Vitamin D has been suggested as a possible adjunctive treatment to ameliorate disease severity in human inflammatory bowel disease. In this study, the effects of diets containing high (D++, 10,000 IU/kg), moderate (D+, 2,280 IU/kg) or no vitamin D (D−) on the severity of dextran sodium sulphate (DSS) colitis in female C57Bl/6 mice were investigated. The group on high dose vitamin D (D++) developed the most severe colitis as measured by blinded endoscopic (p < 0.001) and histologic (p < 0.05) assessment, weight loss (p < 0.001), drop in serum albumin (p = 0.05) and increased expression of colonic TNF-α (p < 0.05). Microbiota analysis of faecal DNA showed that the microbial composition of D++ control mice was more similar to that of DSS mice. Serum 25(OH)D3 levels reduced by 63% in the D++ group and 23% in the D+ group after 6 days of DSS treatment. Thus, high dose vitamin D supplementation is associated with a shift to a more inflammatory faecal microbiome and increased susceptibility to colitis, with a fall in circulating vitamin D occurring as a secondary event in response to the inflammatory process.

Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk.

BACKGROUND:Secreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation.METHODS:Wild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry.RESULTS:KO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p= 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon.CONCLUSIONS:Compared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.

LPS was used as an inducer of pro-inflammatory responses.Results: LPS-stimulated ivMACs and ivDCs pre-treated with retinoids released significantly less TNF (p<0.0001),IL-6 (p<0.05),MIP-1alpha (p<0.05) and MIP-1beta (p<0.0001).ATRA and its derivatives potentiated LPS-induced release of ICAM-1 from ivDCs (p=0.001),but not from ivMACs.Retinoids alone induced the release of VEGF (p<0.05) and IL-10 (p<0.05) from both ivMACs and ivDCs in a concentration-dependent manner.Consistently, we observed that retinoids inhibited LPSinduced release of IL-6, TNF and MIP-1beta in THP-1 cells.In addition ATRA and derivatives induced the release of IL-10 and decreased the production of MIP-1alpha.Retinoids increased the diffusion of FITC-dextran from apical to the basolateral side of Caco2 monolayers in a concentration-dependent manner.Conclusions: Vitamin A and its derivatives have distinct effects on different cell types involved in the inflammatory response in the gut.They inhibited the release of pro-inflammatory cytokines induced by LPS and stimulated the release of anti-inflammatory mediators in macrophages and dendritic cells, alongside with a possible enhancement of intestinal permeability.

Background: Colorectal cancer (CRC) is common, but human sporadic and colitis-associated CRC (CAC) have different pathogeneses. The aim of this study was to identify differences in gene expression levels and inflammatory cell infiltration of tumours from mice models of sporadic and CAC. Methods: Mice used in this study are on a FVB background. Sporadic CRC tumourigenesis was mimicked by giving 6 weekly intraperitoneal (IP) injections of azoxymethane (AOM). CAC was modelled by a single AOM IP injection prior to two cycles of 7 days of dextran sodium sulphate (DSS) in the drinking water followed by 14 days of plain water. Tumours of similar size were harvested from both models at weeks 13 (AOM-alone) and 8 (AOM/DSS) respectively and processed for further analysis. Histological and immunofluorescent (IF) assessment of CD4, CD8a, F4/80, and Ly6G was performed on tumours. Wholegenome microarray (Illumina BeadChip, MouseRef 8 v2) was used to compare gene expression in tumours from both models. Gene ontology analysis was performed using the Database for Annotation, Visualisation and Integrated Discovery. Results: The majority of tumours were polypoid and all were dysplastic. Unlike AOM/DSS, the AOM-alone protocol induced high-grade dysplasic polyps (21% [4/19] vs 0% [0/9]) and 2 flat lesions, one with invading CRC. Microarray analysis identified that 665 genes were differentially expressed between the tumours (p < 0.05), and 398 had increased expression levels in the AOM-alone tumours and included genes associated with cytokine activity, cell division, and the regulation of apoptosis. 267 genes were reduced and were associated with immune response, the lysosome and positive regulation of the immune system. IF assessment of the inflammatory cell infiltrates showed the presence of CD4-positive, CD8a-positive, F4/80-positive and Ly6G-positive cells in all tumours without any significant differences in the cell numbers between the models. Conclusions: AOM and AOM/DSS tumours are histologically different with marked gene-expression differences suggesting a different pathogenesis. All AOM-alone tumours displayed inflammatory cell infiltration suggesting a crucial role for the immune cells in the progression of tumours in both models once tumourigenesis is initiated. Further work is required to confirm histological differences between AOM and AOM/DSS tumours.

