The interaction between epithelial cancer cells and cancer-associated fibroblasts (CAFs) has a major role in cancer progression and eventually in metastasis. In colorectal cancer (CRC), CAFs are present in high abundance, but their origin and functional interaction with epithelial tumor cells has not been elucidated. In this study we observed strong activation of the transforming growth factor-β (TGF-β)/Smad signaling pathway in CRC CAFs, accompanied by decreased signaling in epithelial tumor cells. We evaluated the TGF-β1 response and the expression of target genes including matrix metalloproteinases (MMPs) and plasminogen activator inhibitor (PAI)-1 of various epithelial CRC cell lines and primary CAFs in vitro. TGF-β1 stimulation caused high upregulation of MMPs, PAI-1 and TGF-β1 itself. Next we showed that incubation of CAFs with conditioned medium (CM) from epithelial cancer cells led to hyperactivation of the TGF-β signaling pathway, enhanced expression of target genes like PAI-1, and the expression of α-smooth muscle actin (α-SMA). We propose that the interaction of tumor cells with resident fibroblasts results in hyperactivated TGF-β1 signaling and subsequent transdifferentiation of the fibroblasts into α-SMA-positive CAFs. In turn this leads to cumulative production of TGF-β and proteinases within the tumor microenvironment, creating a cancer-promoting feedback loop.

Disintegration of the colonic epithelial barrier is considered a key event in the initiation and progression of inflammatory bowel and celiac disease. As the primary etiology of these diseases remains unknown, we hypothesized that the trichothecene deoxynivalenol (DON), a fungal metabolite found in grain-based human diets, might be one of the triggers resulting in an impairment of the intestinal tight junction network preceding an inflammatory response. Using horizontal impedance measurements, we demonstrate that DON disintegrates a human Caco-2 cell monolayer within <1 h after exposure to concentrations as low as 1.39 μM. This initial trigger is followed by a decrease in transepithelial resistance and an increased permeability of marker molecules, such as lucifer yellow and FITC-labeled dextran. In parallel, the increase in paracellular transport of FITC-dextran is demonstrated in vivo in B6C3F1 mice, challenged orally with DON. In vitro claudin protein levels are decreased and correlated with a displacement within the cells in vitro and in vivo, accompanied by a compensatory up-regulation of mRNA levels of claudins and their binding partner ZO-1. In treated mice, alterations in villus architecture in the entire intestinal tract resemble the disintegration of the epithelial barrier, a characteristic of chronic inflammatory bowel disease.

ObjectiveProline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD).DesignIn intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies.ResultsIn the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine.ConclusionsThe proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

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There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide L-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema.

Background and AimsAlthough several endoscopic and histopathologic indices are available for evaluating the severity of inflammation in mouse models of colitis, the reliability of these scoring instruments is unknown. Our aim was to evaluate the reliability of the individual items in the existing indices and develop new scoring systems by selection of the most reliable index items.MethodsTwo observers scored the histological slides [n = 224] and endoscopy videos [n = 201] from treated and untreated Interleukin[IL]-10 knock-out and T-cell transferred SCID mice. Intra-rater and inter-rater reliability for endoscopy and histology scores, and each individual item, were measured using intraclass correlation coefficients [ICCs]. The Mouse Colitis Histology Index [MCHI] and Mouse Colitis Endoscopy Index [MCEI] were developed using the most reliable items. Both were correlated to the colon density and to each other and were evaluated for their ability to detect changes in pathobiology.ResultsThe intraclass correlation coefficients (ICCs) for inter-rater agreement (95% CIs) for the total histology and endoscopy scores were 0.90 [0.87–0.92] and 0.80 [0.76–0.84], respectively. The MCHI and MCEI were highly correlated with colon density, with a Spearman Rho = 0.81[0.75–0.85] and 0.73 [0.66–0.79], respectively, and with each other, Spearman Rho = 0.71 [0.63–0.77]. The MCHI and MCEI were able to distinguish between the experimental groups within the models, with pairwise differences between the treated and untreated groups being statistically significant [p < 0.001].ConclusionsThese histological and endoscopic indices are valid and reliable measures of intestinal inflammation in mice, and they are responsive to treatment effects in pre-clinical studies.

ObjectiveMacrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fcγ-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined.DesignWe used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rbhigh T-cell transfer model in combination with several genetic mouse models.ResultsAnti-TNF therapy increased macrophage IL-10 production in an FcγR-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rbhigh T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF.ConclusionThe therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages.

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