed
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(Fig. 7A). In concordance, in vitro mucosal to basolateral passage of FD4 across jejunal segments was significantly increased in jejunal sheets from T. spiralis infected animals compared with non-infected controls (Fig. 7B). These results were further confirmed in in vivo conditions, where intestinal passage of FD4 was increased by 2.5- and 2.2-fold at 14 and 30 days post-infection, respectively, when compared with control conditions (both P occludin > JAM-1 >> tricellulin > ZO-1 > claudin 2.
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Similar order of magnitude in the relative expression levels was maintained after
T. spiralis infection. However, time-dependent changes in the absolute levels of
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expression were detected, and both up-regulation and down-regulation of genes could
be observed depending upon the protein considered. Claudin 2 and ZO-1 showed an up-regulation; while genes encoding for claudin 3, occludin and MLCK were down-
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regulated (Fig. 8). Overall, expression changes were fast in time, as maximal variations were observed between 2 and 6 days post-infection (Fig. 8). JAM-1 and tricellulin
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expression levels were not affected.
The major relative changes in gene expression were observed for claudin 2,
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which was up-regulated by 20-30-fold between days 2 and 6 post-infection. To further understand the functional significance of these changes, we assessed claudin 2 expression in jejunal samples of non-infected and T. spiralis infected animals. In
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control conditions, claudin 2 immunoreactivity was observed restricted to the crypts of the jejunal epithelium (Fig. 9A). In T. spiralis infected animals, claudin 2 immunoreactivity could be observed within the crypts, but also along the villi (Fig. 9B). Semi-quantification of claudin 2 immunoreactivity pointed to a relative increase in protein content by days 2 and 14 post-infection, with normalization by day 30; however, statistical significance was not reached (Fig. 9D). In some cases, unspecific staining was observed in unidentified cells of the lamina propria. Immunoreactivity
disappeared when the primary antibody was omitted (Fig. 9C), thus confirming the specificity of the staining. Gene expression of GLP-2 precursor, proglucagon, showed a clear tendency to be
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down-regulated over time [ANOVA: F(4.19)=2.67, P=0.06; Fig. 8].
4. Discussion
The present study shows time-associated changes in intestinal MC populations and TJ-
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related proteins in a rat model of gut dysfunction induced by T. spiralis infection. Timedependent variations in the expression of MC proteinases indicate an early mucosal
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MC hyperplasia and a later increase in connective tissue MCs. Simultaneously, alterations in the expression of TJ-related proteins, potentially modulated by MC
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proteinases, are associated with an increase of epithelial permeability. One of the main characteristics of the T. spiralis infection model in rats is the presence of a long-lasting postinfectious infiltrate of mucosal MC populating the mucosa-submucosa of the small intestine.14 Here we show that, in addition to the
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previously well-known mucosal MCs hyperplasia, a hyperplasia of connective tissue
MCs (rMCP-6 positive) also occurs in this model. Moreover, we show that infiltration of mucosal and connective tissue MCs occurs at different times along the postinfectious process. Overall, gene expression data suggest that in early phases (by day 2) there is an activation of resident MCs. This early MC activation may trigger the release of mediators and contribute to the recruitment of new MCs, leading to the persistent MC hyperplasia observed from day 6 post-infection. Changes in the expression of rMCP-2,
particularly in LCM-enriched samples, indicate that MC precursors colonize the mucosa-submucosa and differentiate towards a mucosal phenotype in the early postinfectious phase. This coincides with the intestinal phase of the infection and may represent an early defensive response to the presence of parasites in the mucosa.24-26
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At later phases (from day 14 post-infection), MCs appear to infiltrate into external
muscle layers. In this case, the morphology and gene expression analyses point to a
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preferential differentiation towards connective tissue MCs, as indicated by the
selective increase in rMCP-6 expression. At this stage, scattered rMCP-2-positive cells were also observed within the muscle layers, suggesting recruitment into smooth
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muscle of submucosal MCs with an immature phenotype. These rMCP-2-expressing MC may be precursors of fully differentiated rMCP-6-positive connective tissue MCs.27Co-expression of rMCP-2/rMCP-6, while cells migrate across the muscle to generate
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29
mature connective tissue MCs, is similar to that observed in mouse MCs, which co-
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express mouse mast cell proteinases 2 and 6 in the mucosa after T. spiralis infection.30 As in the epithelium,24 MCs infiltrating the muscle layers might represent a defensive response towards the penetration of T. spiralis larvae into the gut wall. It is reasonable
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to assume that MCs infiltrate contribute to smooth muscle long lasting hyperplasia and hypercontractile responses observed in infected animals.18,19 Some overlapping in proteinase expression was observed between mucosal and
connective tissue MCs. Indeed, both rMCP-1 and rMCP-2 were detected in LCM samples from lamina propria, submucosa and smooth muscle. This agrees with data showing the expression of chymases (rMCP-1 and mMCP-5), classically associated with connective tissue MCs, in both mucosal and transitional populations of MCs32-34 and might be related with the common origin of connective tissue and mucosal MCs. Taken
together, these observations support the view that tissue location of MCs alone does not fully predict the phenotypic pattern of proteinases expression.31,32 According to our data, only the expression of the tryptase rMCP-6 seems to be fully specific of mature connective tissue MCs, suggesting that this proteinase is a selective phenotypic marker
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for these cells.