findings. Endoscopic activity was classified as active and quiescent disease, based on the endoscopic Mayo Subscore (UC) and SES-CD (CD) index. In cases with active disease, biopsies were taken from both endoscopically affected and non-affected mucosa. Following pathologist’s criteria, biopsies were classified as normal, affected mucosa, unspecific inflammation, and quiescent. Results: 67 biopsies from 52 patients with histological diagnosis of IBD (31 UC and 21 EC) and 19 from subjects without IBD were studied. 38.6% cases had active disease. MV count was lower in samples from patients without IBD (Table 1; p < 0.0001), even when comparing only normal mucosa. Patients with UC showed the highest count of Ang1+MV in both cases (Table 2). MV density was also increased in samples with endoscopic activity (52.3±19.5 vs 41.7±15.8; P= 0.02) but it did not correlate with histology.

Background: Colorectal cancer (CRC) is common, but human sporadic and colitis-associated CRC (CAC) have different pathogeneses. The aim of this study was to identify differences in gene expression levels and inflammatory cell infiltration of tumours from mice models of sporadic and CAC. Methods: Mice used in this study are on a FVB background. Sporadic CRC tumourigenesis was mimicked by giving 6 weekly intraperitoneal (IP) injections of azoxymethane (AOM). CAC was modelled by a single AOM IP injection prior to two cycles of 7 days of dextran sodium sulphate (DSS) in the drinking water followed by 14 days of plain water. Tumours of similar size were harvested from both models at weeks 13 (AOM-alone) and 8 (AOM/DSS) respectively and processed for further analysis. Histological and immunofluorescent (IF) assessment of CD4, CD8a, F4/80, and Ly6G was performed on tumours. Wholegenome microarray (Illumina BeadChip, MouseRef 8 v2) was used to compare gene expression in tumours from both models. Gene ontology analysis was performed using the Database for Annotation, Visualisation and Integrated Discovery. Results: The majority of tumours were polypoid and all were dysplastic. Unlike AOM/DSS, the AOM-alone protocol induced high-grade dysplasic polyps (21% [4/19] vs 0% [0/9]) and 2 flat lesions, one with invading CRC. Microarray analysis identified that 665 genes were differentially expressed between the tumours (p < 0.05), and 398 had increased expression levels in the AOM-alone tumours and included genes associated with cytokine activity, cell division, and the regulation of apoptosis. 267 genes were reduced and were associated with immune response, the lysosome and positive regulation of the immune system. IF assessment of the inflammatory cell infiltrates showed the presence of CD4-positive, CD8a-positive, F4/80-positive and Ly6G-positive cells in all tumours without any significant differences in the cell numbers between the models. Conclusions: AOM and AOM/DSS tumours are histologically different with marked gene-expression differences suggesting a different pathogenesis. All AOM-alone tumours displayed inflammatory cell infiltration suggesting a crucial role for the immune cells in the progression of tumours in both models once tumourigenesis is initiated. Further work is required to confirm histological differences between AOM and AOM/DSS tumours.

