Silvia Melgar

Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease.

Dextran sulfate sodium (DSS)-induced colitis is one of the most frequently used rodent models for inflammatory bowel disease (IBD). The aim of this study was to validate the murine DSS-induced colitis model using four therapeutic agents for IBD. C57BL/6 mice were exposed to 3% DSS for 5days followed by 7-9 days of water (acute inflammation) or 20-31 days of water (chronic phase). Clinical symptoms, plasma and colonic inflammatory markers and histology were assessed for the efficacy of cyclosporine A (CsA), methotrexate or anti-IL-12p40 in acute colitis and of anti-IL-12p40 or an agonistic anti-CD3 antibody in chronic colitis. Cyclosporine A and anti-IL-12p40 (in the acute phase) and anti-CD3 (in the chronic phase) treatment attenuated local cytokine levels, improved clinical symptoms (CsA and anti-IL-12p40) and histology (CsA and anti-CD3). Further, anti-IL-12p40 treatment was partly efficacious in the chronic phase, whereas methotrexate showed no efficacy in the acute colitis. Thus, three of the current tested agents showed efficacy in the disease model, arguing that DSS-induced colitis can be used as a relevant model for the translation of mice data to human disease.

Characterisation of mucosal lymphoid aggregates in ulcerative colitis: immune cell phenotype and TcR-γδ expression

BACKGROUND AND AIMS A histopathological feature considered indicative of ulcerative colitis (UC) is the so-called basal lymphoid aggregates. Their relevance in the pathogenesis of UC is, however, unknown. We have performed a comprehensive analysis of the immune cells in these aggregates most likely corresponding to the lymphoid follicular hyperplasia also described in other colitides. METHODS Resection specimens of UC and normal colon were analysed by immunomorphometry, immunoflow cytometry, and immunoelectron microscopy, using a large panel of monoclonal antibodies. RESULTS (1) In all cases of UC, colonic lamina propria contained numerous basal aggregates composed of lymphocytes, follicular dendritic cells, and CD80/B7.1 positive dendritic cells. (2) CD4+CD28−αβ T cells and B cells were the dominant cell types in the aggregates. (3) The aggregates contained a large fraction of cells that are normally associated with the epithelium: that is, γδ T cells (11 (7)%) and αEβ7 +cells (26 (13)%). The γδ T cells used Vδ1 and were CD4−CD8−. Immunoelectron microscopy analysis demonstrated TcR-γδ internalisation and surface downregulation, indicating that the γδ T cells were activated and engaged in the disease process. (4) One third of cells in the aggregates expressed the antiapoptotic protein bcl-2. CONCLUSIONS Basal lymphoid aggregates in UC colon are a consequence of anomalous lymphoid follicular hyperplasia, characterised by abnormal follicular architecture and unusual cell immunophenotypes. The aggregates increase in size with severity of disease, and contain large numbers of apoptosis resistant cells and activated mucosal γδ T cells. The latter probably colonise the aggregates as an immunoregulatory response to stressed lymphocytes or as a substitute for defective T helper cells in B cell activation. γδ T cells in the aggregates may be characteristic of UC.

Over‐expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis

Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohns disease and control patients were analysed for cytokine mRNA levels by real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)‐2, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β1, but not IL‐4, IL‐5 or IL‐10. In UC, a highly significant increase in IL‐10 mRNA levels in T lymphocytes and an increased frequency of IL‐10 positive cells was seen in colon. IL‐10 mRNA levels were also elevated in T lymphocytes of the non‐inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL‐10 mRNA. IL‐2, IFN‐γ and TNF‐α mRNA levels were decreased in colonic T lymphocytes, and virtually no IL‐2, IFN‐γ, TNF‐α or TGF‐β positive cells were detected in basal lymphoid aggregates. However, scattered IL‐10 positive cells were found here. Lamina propria outside the aggregates contained IL‐10‐, IFN‐γ, TNF‐α and TGF‐β but not IL‐2 positive cells. T cells of UC patients did not express IL‐4 or IL‐5. Taken, together the data suggest a generalized activation of IL‐10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL‐10.