The variable pattern of expression of substrate-specific MC proteinases may be
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related to functional changes along the postinfectious phase. One of the major effects of substrate cleavage mediated by MC proteinases is the modulation of epithelial permeability, by controlling the expression of TJ transmembrane components.8,9,24 In
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this respect, our studies show a time-related down-regulation of transmembrane junctional proteins with adhesive properties, namely claudin 3 and occludin, and an
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over-expression of the pore-forming protein claudin 2. In particular, the lack of occludin would result in an increase of paracellular permeability. Occludin expression
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has been associated with the release of the barrier-enhancement factor GLP-2,16 and its precursor proglucagon, which tended to be down-regulated after the infection. In addition, mucosal MCs hyperactivity and increased rMCP-2 release would favor
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occludin degradation.8,14,18 This is in agreement with a down-regulation of occludin expression and the subsequent increase in epithelial permeability observed in functional studies, both in vivo and in vitro. In the same way, the up-regulation of claudin 2 might also contribute to functional alterations in epithelial barrier function, particularly at early states of the infectious process. In this case, gene expression results were accompanied by a slight increase in the amount of claudin 2 immunoreactivity in the jejunal epithelium, with a widespread distribution along the villi. These data are consistent with the contribution of claudin 2 to the increased
epithelial permeability. Interestingly, similar structural and functional alterations have been observed in patients with inflammatory and functional gastrointestinal disorders.35-38 Altered epithelial permeability facilitates passage of luminal antigens,
including the maintenance of a persistent MCs infiltrate.
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thus contributing to the maintenance of a state of chronic, low-grade, inflammation,
While mucosal MCs-derived proteinases can be important modulating barrier
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function, a role for connective tissue MCs-derived proteinases seems least clear. Nevertheless, proteinases released within the smooth muscle and the myenteric plexus may modulate the activity of proteinase-activated receptors (PAR), leading to
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altered sensory and motor functions. Indeed, we previously demonstrated altered motor responses and changes in the expression of PAR-2 during T. spiralis infection in
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rats15, thus suggesting that connective tissue MCs might play a functional role. In summary, the present study reveals that, in addition to mucosal MCs,
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connective tissue MCs are also a significant component of the response to T. spiralis infection in rats. There is a differential time profile in the infiltration of mucosal and connective tissue MCs as well as in the expression of MC serine proteinases in the rat
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jejunum after T. spiralis infection. MC infiltrates are likely to be active components in the epithelial and motor defensive responses generated against the infection. We also show that long-lasting postinfectious increases in intestinal permeability reflect a dysregulation in the expression of TJ transmembrane proteins, which may be attributed, in part, to the effects of MC-derived proteinases. From a translational point of view, these observations suggest that MC proteinases and TJs represent potential pharmacological targets for the treatment of inflammatory and functional gastrointestinal disorders.
Conflict of interest The authors have no conflict of interest to declare.
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Acknowledgements and Author Contributions A. Acosta, E. Martínez, F. Jardí, M. Aguilera, V. Grinchuk, L. Notari, S. Yan, A. Zhao and
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S. Bartolomé are thanked for their assistance in different parts of this work. This study was supported by grants 2014SGR00789 from the Generalitat de Catalunya (Spain), and BFU2009-08229 and BFU2010-15401 from Ministerio de Ciencia e Innovación
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(Spain). JAF-B personal support: FPU program (AP2007–01619) from Ministerio de Ciencia e Innovación (Spain).
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JAF-B designed and performed experiments, analyzed data and wrote the paper. JE designed and performed experiments and analyzed data. VM designed and
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performed experiments, analyzed data and wrote the paper. PV designed experiments
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and wrote the paper. TS-D designed experiments and wrote the paper.
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mucosal mast cell granule chymase, rat mast cell protease-II, during anaphylaxis is associated with the rapid development of paracellular permeability to macromolecules in rat jejunum. J Exp Med 1995;182:1871-1881.
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Scudamore CL, Jepson MA, Hirst BH, Miller HR. The rat mucosal mast cell chymase, RMCP-II, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and occludin. Eur J Cell Biol
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10. McDermott JR, Bartram RE, Knight PA, Miller HR, Garrod DR, Grencis RK. Mast cells disrupt epithelial barrier function during enteric nematode infection. Proc Natl
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11. Cenac N, Coelho AM, Nguyen C, Compton S, Andrade-Gordon P, MacNaughton WK, Wallace JL, Hollenberg MD, Bunnett NW, Garcia-Villar R, Bueno L, Vergnolle N. Induction of intestinal inflammation in mouse by activation of proteinase-
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activated receptor-2. Am J Pathol. 2002;161:1903-1915.
12. Bueno L, Fioramonti J. Protease-activated receptor 2 and gut permeability: A review. Neurogastroenterol Motil 2008;20:580-587.
13. Martinez-Augustin O, Romero-Calvo I, Suarez MD, Zarzuelo A, de Medina FS. Molecular bases of impaired water and ion movements in inflammatory bowel diseases. Inflamm Bowel Dis 2009;15:114-127.
14. Fernandez-Blanco JA, Barbosa S, Sanchez de Medina F, Martinez V, Vergara P. Persistent epithelial barrier alterations in a rat model of postinfectious gut dysfunction. Neurogastroenterol Motil 2011;23:e523-33.
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15. Fernández-Blanco JA, Hollenberg MD, Martínez V, Vergara P. PAR-2-mediated control of barrier function and motility differs between early and late phases of
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postinfectious gut dysfunction in the rat. Am J Physiol Gastrointest Liver Physiol 2013;304:G390-400.
16. Cani PD, Possemiers S, Van de Wiele T, Guiot Y, Everard A, Rottier O, Geurts L,
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Naslain D, Neyrinck A, Lambert DM, Muccioli GG, Delzenne NM. Changes in gut microbiota control inflammation in obese mice through a mechanism involving
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GLP-2-driven improvement of gut permeability. Gut 2009;58:1091-1103.
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17. Castro GA, Fairbairn D. Carbohydrates and lipids in Trichinella spiralis larvae and their utilization in vitro. J Parasitol 1969;55:51-58.
18. Serna H, Porras M, Vergara P. Mast cell stabilizer ketotifen [4-(1-methyl-4fumarate]
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piperidylidene)-4h-benzo[4,5]cyclohepta[1,2-b]thiophen-10(9H)-one
prevents mucosal mast cell hyperplasia and intestinal dysmotility in experimental Trichinella spiralis inflammation in the rat. J Pharmacol Exp Ther 2006;319:11041111.
19. Torrents D, Vergara P. In vivo changes in the intestinal reflexes and the response to CCK in the inflamed small intestine of the rat. Am J Physiol Gastrointest Liver Physiol 2000;279:G543-51.