Background Patients with inflammatory bowel diseases (IBD) develop anaemia of inflammation (AI) due to disturbances in iron homeostasis that limits the availability of iron for erythropoiesis. Aims In this study, the effects of colonic inflammation and dietary iron levels on liver iron homeostasis were investigated in a mouse model of colitis. Methods Colonic inflammation was induced by the administration of dextran sodium sulphate (DSS) to mice fed either an iron-supplemented (1%) or control iron (0.01%) diet. Liver and plasma iron concentrations as well as plasma transferrin saturation were measured biochemically. Liver gene expression was determined by real-time PCR and plasma IL-6 levels were measured by ELISA. Results DSS-induced colonic inflammation increased plasma IL-6 levels. Dietary iron supplementation further enhanced colonic inflammation and plasma IL-6 levels (p < 0.0001). Liver iron and plasma transferrin saturation were elevated in dietary iron-supplemented mice. Post-DSS treatment, liver iron levels increased (p < 0.01) and transferrin saturation decreased (p < 0.01) in mice fed the iron-supplemented and control iron diets, consistent with the presence of AI. Liver expression of the iron regulatory genes, hepcidin (Hamp1) and inhibitor of DNA binding 1 (Id1), was upregulated by dietary iron (p < 0.01) but unexpectedly downregulated by DSS treatment (p < 0.05). Smad7 gene expression was decreased in DSStreated mice and Bmp6 expression was increased by dietary iron supplementation (p < 0.001). Dietary iron supplementation decreased the gene expression of the iron importer transferrin receptor 1 (Tfr1), congruent with the iron-dependent regulation of Tfr1 and expression and further diminished by DSS treatment (p < 0.05). Gene expression of the iron importer Zip14 was increased (p < 0.05) whilst that of the iron exporter ferroportin 1A was decreased (p < 0.0001) with DSS treatment, consistent with the retention of iron by the liver. Conclusion The perturbations in iron homeostasis resulting from increased colonic inflammation observed in this study are consistent with AI. The regulatory pathways for the changes seen, however, are unclear. The lack of induction of Hamp1 expression by plasma IL-6 levels suggests that other regulatory signals may impede hepcidin induction by inflammation. A possible candidate is the erythroid signal as increased erythropoietic activity is known to be a strong negative regulator of hepcidin.

Chronic intestinal inflammation and high dietary iron are associated with a greater risk of developing colorectal cancer. The aim of this study was to investigate the role of IL-6 in iron-induced colonic inflammation and tumourigenesis in a mouse model of colorectal cancer. Methods: Mice, fed either an iron-supplemented (1% carbonyl iron) or control (0.01% iron) diet, were treated with dextran sodium sulphate (DSS) and azoxymethane (AOM) to induce intestinal inflammation and cancer. Intestinal inflammation and tumour development were assessed using high-resolution video endoscopy at multiple time-points. Colonic inflammation and tumours were examined histologically and gene expression by real-time PCR. Results: Seven days post-AOM/DSS treatment, intestinal inflammation was more severe in iron-loaded mice (p < 0.05). Colonic pro-inflammatory cytokines, IL-6, IFN-γ and TNFα gene expression increased with AOM/ DSS treatment confirming the presence of intestinal inflammation (p < 0.05). Dietary iron loading had an additive effect on the gene expression of IL-6 and other members of this family, IL-11 and IL-17a, in AOM/ DSS-treated mice. STAT3 phosphorylation was increased in AOM/DSS treated mice and this was further enhanced with dietary iron loading. Five weeks after AOM/DSS treatment, iron-loaded mice developed a greater number and larger-sized colonic tumours compared to control mice (p < 0.05). Dietary iron-loading also induced an additive effect on tumour IL-6 gene expression in AOM/DSS-treated mice (p < 0.05). Expression of cellular iron import genes DMT1, Zip14a and Tfr1 was increased and cellular export gene ferroportin was reduced in colonic tumours compared to non-tumour tissue from the same animal (p < 0.05), suggesting increased iron uptake by tumours may promote growth. Summary/Conclusions: Dietary iron-loading promoted colonic inflammation and tumour formation. The mechanistic basis for the interaction between iron, inflammation and colorectal cancer may involve IL-6/ STAT3 signalling.