INTESTINAL DELIVERY OF NON-VIRAL GENE THERAPEUTICS: PHYSIOLOGICAL BARRIERS AND PRECLINICAL MODELS

The future of nucleic acid-based therapeutics is dependent on achieving successful delivery. Recently, there has been an increasing interest in delivery via the gastrointestinal tract. Gene therapy via this route has many advantages, including non-invasive access and the versatility to treat local diseases, such as inflammatory bowel disease, as well as systemic diseases, such as haemophilia. However, the intestine presents several distinct barriers and, therefore, the design of robust non-viral delivery systems is key to future success. Several non-viral delivery strategies have provided evidence of activity in vivo. To facilitate the design of more efficient and safe gene medicines, more physiologically relevant models, at both the in vitro and in vivo levels, are essential.

Magnetic resonance imaging of experimental mouse colitis and association with inflammatory activity

Background: Ulcerative colitis and Crohns disease are the major chronic inflammatory bowel diseases affecting the gastrointestinal tract in humans. Imaging techniques such as endoscopy and computed tomography are used to monitor disease activity. Magnetic resonance imaging (MRI) is emerging as a diagnostic modality, and studies have shown that MRI can be used in the diagnostic procedure of patients with inflammatory bowel disease. The aim of the present study was to investigate the role of MRI in quantitatively reflecting inflammation in an experimental mouse colitis model. Methods: Colonic inflammation was induced by exposing mice to dextran sulfate sodium. MRI was used to assess colon wall thickness, T2‐weighted (T2w) signal, and contrast‐enhanced T1‐weighted (T1w) signal in inflamed and healthy animals in vivo. Haptoglobin and interleukin‐1&bgr; served as systemic and local inflammatory markers, and macroscopic ex vivo scoring of the colon was performed to assess colonic inflammation. Results: Dextran sulfate sodium‐exposed animals displayed increased levels of inflammatory markers and higher inflammatory score compared with healthy animals. Colon wall thickness and contrast‐enhanced T1w signal were significantly increased in dextran sulfate sodium‐exposed compared with healthy animals. In addition, the T2w signal was positively correlated with haptoglobin levels and colon wall thickness in the inflamed animals. Conclusions: Our results show that MRI can be used to depict healthy and inflamed mouse colon and that the T2w signal, contrast‐enhanced T1w signal, and colon wall thickness may be used to characterize inflammation in experimental colitis. These potential biomarkers may be useful in the evaluation of putative drugs in longitudinal studies in both mice and humans.

Mechanism of protection of transepithelial barrier function by Lactobacillus salivarius: strain dependence and attenuation by bacteriocin production

Enhanced barrier function is one mechanism whereby commensals and probiotic bacteria limit translocation of foreign antigens or pathogens in the gut. However, barrier protection is not exhibited by all probiotic or commensals and the strain-specific molecules involved remain to be clarified. We evaluated the effects of 33 individual Lactobacillus salivarius strains on the hydrogen peroxide (H(2)O(2))-induced barrier impairment in human epithelial Caco-2 cells. These strains showed markedly different effects on H(2)O(2)-induced reduction in transepithelial resistance (TER). The effective strains such as UCC118 and CCUG38008 attenuated H(2)O(2)-induced disassembly and relocalization of tight junction proteins, but the ineffective strain AH43324 did not. Strains UCC118 and CCUG38008 induced phosphorylation of extracellular signal-regulated kinase (ERK) in Caco-2 cells, and the ERK inhibitor U0126 attenuated the barrier-protecting effect of these strains. In contrast, the AH43324 strain induced phosphorylation of Akt and p38, which was associated with an absence of a protective effect. Global transcriptome analysis of UCC118 and AH43324 revealed that some genes in a bacteriocin gene cluster were upregulated in AH43324 under TER assay conditions. A bacteriocin-negative UCC118 mutant displayed significantly greater suppressive effect on H(2)O(2)-induced reduction in TER compared with wild-type UCC118. The wild-type strain augmented H(2)O(2)-induced phosphorylation of Akt and p38, whereas a bacteriocin-negative UCC118 mutant did not. These observations indicate that L. salivarius strains are widely divergent in their capacity for barrier protection, and this is underpinned by differences in the activation of intracellular signaling pathways. Furthermore, bacteriocin production appears to have an attenuating influence on lactobacillus-mediated barrier protection.

Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis

Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohns disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohns disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.

Human small intestinal mucosa harbours a small population of cytolytically active CD8+ alphabeta T lymphocytes.

Intraepithelial lymphocytes (IEL) in normal human small intestine exhibit cytotoxicity. This study was undertaken to characterize the effector cells and their mode of action. Freshly isolated jejunal IEL and lamina propria lymphocytes (LPL), as well as IEL and LPL depleted of CD4+, CD8+ and T‐cell receptor (TCR)‐γδ+ cells were used as effector cells in anti‐CD3‐mediated redirected cytotoxicity against a murine FcγR‐expressing cell line. Effector cell frequencies were estimated by effector to target cell titration and limiting dilution. The capacity of IEL and LPL to kill a Fas‐expressing human T‐cell line was also analysed. T‐cell subsets were analysed for perforin, granzyme B, Fas‐ligand (FasL), tumour necrosis factor‐α (TNF‐α) and TNF‐related apoptosis inducing ligand (TRAIL) mRNA expression by reverse transcription–polymerase chain reaction (RT‐PCR). Frequencies of IEL expressing the perforin and FasL proteins were determined by immunomorphometry. Both IEL and LPL exhibited significant Ca2+‐dependent, anti‐CD3‐mediated cytotoxicity, ≈ 30% specific lysis at the effector to target cell ratio 100. The cytotoxic cells constituted, however, only a small fraction of IEL and LPL (≈ 0·01%). CD8+ TCR‐αβ+ cells accounted for virtually all the cytotoxicity and expressed mRNA for all five cytotoxic proteins. The frequency of granzyme B‐expressing samples was higher in CD8+ cells than in CD4+ cells (P<0·05 and <0·01 for IEL and LPL, respectively). In addition, both IEL and LPL exhibited significant spontaneous anti‐CD3‐independent cytotoxicity against Fas‐expressing human T cells. This killing was mediated by Fas–FasL interaction. On average, 2–3% of the IEL expressed perforin and FasL. We speculate that CD8+ memory cells accumulate in the jejunal mucosa and that the CD8+ TCR‐αβ+ lymphocytes executing TCR/CD3‐mediated, Ca2+‐dependent cytotoxicity are classical cytotoxic T lymphocytes ‘caught in the act’ of eliminating infected epithelial cells through perforin/granzyme exocytosis. The observed Fas/FasL‐mediated cytotoxicity may be a reflection of ongoing down‐regulation of local immune responses by ‘activation‐induced cell death’.

Psychological stress reactivates dextran sulfate sodium-induced chronic colitis in mice

Inflammatory bowel disease (IBD) is a chronic condition with alternating active and quiescent phases of inflammation. Stress has been suggested as a factor triggering a relapse of IBD. We investigated the role of repetitive psychological stress [water avoidance stress (WAS)] in reactivating colonic inflammation in a murine model of dextran sulfate sodium (DSS)-induced chronic colitis. Colitis was induced in C57BL/6 female mice by exposure to 3% DSS (5 days). During chronic inflammation (day 34), mice underwent repetitive WAS (1 h/day/7 days) and were given a sub-threshold concentration of DSS (1%, 5 days) or normal water to drink. At euthanasia (day 40), inflammatory parameters were assessed (colon inflammatory score, levels of inflammatory markers and histology). Mice with chronic colitis exposed to WAS had higher macroscopic and microscopic colonic inflammatory scores and levels of inflammatory markers (mainly IL-1β, IL12p40 and CCL5) than non-stressed mice. Inflammatory responses were further enhanced by the presence of a sub-threshold concentration of DSS (1%). In mice without chronic inflammation, neither WAS nor 1% DSS, individually or in combination, elicited any inflammation. Hence stress, per se, reactivates a quiescent chronic inflammation, but does not initiate inflammation in healthy mice. Stress should be regarded as an environmental factor triggering IBD relapses in humans.