20. Morimoto M, Morimoto M, Whitmire J, Star RA, Urban JF Jr, Gause WC. Laser Capture Microdissection (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cells. Cold Spring Harb Protoc 2006;
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doi:10.1101/pdb.prot4107.
21. Morimoto M, Morimoto M, Whitmire J, Star RA, Urban JF Jr, Gause WC. cDNA
Harb Protoc 2006; doi:10.1101/pdb.prot4108
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Synthesis and Real-Time PCR Using RNA from Laser-Captured Cells. Cold Spring
22. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)
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method. Nat Protoc 2008;3:1101-1108.
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23. Tanovic A, Jimenez M, Fernandez E. Changes in the inhibitory responses to electrical field stimulation of intestinal smooth muscle from Trichinella spiralis
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24. McDermott JR, Bartram RE, Knight PA, Miller HR, Garrod DR, Grencis RK. Mast cells disrupt epithelial barrier function during enteric nematode infection. Proc Natl
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Acad Sci U S A 2003;100:7761-7766.
25. Stewart GL, Na H, Smart L, Seelig LL Jr. The temporal relationship among antiparasite immune elements expressed during the early phase of infection of the rat with Trichinella spiralis. Parasitol Res 1999;85:672-677.
26. Suzuki T, Sasaki T, Takagi H, Sato K, Ueda K. The effectors responsible for gastrointestinal nematode parasites, Trichinella spiralis, expulsion in rats. Parasitol Res 2008;103:1289-1295.
27. Kitamura Y, Kanakura Y, Sonoda S, Asai H, Nakano T. Mutual phenotypic changes between connective tissue type and mucosal mast cells. Int Arch Allergy Appl Immunol 1987;82:244-248.
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28. MacDonald AJ, Pick J, Bissonnette EY, Befus AD. Rat mucosal mast cells: The cultured bone marrow-derived mast cell is biochemically and functionally
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analogous to its counterpart in vivo. Immunology 1998;93:533-539.
29. Pennock JL, Grencis RK. In vivo exit of c-kit+/CD49d(hi)/beta7+ mucosal mast cell precursors from the bone marrow following infection with the intestinal
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nematode Trichinella spiralis. Blood 2004;103:2655-2660.
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30. Friend DS, Ghildyal N, Gurish MF, Hunt J, Hu X, Austen KF, Stevens RL. Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected
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with Trichinella spiralis. J Immunol 1998;160:5537-5545.
31. Hyland NP, Julio-Pieper M, O'Mahony SM, Bulmer DC, Lee K, Quigley EM, Dinan TG, Cryan JF. A distinct subset of submucosal mast cells undergoes hyperplasia
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following neonatal maternal separation: A role in visceral hypersensitivity? Gut 2009;58:1029-30; author reply 1030-1.
32. Kruger PG, Huntley JF, MacKellar A, Roli J, Newlands GF. Mast cell and mast cell granule phenotypes in normal and Nippostrongylus-infected rats. A qualitative laser confocal microscopic study. APMIS 1997;105:229-237.
33. Friend DS, Ghildyal N, Austen KF, Gurish MF, Matsumoto R, Stevens RL. Mast cells that reside at different locations in the jejunum of mice infected with Trichinella
spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype. J Cell Biol 1996;135:279-290.
34. Lutzelschwab C, Lunderius C, Enerback L, Hellman L. A kinetic analysis of the
brasiliensis-infected rats. Eur J Immunol 1998;28:3730-3737.
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expression of mast cell protease mRNA in the intestines of Nippostrongylus
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35. Prasad S, Mingrino R, Kaukinen K, Hayes KL, Powell RM, MacDonald TT, Collins JE. Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic
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epithelial cells. Lab Invest 2005;85:1139-1162.
36. Zeissig S, Bürgel N, Günzel D, Richter J, Mankertz J, Wahnschaffe U, Kroesen AJ,
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Zeitz M, Fromm M, Schulzke JD. Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active
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crohn's disease. Gut 2007;56:61-72.
37. Kucharzik T, Walsh SV, Chen J, Parkos CA, Nusrat A. Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial
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intercellular junction proteins. Am J Pathol 2001;159:2001-2009.
38. Martínez C, Lobo B, Pigrau M, Ramos L, González-Castro AM, Alonso C, Guilarte M, Guilá M, de Torres I, Azpiroz F, Santos J, Vicario M. Diarrhoea-predominant irritable bowel syndrome: An organic disorder with structural abnormalities in the jejunal epithelial barrier. Gut 2013;62:1160-1168.
Figure Legends Figure 1. Representative microphotographs showing hematoxilin and eosin-stained jejunal slices from a non-infected control (A) and T. spiralis infected animals at post-
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infection days 2 (B), 14 (C) and 30 (D). At day 2 post-infection (B) affectation of the epithelium (villi destruction and inflammatory infiltrate) with slight thickening of the
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muscle layers can be already observed. By day 14 post-infection (C) a generalized destruction of the epithelium with abundant inflammatory infiltrate and thickening of the muscle layers can be observed. At day 30 post-infection (D), notice the restoration
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of the epithelial integrity (without evidence of inflammatory infiltrate but with an increase in the density of globet cells), and the persistent thickening of the muscle
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layers. See also Table 2 for detailed hystopathological scores. Sacle bar: 400 µm.
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Figure 2. Effects of T. spiralis infection on interleukin 6 (IL-6) gene expression. Relative expression of IL-6 mRNA in jejunal mucosa-submucosa (A) and external muscle-serosa samples (B) from control and previously infected rats. Data are mean±SEM of 4-5
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animals per group. *, ***: P> rMCP-8 ≥ rMCP-1 >
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T . s p ir a lis - i n f e c t e d
Journal of Crohn's and Colitis Advance Access published March 28, 2015
Changes in epithelial barrier function in response to parasitic infection: implications for IBD pathogenesis Joan Antoni Fernández-Blancoa, Javier Estéveza, Terez Shea-Donohueb, Vicente
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Martíneza,c,d and Patri Vergaraa,c,d a
Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de
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Barcelona, Barcelona, Spain. bUniversity of Maryland School of Medicine, Department
of Medicine, Division of Gastroenterology & Hepatology and Mucosal Biology Research Center, Baltimore, MD, USA. cInstituto de Neurociencias; Universitat Autònoma de Barcelona, Barcelona, Spain.
d
Centro de Investigación Biomédica en Red de
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Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid,
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Spain.