Background SPARC is a matricellular protein involved in tissue remodelling, cell migration and angiogenesis, while forkhead box P3 (FOXP3) protein functions as a transcription factor involved in immune cell regulation. Both SPARC and FOXP3 can play an anti-tumorigenic role in cancer progression. The aim was to determine if SPARC, FOXP3, CD8 and CD45RO expression levels are associated with colorectal cancer (CRC) stage, disease outcome and long-term cancer-specific survival (CSS) in stage II and III CRC. Methods and Findings SPARC expression was initially assessed in 120 paired normal and stage I-IV CRCs. Subsequently, approximately 1000 paired patient samples of stage II or III CRCs in tissue microarrays were stained for SPARC, FOXP3, CD8 or CD45RO. Proportional hazards modelling assessed correlations between these markers and clinicopathological data, including disease outcome and cancer specific survival (CSS). Both SPARC and FOXP3 expression were significantly greater in CRC than normal colon (p<0.0001). High SPARC expression correlated with good disease outcome (≥60 mths without disease recurrence, p = 0.0039) and better long-term CSS in stage II CRC (<0.0001). In stage III CRC, high SPARC expression correlated with better long-term CSS (p<0.0001) and less adjuvant chemotherapy use (p = 0.01). High FOXP3 correlated with a good disease outcome, better long-term CSS and less adjuvant chemotherapy use in stage II (p<0.0037, <0.0001 and p = 0.04 respectively), but not in stage III CRC. High CD8 and CD45RO expression correlated with better disease outcome in stage II CRC, and better CSS, but the differences were not as marked as for SPARC and FOXP3. Conclusions These data suggest that high SPARC and FOXP3 are associated with better disease outcome in stage II CRC and may be prognostic indicators of CSS. Further assessment of whether these markers predict patients at high risk of recurrence with stage II CRC and functional studies of these effects are underway

The carcinogenic potential of iron in colorectal cancer (CRC) is not fully understood.Iron is able to undergo reduction and oxidation, making it important in many physiological processes.This inherent redox property of iron, however, also renders it toxic when it is present in excess.Iron-mediated generation of reactive oxygen species via the Fenton reaction, if uncontrolled, may lead to cell damage as a result of lipid peroxidation and oxidative DNA and protein damage.This may promote carcinogenesis through increased genomic instability, chromosomal rearrangements as well as mutations of proto-oncogenes and tumour suppressor genes.Carcinogenesis is also affected by inflammation which is exacerbated by iron.Population studies indicate an association between high dietary iron intake and CRC risk.In this editorial, we examine the link between iron-induced oxidative stress and inflammation on the pathogenesis of CRC.

Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemo-chromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our marine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively up-regulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NF kappa B) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NF kappa B subunit by RNA interference. Conclusion: TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NF kappa B-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration. (HEPATOLOGY 2010;52:291-302)

Background: Experimental and clinical studies suggest an association between SIBO and NASH.Liver injury and fibrosis could be related to exposure to a bacterial burden, notably endotoxins, of intestinal origin.Moreover, the gut microbiota and endotoxemia have been described to play a contributory role in metabolic dysfunction, particularly insulin resistance, which is a key factor in NASH development.The extracellular receptor molecules for LPS are Lipopolysaccharide Binding Protein (LBP), CD14, toll-like receptor-4 (TLR-4) and myeloid differentiation-2 (MD-2).Endotoxaemia also induces proinflammatory cytokine release from immune cells.Aim: To investigate the prevalence of small intestinal bacterial overgrowth (SIBO) and relationships to LPS receptors and systemic cytokines in NASH.Methods: 18 NASH patients (8 males and 10 females) and 16 age-and gender-matched healthy volunteers were studied.SIBO was assessed by the lactulose breath hydrogen test (LHBT), plasma LBP levels were measured by ELISA and expression (as a %) of TLR-2 and 4 on CD14 positive cells determined by flow cytometry.Proinflammatory mediators (IL-1β, IL-6, IL-8 and TNFα) were measured in plasma by MSD (Meso Scale Discovery).Results: SIBO was more common in NASH patients (71%) than in control subjects (35.7%).This was mainly due to H2 production which was significantly greater in NASH than control at time points 45, 60, 75 and 90 minutes during the LHBT (p=0.0241,0.0476, 0.0470 and 0.0139 respectively).On the other hand, CH4 production was similar in NASH and control groups.No significant differences were detected between NASH and controls for LBP levels, p=0.5448.TLR-4/ MD-2 expression on CD14 positive was significantly higher among NASH patients than in control subjects: % expression, mean ± SEM, NASH vs control: 20.95± 2.91% vs 12.73 ± 2.29%, p=0.048 while TLR-2 expression was not significantly different.Among the cytokines studied, IL-8 levels were significantly higher in patients than control (p=0.0413) and correlated positively with TLR-4 (r=0.5123,p=0.0355) expression but not with TLR-2 expression.Conclusion: Small intestinal bacterial overgrowth is common in NASH, largely related to excess production of H2 and is associated with increased circulating levels of IL-8 and upregulation of TLR-4/MD2.