Joan Antoni Fernandez-Blanco

Persistent epithelial barrier alterations in a rat model of postinfectious gut dysfunction

Background  Mucosal mast cells (MMCs), epithelial barrier function (EBF) and the enteric nervous system (ENS) are interactive factors in the pathophysiology of functional gastrointestinal disorders. We characterized postinfectious EBF alterations in the Trichinella spiralis infection model of MMC‐dependent intestinal dysfunction in rats.

Changes in Epithelial Barrier Function in Response to Parasitic Infection: Implications for IBD Pathogenesis

BACKGROUND AND AIMS Mast cells [MCs] are implicated in epithelial barrier alterations that characterize inflammatory and functional bowel disorders. In this study, we describe mast cell proteinases [chymases and tryptases] and tight junction [TJ] proteins kinetics in a rat model of postinfectious gut dysfunction. METHODS Jejunal tissues of control and -infected rats were used. Inflammation-related changes in MCs and the expression of TJ-related proteins were evaluated by immunostaining and reverse transcription-quantitative polymerase chain reaction. Epithelial barrier function was assessed in vitro (Ussing chambers) and in vivo. RESULTS After infection, intestinal inflammation was associated with a generalized overexpression of MC chymases, peaking between Days 6 and 14. Thereafter, a 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 down-regulated whereas 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 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 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.

Plasticity of dorsal root ganglion neurons in a rat model of post-infectious gut dysfunction: potential implication of nerve growth factor

Abstract Objective. Intestinal infections are suggested as a risk factor for the development of irritable bowel syndrome (IBS)-like visceral hypersensitivity. The mechanisms implicated might involve long-term changes in visceral afferents, with implication of nerve growth factor (NGF). We explored plastic changes in dorsal root ganglia (DRGs) receiving innervation from the gut and the potential implication of NGF in a rat model of IBS-like post-infectious gut dysfunction. Materials and methods. Rats were infected with Trichinella spiralis larvae. Thirty days post-infection, inflammatory markers, including interleukins (ILs) and mucosal mast cell infiltration (rat mast cell protease II [RMCPII]), and NGF and TrkA expression was determined in the jejunum and colon (RT-qPCR). In the same animals, morphometry (neuronal body size) and NGF content (immunofluorescence) were assessed in thoracolumbar DRG neurons. Results. In infected animals, a low-grade inflammatory-like response, characterized by up-regulated levels of RMCPII and IL-6, was observed in the jejunum and colon. TrkA expression was increased in the jejunum, whereas the colon showed a slight reduction. NGF levels remained unaltered regardless the gut region. Overall, the mean cross-sectional area of DRG neurons was increased in T. spiralis-infected animals, with a reduction in both TrkA and NGF staining. Conclusions. Results suggest that during T. spiralis infection in rats, there is a remodeling of sensory afferents that might imply a NGF-mediated mechanism. Plastic changes in sensory afferents might mediate the long-lasting functional alterations that characterize this model of IBS. Similar mechanisms might be operating in patients with post-infectious-IBS.

Persistent alterations in colonic afferent innervation in a rat model of postinfectious gut dysfunction: Role for changes in peripheral neurotrophic factors

Visceral hypersensitivity in the inflamed gut is related partly to the effects of peripheral neurotrophic factors (NTFs) on local afferent neurons. However, alterations in sensory afferents of distant areas remain unexplored. Using the Trichinella spiralis infection model, which causes a jejunitis, we investigated the remodeling of colonic afferents and the potential role of NTFs.

Mo2036 Remodeling of Colonic Sensory Afferents in a Rat Model of Post-Infectious Gut Dysfunction: Implication of Neurotrophic Factors