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Running title: Mast cells and tight junctions in gut barrier dysfunction
Keywords: Epithelial permeability; Mast cell proteinases; Post-infectious; Tight junction; Trichinella spiralis.
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Address for correspondence
Vicente Martínez
Unit of Physiology – Veterinary School Department of Cell Biology, Physiology and Immunology Universitat Autònoma de Barcelona 08193 – Bellaterra, Barcelona Spain Phone: +34 93 581 3834 FAX: +34 93 581 2006 e-mail: [email protected]
Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
Abbreviations: Cq: Quantification cycle FD4: 4KD Fluorescein Isothiocyanate-Labeled Dextran G: Conductance
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GLP-2: Glucagon-like peptide 2 IBD: Inflammatory bowel disease
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IBS: Irritable bowel syndrome IL-6: Interleukin 6 JAM-1: Junctional adhesion molecule 1
MC: Mast cell
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MLCK: Myosin light chain kinase
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LCM: Laser capture microdissection
PAR: Proteinase-activated receptor
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rMCP: Rat mast cell proteinase TJ: Tight junction
VCU: Villus-crypt unit
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ZO: Zonula occludens
Abstract Background and Aims: Mast cells (MCs) are implicated in epithelial barrier alterations that characterize inflammatory and functional bowel disorders. In this study, we
proteins kinetics in a rat model of postinfectious gut dysfunction.
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describe mast cell proteinases (chymases and tryptases) and tight junction (TJ)
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Methods: Jejunal tissues of control and Trichinella spiralis-infected rats were used. Inflammation-related changes in MCs and the expression of TJ-related proteins were evaluated by immunostaining and RT-qPCR. Epithelial barrier function was assessed in vitro (Ussing chambers) and in vivo.
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Results: After infection, intestinal inflammation was associated with a generalized overexpression of MC chymases, peaking between days 6 and 14. Thereafter, a
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mucosal MC hyperplasia and a late increase in connective tissue MC counts were observed. From day 2 post-infection, TJ proteins occludin and claudin-3 expression was
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down-regulated while the pore-forming protein claudin-2 was overexpressed. The expression of proglucagon, precursor of the barrier-enhancing factor glucagon-like peptide-2, was reduced. These changes were associated with an increase in epithelial
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permeability, both in vitro and in vivo.
Conclusions: Proteinases expression and location of mucosal and connective tissue MCs indicate a time-related pattern in the maturation of intestinal MCs following T. spiralis infection. Altered expression of TJ-related proteins is consistent with a loss of epithelial tightness, and provides a molecular mechanism for the enhanced epithelial
permeability observed in inflammatory conditions of the gut.
1. Introduction Mast cells (MCs) participate in the pathophysiology of inflammatory and functional gut disorders. Indeed, an increased number of intestinal MCs, together with a persistent state of activation, have been described for both inflammatory bowel disease (IBD)
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and irritable bowel syndrome (IBS).1-3 MC mediators can modulate a variety of
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secretomotor and sensory functions within the gut, including epithelial barrier function. There is evidence that MCs participate in the enhancement of intestinal permeability associated with IBD and IBS.4
Regardless of the species studied, mature MCs are classified taking into account
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their tissue location (mucosa vs. connective tissue) and their enzymatic characteristics, particularly their content of serine proteinases (chymases and tryptases). Accordingly,
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in rodents, MCs are usually classified as mucosal MCs or connective tissue MCs. In the intestine, mucosal MCs are preferentially located in the epithelium and the lamina
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propria, whereas connective tissue MCs are situated in the submucosa and the serosa layers. In the rat, mucosal MCs express predominantly chymases; in particular, rat mast cell proteinase (rMCP)-2, -3, -4 and the three members of the rMCP-8 subfamily
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(rMCP-8, -9 and -10). In contrast, rat connective tissue MCs express two chymases, rMCP-1 and -5, and two tryptases, rMCP-6 and -7.5,6 Since each proteinase cleaves different target substrates, the profile of serine
proteinases expressed by MCs conditions the potential biological effects to be observed in states of cell activation-degranulation.5,6 In the gut, one of the main effects of MC proteinases is to modulate epithelial permeability.7-11 MC proteinases are able to directly alter the expression and location of epithelial tight junction (TJ) proteins.8,10 At the same time, they also regulate TJs indirectly, by their effects over scaffolding
proteins, like the zonula occludens (ZO) proteins, and the cytoskeleton. 8,12 Overall, MC activation and the associated proteinases release disrupt the epithelial barrier leading to an increase in epithelial permeability. Within the gut, increased epithelial permeability might act as a defensive
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mechanism, favoring the movement of fluids to the lumen and facilitating intestinal
motility, as a way to eliminate potentially harmful luminal contents.10,13 However, in
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parallel, epithelial barrier disruption may also facilitate passage of luminal antigens across the mucosal barrier, activating the immune system and generating an inflammatory-like state.4 Therefore, MC proteinases-mediated effects on the epithelial
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barrier may contribute to the underlying pathophysiological alterations observed in inflammatory and functional bowel disorders.
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The main goal of the present study was to characterize the changes in MC populations and epithelial barrier function in a rat model of postinfectious gut
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dysfunction. Trichinella spiralis infection in the rat is an accepted model of inflammatory and functional gastrointestinal diseases characterized by the presence of jejunal MC hyperplasia and epithelial barrier dysfunctions.14,15 During acute
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inflammation and in the recovery postinfectious phase (days 2 to 30 post-infection), we assessed the kinetics of MC hyperplasia. In this regard, we emphasized the study of the appearance of phenotypically differentiated MC populations and time-related changes in MC serine proteinases gene expression. In addition, we assessed infectionassociated epithelial barrier alterations, both in vitro and in vivo, and changes in the expression of TJ-related proteins, as a possible underlying mechanism. Finally, we evaluated the expression of proglucagon, as precursor of the barrier-enhancing factor glucagon-like peptide 2 (GLP-2).16
2. Materials and Methods 2.1 Animals Adult male OFA Sprague-Dawley rats (7-8 weeks old, 250-275 g; Charles River Laboratories, Lyon, France) were used. Rats were housed under conventional
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conditions in a light (12h/12h light-dark cycle) and temperature controlled (20-22°C)
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room, with access to tap water and laboratory rat chow ad libitum. Animals were kept
in groups of two to three per cage. All experimental procedures were approved by the Ethics Committee of the Universitat Autònoma de Barcelona and the Generalitat de
2.2 Trichinella spiralis Infection
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Catalunya (protocols 5352 and 5564).
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Muscle-stage larvae of T. spiralis were obtained from infected CD1 mice as previously described.17-19 Rats were infected at 8-9 weeks of age by administration of 7.500 T.
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spiralis larvae suspended in 1 mL of saline by oral gavage and studies were performed on days 2, 6, 14 and 30 post-infection. Age- and time-matched rats dosed orally with 1 mL of saline were used as controls. During this time, animals were regularly monitored
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for clinical signs and body weight changes. Normal course of the infection was confirmed by a significant decrease of body weight after T. spiralis infection compared
with controls, with a peak reduction on days 8-10 and a subsequent increase over time, as previously described by us.14,15,19
2.3 Tissue Sampling At the time of the experiments, animals were euthanatized by decapitation, except otherwise stated. A laparotomy was performed and jejunal samples (beginning 10 cm
distal to the ligament of Treitz) were excised. Immediately, the intestines were flushed and placed in ice cold oxygenated Krebs buffer [(in mM): 115.48 NaCl, 21.90 NaHCO3; 4.61 KCl; 1.14 NaH2PO4; 2.50 CaCl2; 1.16 MgSO4 (pH: 7.3-7.4)] containing 10mM glucose. Jejunal segments were used to perform in vitro epithelial barrier function
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studies or preserved for morphological or molecular biology studies (see below). In
some cases, the mucosa and submucosa ( 2cm) were scraped off with blunt-edged
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glass slides obtaining the mucosa-submucosa and underlying smooth muscle-serosa samples separately. For laser capture microdissection (LCM) studies, jejunal samples were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe B.V.,
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Zoeterwoude, The Netherlands) in a cryomold (Peel-A-Way disposable embedding molds S-22; Polysciences Inc., Warrington, PA., USA). The mucosa-submucosa and
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smooth muscle-serosa samples and the cryoblocks were frozen in liquid nitrogen and stored at −80°C until analysis. Samples for histological and immunostaining studies
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were obtained and fixed in 4% paraformaldehyde in phosphate buffer for 24 h. Thereafter, fixed samples were processed routinely for paraffin embedding and 5 µm
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sections were obtained for H&E staining or immunohistochemistry.
2.4 Histopathological Studies H&E-stained slides were evaluated in a blinded fashion by two independent investigators. A histological score based on the epithelial structure (0-3), presence of edema (0-3), presence of ulcerations (0-3), presence of inflammatory infiltrate (0-3), relative density of goblet cells (0-3) and relative thickness of the intestinal wall (0-3) was assigned to each animal (maximal score of 18).
2.5 Epithelial Barrier Function Measurements 2.5.1 In vitro assessment of epithelial barrier function (Ussing chambers)
Jejunal
segments stripped of the outer muscle layers and myenteric plexus were mounted in Ussing chambers, following procedures previously described.14,15 Tissues were bathed
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bilaterally with 5 mL of 37°C oxygenated Krebs buffer. The basolateral buffer contained
10 mM glucose, osmotically balanced with 10 mM mannitol in the apical buffer. A zero
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potential difference was maintained injecting the required short-circuit current in each
moment. A voltage step of 1mV was applied every 5 min and the change in shortcircuit current was used to calculate tissue conductance (G) by Ohm’s law, as a
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measure of intestinal permeability. Tissues were allowed to stabilize for 15-25 min before baseline values were recorded. Data were digitized with an analog-to-digital
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converter and measurements were recorded and analyzed with Acqknowledge computer software. G was normalized for the exposed surface area (0.67 cm2).
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Paracellular permeability was further assessed by measuring mucosal to basolateral flux of fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4kD (FD4; Sigma Aldrich, St Louis, MO, USA). After stabilization,
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FD4 was added to the mucosal reservoir to a final concentration of 2.5x10-4 M. Basolateral samples (250 μL; replaced by 250 µL of appropriate buffer solution) were taken for subsequent fluorescence measurement (Infinite F200; Tecan, Crailsheim, Germany), with an excitation wavelength of 485 nm and an emission wavelength of 535 nm, against a standard curve. Readings are expressed as a percentage (%) of the total amount of FD4 added to the mucosal reservoir. Hydroelectrolytic transport capacity of the tissues was tested at the end of the permeability experiments by assessing responses to CCh (10 -4 M) added to the
basolateral side. Tissues with abnormal baseline values of electrophysiological parameters or with a lack of response to CCh were considered damaged and were excluded. Overall, these represented less than 5% of the preparations tested.
Non-infected controls and T.
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2.5.2 In vivo assessment of epithelial barrier function
spiralis-infected animals (at days 14 and 30 post-infection) were dosed orally with FD4
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(10 mg/rat, 0.2 mL) and returned to their home cages. Thirty minutes later, each animal was deeply anesthetized with isoflurane (Isoflo®, Esteve Veterinaria, Barcelona, Spain) and a blood sample was obtained by intracardiac puncture. Samples were
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maintained in ice in a dark box until processing. Serum was obtained by centrifugation (3000xg, 10 min, 4°C) and the concentration of FD4 (µg/mL) determined by
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fluorimetry, as described above for in vitro experiments.