Background: Inflammation-induced visceral hypersensitivity is associated, at least partially, to peripheral sensitization of sensory afferents by locally released neurotrophic factors. Aims: Here, we explore the potential role of neurotrophic factors in long-term changes in visceral afferents innervating non-primary areas of inflammation (colon) in a rat model of Trichinella spiralis (TS)-induced gut dysfunction. Methods: Inflammatory-like changes, mucosal mast cells (MMCs) dynamics, and expression of nerve growth factor (NGF) and glial cell linederived neurotrophic factors (GDNF, artemin and neurturin) were determined in the colon of control and TS-infected rats, up to day 30 post-infection (PI). Markers of sensory afferents (high affinity nerve growth factor receptor, TrkA; GDNF family receptor alpha 3, GFRα3; calcitonin gene-related peptide, CGRP; and transient receptor potential vanilloid channel1, TRPV1) were assessed in thoracolumbar (T12-L2, TL) and lumbosacral (L6-S2, LS) DRGs. Functionality of colonic afferents at 14 and 30 days PI was determined by assessing changes in TRPV1 levels in TL and LS DRGs following intracolonic capsaicin. Results: TS infection induced a primary jejunitis with concomitant inflammatory-like changes in the colon: an increase in histological scores, IL-13 up-regulation and MMC infiltration (Table 1). Inflammation was partly resolved at day 30 PI, except for a persistent MMC infiltrate (Table 1). Colonic expression of neurotrophic factors showed a transient reduction by day 14 PI (25%50%), with the exception of artemin that showed a biphasic response with an up-regulation (by 30%; P<0.05) at day 30 PI (Table 2). Overall, TS infection did not affect DRG expression of none of the genes assessed, except for a transient down-regulation of TPRV1 in TL DRGs at day 14 PI. Afferent stimulation with intracolonic capsaicin induced a down-regulation of TRPV1 expression in TL and LS DRGs, an effect significantly enhanced in LS DRGs of TSinfected rats, at either 14 or 30 days PI (Table 2). Intracolonic capsaicin also up-regulated peripheral artemin in non-infected rats, an effect absent in TS-infected animals at day 30 PI (Table 2). Capsaicin-induced changes in colonic expression of artemin correlated negatively with the down-regulation of TRPV1 in LS DRGs. Conclusions: During intestinal inflammation, changes in expression of neurotrophic factors and remodeling of visceral afferents are observed outside the primary region affected by the inflammatory insult. These observations suggest that extended remodelation of sensory afferents, probably related to local changes in neurotropins, might occur during inflammatory conditions of the gut. Similar mechanisms might be operating in states of widespread alterations of visceral sensitivity. Table 1 Colonic inflammation-related changes during T. spiralis infection

114 Altered Expression of Tight Junction-Related Proteins and Increased Intestinal Permeability in a Rat Model of Post-Infectious Gut Dysfunction

Background & Aims: Reduced Kitl from the microenvironment and consequent loss of Kit expression contributes to ICC depletion e.g. in diabetic gastroparesis. It has been hypothesized that ICC that have lost Kit may be able to regain their phenotype. However, the fate of ICC has been difficult to track. Previously we developed conditionally immortalized cell lines (e.g., ICL2A) from Kithigh, hematopoietic marker-negative cells purified from gastric muscles of H-2Kb-tsA58 mice and used them to characterize the effects of chronic Kitl deprivation (DDW 2009, #270). These cells, while maintaining ICC-like morphology and ultrastructure, underwent progressive dedifferentiation resulting in a Kitlow/-Ano1low/-Cd44+Cd34phenotype and aberrant electrical activity consisting of small, highly irregular oscillations and rare, abnormally long slow waves. Resupplying membrane-bound Kitl did not increase ICCspecific gene expression. Here, we investigated whether epigenetic silencing could underlie these changes and whether pharmacological de-repression could restore ICC phenotype and function. Methods: ICL2A cells were treated with 2.5μM adenosine dialdehyde (Adox), an Ezh2 histone lysine methyltransferase inhibitor (HKMTI); 10μM BIX-01294, a G9a HKMTI; 1μM SAHA, a class I-II histone deacetylase inhibitor; or 5μM RG-108, a DNA methyltransferase inhibitor. Gene expression was studied by qRT-PCR and flow cytometry. Electrical activity was studied by whole-cell patch clamp in the current clamp mode. Occupancy of the Kit promoter (-1 to -1000 bp) by Ezh2 and Suz12, members of PcG repressive complex (PRC) 2, Ring1b, the histone ubiquityltransferase component of PRC1, as well as by the PRC2-established mark H3K27me3 (histone H3 trimethylated on K27) and the PRC1dependent mark H2AK119ub1 (monoubiquitylated histone H2A K119) was quantified by ChIP. Results: BIX-0129, SAHA and RG-108 applied for 2 weeks did not stimulate Kit protein expression. In contrast, Adox increased both Kit mRNA (P=0.001) and cell surface Kit and Cd44 expression. Ano1 mRNA also increased significantly (P,0.001), although its levels remained low. The role of PcG-mediated repression was verified by detecting occupancy of the 1000-bp Kit promoter by Ezh2, Suz12, Ring1b, H3K27me3 and H2AK119ub1. Adox reduced H3K27me3 and H2AK119ub1 occupancy by 47±6% and 33±3%, respectively. In Adox-treated ICL2A cells, current clamp recordings revealed steady, rhythmic, slow wavelike oscillations in 3 of 15 cells that were not seen in 64 control cells. Conclusions: Similarly to KitlowCd34+ ICC stem cells, Kit expression is repressed by PcG proteins in ICC chronically deprived of Kitl signaling. Inhibition of PcG activity may help restore ICC and their function in conditions that negatively impact the ICCmicroenvironment such as diabetic gastroparesis. Grant support: NIH DK58185.