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2.6 Immunohistochemistry for rat mast cell proteinases 2 (rMCP-2) and 6 (rMCP-6) and mast cells counts
Immunodetection of rMCP-2 and rMCP-6 was carried out on jejunal sections following
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standard immunohistochemical procedures using the monoclonal antibodies MS-RM4 (1:500; Moredun Animal Health, Edinburgh, UK) and sc-32473 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively, as previously described.14,15 Specificity of the staining was confirmed by omission of the primary antibody. Stained mucosal MCs (rMCP-2 positive) were counted in at least 20 well-oriented
villus-crypt units (VCU) per animal, at X400 magnification, and cell counts expressed as mucosal MCs/VCU. The total number of stained connective tissue MCs (rMCP-6 positive) in the submucosa, external smooth muscle and serosa areas was determined
in two complete tissue sections of the jejunum for each animal (X600). Connective tissue MC counting was normalized for the surface area of submucosa, external smooth muscle and serosa layers, as evaluated in digital images. Cell counting was performed using an Axioskop 40 microscope (Carl Zeiss, Jena, Germany; equipped with
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a digital camera, Zeiss AxioCam MRm; image analysis software: Zeiss Axiovision
on coded slides to avoid observer’s bias.
2.7 Immunohistochemistry for claudin 2
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Release 4.8.1). Counting and analysis of all data were performed in a blinded manner
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For claudin 2 immunohistochemistry, tissue sections were rehydrated, and microwave antigen retrieval was performed (10 mM Tris Base, 1 mM EDTA solution, 10 mM, pH 9;
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2 cycles of 5 min, 800 W). Thereafter, samples were incubated for 40 min in H 2O2 (5%), for inhibiting endogenous peroxidases, followed by a 30 min incubation with horse
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serum. Subsequently, slides were incubated (overnight, 4°C) with mouse monoclonal anti-claudin 2 antibody (1:2000; ref.: 32-5600. Invitrogen, Camarillo, CA, USA). Finally, sections were incubated (overnight, 5°C) with biotinylated horse anti-mouse IgG
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(1:200; Santa Cruz Biotechnology). The avidin/peroxidase kit (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) was used for detection, and the antigenantibody complexes were revealed using 3,3’-diaminobenzidine (SK-4100 DAB; Vector Laboratories). Sections were counterstained with hematoxylin. Specificity of the staining was confirmed by omission of the primary antibody. Coded slides were observed with an Axioskop 40 microscope. Claudin 2 immunoreactivity was quantified applying a semiquantitative score (0: no immunoreactivity; 1: some immunoreactivity restricted to the epithelial crypts; 2:
intense immunoreactivity restricted to the majority/all epithelial crypts; 3: intense immunoreactivity within the epithelial crypts and also extending to some of the villi; 4: intense immunoreactivity within the epithelial crypts and extending along the majority of the villi). For quantification, 15 randomly selected fields covering the whole
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thickness of the jejunal mucosa, from at least two tissue sections for animal, were
scored by two independent observers. A final score (0-4) for each animal was
performed on coded slides to avoid any bias.
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2.8 Laser capture microdissection (LCM)
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calculated as the mean of the scores assigned by each observer. All procedures were
LCM was performed as previously described20, with minor modifications. Briefly, 4 µm
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sections were obtained from jejunal cryoblocks using a cryostat and placed in plain uncoated pre-cleaned slides (Corning Inc., Corning, NY, USA) maintained in dry ice, and
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stored at -80°C until needed. Cryosectioned tissues were stained with H&E and dehydrated. Thereafter, LCM was performed with an ArcturusXT microdissection system (Arcturus Engineering Inc., Mountain View, CA, USA). Cells were captured from
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the lamina propria, the submucosa and the external smooth muscle layers, transferred to CapSure Macro LCM Caps (LCM 0211; Arcturus Engineering Inc.) and processed
immediately (see below).
2.9 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Total RNA extractions from jejunal mucosa-submucosa and smooth muscle-serosa samples were performed using TRI reagent and the Ribopure RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA). RNA was quantified by Nanodrop (Nanodrop
Technologies, Rockland, DE, USA) and 1 µg of RNA was reverse-transcribed in a 20 µL reaction volume for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For LCM samples, RNA was extracted and reverse-transcribed following
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previously described methods20,21 , with minor modifications. Briefly, thermoplastic films containing microdissected cells were incubated with RNA denaturing buffer
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(Stratagene, La Jolla, CA, USA) and β-mercaptoethanol. Then the phenol-chloroformbased method was used for the extraction and RNA was precipitated with sodium acetate aided by glycogen and ice-cold isopropanol. Thereafter, RNA was reverse-
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transcribed with the SuperScript II reverse transcriptase (Invitrogen, Grand Island, NY, USA) using dNTP (0.5 mM each) and random primers (10ng/μl; Hexanucleotide Mix,
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10x conc.; Roche, Penzberg, Germany). Complementary DNA obtained was then preamplified with the TaqMan® preAmp Master Mix Kit and validated TaqMan®
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probes (Applied Biosystems; Table 1), following manufacturers' instructions. The temperature profile was 25°C for 10 min, 42°C for 50 min and 70°C for 15 min for reverse transcription and 95°C for 10 min; 14 cycles of 95°C for 15 s, 60°C for 4 min for
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cDNA preamplification.
Validated TaqMan® gene expressions assays with hydrolysis probes for rat mast
cell proteinases, epithelial barrier function-related proteins, interleukin-6 (IL-6) and reference genes were used (Applied Biosystems) (Table 1). PCR reaction mixtures were transferred to clear 384-well reaction plates (HSP-3801; Bio-Rad, Barcelona, Spain; 20 µL/well), sealed by adhesives and incubated on a CFX384 Touch real-time PCR
detection system (Bio-Rad). Amplification conditions were as follows: 95°C for 10 min; 40 cycles of 95°C for 15 s, 60°C for 1min. Fluorescence signals measured during
amplification were processed after amplification. Bio-Rad CFX Manager 2.1 software was used to obtain the quantification cycle (Cq) for each sample. Each sample was run in triplicate and data were analyzed by the comparative Cq method (2 -∆∆Cq), as previously described.22 Rps18 and Actb were tested as reference genes. Actb
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expression levels were used for normalizing the mRNA levels of the target gens
because of their constancy across the different experimental groups. Controls of
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analytical specificity included omission of reverse transcriptase, to exclude contamination with genomic DNA, and no-template controls, omitting the cDNA.
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2.10 Statistical analysis
All data are expressed as mean ± SEM. A robust analysis (one iteration) was used to
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obtain mean ± SEM for RT-qPCR data. Comparisons between multiple groups were performed using a one-way ANOVA, a two-way ANOVA or a Kruskal-Wallis ANOVA, as
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appropriate; followed, when necessary, by a Newman-Keuls or a Dunns multiple comparisons test. In all cases, results were considered statistically significant when
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prMCP-9>rMCP-2>rMCP-6. A similar pattern was detected in smooth muscle-serosa samples (Fig. 5); however, in this case, expression of rMCP-6 was up-regulated, showing a progressive time-
related increase, with the highest expression levels detected by day 30 post-infection (Fig. 5). Overall, relative expression changes were: rMCP-1>rMCP-4>rMCP-6~rMCP8>rMCP-9>rMCP-5~rMCP-10>rMCP-2. Gene expression analysis in LCM samples obtained from the lamina propria, the
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submucosa or the smooth muscle, in which areas containing MCs were selected, were, in general, in consonance with results from whole tissue samples. LCM samples
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confirmed and up-regulation of rMCP-1 in lamina propria and submucosa and of rMCP2 in submucosa (where mucosal MCs are preferentially found) (Fig. 6). Similarly, LCM samples from the muscle layers confirmed the up-regulation on rMCP-6 observed in
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smooth muscle-serosa samples. Moreover, although rMCP-6 was not detected in control conditions, this proteinase was expressed respectively in 2 and 4 (out of 5) of
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(Fig. 6).
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the lamina propria and the submucosa samples obtained from T. spiralis-infected rats
3.4 Effects of T. spiralis infection on epithelial barrier function in vitro and in vivo At days 14 and 30 post-infection, an increase in epithelial conductance was observed
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(Fig. 7A). In concordance, in vitro mucosal to basolateral passage of FD4 across jejunal segments was significantly increased in jejunal sheets from T. spiralis infected animals compared with non-infected controls (Fig. 7B). These results were further confirmed in in vivo conditions, where intestinal passage of FD4 was increased by 2.5- and 2.2-fold at 14 and 30 days post-infection, respectively, when compared with control conditions (both P occludin > JAM-1 >> tricellulin > ZO-1 > claudin 2.
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Similar order of magnitude in the relative expression levels was maintained after
T. spiralis infection. However, time-dependent changes in the absolute levels of
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expression were detected, and both up-regulation and down-regulation of genes could
be observed depending upon the protein considered. Claudin 2 and ZO-1 showed an up-regulation; while genes encoding for claudin 3, occludin and MLCK were down-
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regulated (Fig. 8). Overall, expression changes were fast in time, as maximal variations were observed between 2 and 6 days post-infection (Fig. 8). JAM-1 and tricellulin
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expression levels were not affected.
The major relative changes in gene expression were observed for claudin 2,
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which was up-regulated by 20-30-fold between days 2 and 6 post-infection. To further understand the functional significance of these changes, we assessed claudin 2 expression in jejunal samples of non-infected and T. spiralis infected animals. In
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control conditions, claudin 2 immunoreactivity was observed restricted to the crypts of the jejunal epithelium (Fig. 9A). In T. spiralis infected animals, claudin 2 immunoreactivity could be observed within the crypts, but also along the villi (Fig. 9B). Semi-quantification of claudin 2 immunoreactivity pointed to a relative increase in protein content by days 2 and 14 post-infection, with normalization by day 30; however, statistical significance was not reached (Fig. 9D). In some cases, unspecific staining was observed in unidentified cells of the lamina propria. Immunoreactivity
disappeared when the primary antibody was omitted (Fig. 9C), thus confirming the specificity of the staining. Gene expression of GLP-2 precursor, proglucagon, showed a clear tendency to be
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down-regulated over time [ANOVA: F(4.19)=2.67, P=0.06; Fig. 8].
4. Discussion
The present study shows time-associated changes in intestinal MC populations and TJ-
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related proteins in a rat model of gut dysfunction induced by T. spiralis infection. Timedependent variations in the expression of MC proteinases indicate an early mucosal
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MC hyperplasia and a later increase in connective tissue MCs. Simultaneously, alterations in the expression of TJ-related proteins, potentially modulated by MC
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proteinases, are associated with an increase of epithelial permeability. One of the main characteristics of the T. spiralis infection model in rats is the presence of a long-lasting postinfectious infiltrate of mucosal MC populating the mucosa-submucosa of the small intestine.14 Here we show that, in addition to the
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previously well-known mucosal MCs hyperplasia, a hyperplasia of connective tissue
MCs (rMCP-6 positive) also occurs in this model. Moreover, we show that infiltration of mucosal and connective tissue MCs occurs at different times along the postinfectious process. Overall, gene expression data suggest that in early phases (by day 2) there is an activation of resident MCs. This early MC activation may trigger the release of mediators and contribute to the recruitment of new MCs, leading to the persistent MC hyperplasia observed from day 6 post-infection. Changes in the expression of rMCP-2,
particularly in LCM-enriched samples, indicate that MC precursors colonize the mucosa-submucosa and differentiate towards a mucosal phenotype in the early postinfectious phase. This coincides with the intestinal phase of the infection and may represent an early defensive response to the presence of parasites in the mucosa.24-26
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At later phases (from day 14 post-infection), MCs appear to infiltrate into external
muscle layers. In this case, the morphology and gene expression analyses point to a
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preferential differentiation towards connective tissue MCs, as indicated by the
selective increase in rMCP-6 expression. At this stage, scattered rMCP-2-positive cells were also observed within the muscle layers, suggesting recruitment into smooth
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muscle of submucosal MCs with an immature phenotype. These rMCP-2-expressing MC may be precursors of fully differentiated rMCP-6-positive connective tissue MCs.27Co-expression of rMCP-2/rMCP-6, while cells migrate across the muscle to generate
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29
mature connective tissue MCs, is similar to that observed in mouse MCs, which co-
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express mouse mast cell proteinases 2 and 6 in the mucosa after T. spiralis infection.30 As in the epithelium,24 MCs infiltrating the muscle layers might represent a defensive response towards the penetration of T. spiralis larvae into the gut wall. It is reasonable
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to assume that MCs infiltrate contribute to smooth muscle long lasting hyperplasia and hypercontractile responses observed in infected animals.18,19 Some overlapping in proteinase expression was observed between mucosal and
connective tissue MCs. Indeed, both rMCP-1 and rMCP-2 were detected in LCM samples from lamina propria, submucosa and smooth muscle. This agrees with data showing the expression of chymases (rMCP-1 and mMCP-5), classically associated with connective tissue MCs, in both mucosal and transitional populations of MCs32-34 and might be related with the common origin of connective tissue and mucosal MCs. Taken
together, these observations support the view that tissue location of MCs alone does not fully predict the phenotypic pattern of proteinases expression.31,32 According to our data, only the expression of the tryptase rMCP-6 seems to be fully specific of mature connective tissue MCs, suggesting that this proteinase is a selective phenotypic marker
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for these cells.
The variable pattern of expression of substrate-specific MC proteinases may be
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related to functional changes along the postinfectious phase. One of the major effects of substrate cleavage mediated by MC proteinases is the modulation of epithelial permeability, by controlling the expression of TJ transmembrane components.8,9,24 In
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this respect, our studies show a time-related down-regulation of transmembrane junctional proteins with adhesive properties, namely claudin 3 and occludin, and an
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over-expression of the pore-forming protein claudin 2. In particular, the lack of occludin would result in an increase of paracellular permeability. Occludin expression
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has been associated with the release of the barrier-enhancement factor GLP-2,16 and its precursor proglucagon, which tended to be down-regulated after the infection. In addition, mucosal MCs hyperactivity and increased rMCP-2 release would favor
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occludin degradation.8,14,18 This is in agreement with a down-regulation of occludin expression and the subsequent increase in epithelial permeability observed in functional studies, both in vivo and in vitro. In the same way, the up-regulation of claudin 2 might also contribute to functional alterations in epithelial barrier function, particularly at early states of the infectious process. In this case, gene expression results were accompanied by a slight increase in the amount of claudin 2 immunoreactivity in the jejunal epithelium, with a widespread distribution along the villi. These data are consistent with the contribution of claudin 2 to the increased
epithelial permeability. Interestingly, similar structural and functional alterations have been observed in patients with inflammatory and functional gastrointestinal disorders.35-38 Altered epithelial permeability facilitates passage of luminal antigens,
including the maintenance of a persistent MCs infiltrate.
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thus contributing to the maintenance of a state of chronic, low-grade, inflammation,
While mucosal MCs-derived proteinases can be important modulating barrier
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function, a role for connective tissue MCs-derived proteinases seems least clear. Nevertheless, proteinases released within the smooth muscle and the myenteric plexus may modulate the activity of proteinase-activated receptors (PAR), leading to
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altered sensory and motor functions. Indeed, we previously demonstrated altered motor responses and changes in the expression of PAR-2 during T. spiralis infection in
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rats15, thus suggesting that connective tissue MCs might play a functional role. In summary, the present study reveals that, in addition to mucosal MCs,
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connective tissue MCs are also a significant component of the response to T. spiralis infection in rats. There is a differential time profile in the infiltration of mucosal and connective tissue MCs as well as in the expression of MC serine proteinases in the rat
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jejunum after T. spiralis infection. MC infiltrates are likely to be active components in the epithelial and motor defensive responses generated against the infection. We also show that long-lasting postinfectious increases in intestinal permeability reflect a dysregulation in the expression of TJ transmembrane proteins, which may be attributed, in part, to the effects of MC-derived proteinases. From a translational point of view, these observations suggest that MC proteinases and TJs represent potential pharmacological targets for the treatment of inflammatory and functional gastrointestinal disorders.
Conflict of interest The authors have no conflict of interest to declare.
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Acknowledgements and Author Contributions A. Acosta, E. Martínez, F. Jardí, M. Aguilera, V. Grinchuk, L. Notari, S. Yan, A. Zhao and
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S. Bartolomé are thanked for their assistance in different parts of this work. This study was supported by grants 2014SGR00789 from the Generalitat de Catalunya (Spain), and BFU2009-08229 and BFU2010-15401 from Ministerio de Ciencia e Innovación
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(Spain). JAF-B personal support: FPU program (AP2007–01619) from Ministerio de Ciencia e Innovación (Spain).
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JAF-B designed and performed experiments, analyzed data and wrote the paper. JE designed and performed experiments and analyzed data. VM designed and
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performed experiments, analyzed data and wrote the paper. PV designed experiments
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and wrote the paper. TS-D designed experiments and wrote the paper.
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Figure Legends Figure 1. Representative microphotographs showing hematoxilin and eosin-stained jejunal slices from a non-infected control (A) and T. spiralis infected animals at post-
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infection days 2 (B), 14 (C) and 30 (D). At day 2 post-infection (B) affectation of the epithelium (villi destruction and inflammatory infiltrate) with slight thickening of the
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muscle layers can be already observed. By day 14 post-infection (C) a generalized destruction of the epithelium with abundant inflammatory infiltrate and thickening of the muscle layers can be observed. At day 30 post-infection (D), notice the restoration
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of the epithelial integrity (without evidence of inflammatory infiltrate but with an increase in the density of globet cells), and the persistent thickening of the muscle
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layers. See also Table 2 for detailed hystopathological scores. Sacle bar: 400 µm.
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Figure 2. Effects of T. spiralis infection on interleukin 6 (IL-6) gene expression. Relative expression of IL-6 mRNA in jejunal mucosa-submucosa (A) and external muscle-serosa samples (B) from control and previously infected rats. Data are mean±SEM of 4-5
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animals per group. *, ***: P> rMCP-8 ≥ rMCP-1 >
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