Myeloid-derived suppressor cells (MDSC), emerging during tumor growth or chronic inflammation play a critical role in regulating T cell function. However, mechanisms governing the generation of these cells remain unclear, and need to be further defined. Using a DSS-induced colitis and recovery model, we characterized the dynamic changes within myeloid compartments and the emergence of MDSC during active and resolution phases of inflammation. We show that the immature myeloid compartment expands in bone marrow (BM) specifically at the resolution phase of inflammation during colitis transition to recovery. Additionally, we found enhanced levels of IL-17 in the serum of colitis mice tightly correlates with expansion of the IMC compartment, and is likely the factor responsible for expansion of these cells. Our study also determined that the expanded population of myeloid cells underwent a functional reprogramming event. In particular, two major functional changes occurred when colitic mice were allowed to recover: 1) CD11b+Gr-1+ myeloid cells in bone marrow and spleen acquired T cell suppressive functions, and 2) acquired the ability to enter into circulation from BM, confirming previously reported characteristics of MDSC. Additionally, we determined that acquired migratory capability in the low density myeloid cells isolated from resolution time points was due to enhanced surface expression of chemokine receptor CXCR2. Furthermore, we determined that after mobilization of MDSC from the bone marrow, these cells collected in the T cell-rich spleens, where they effectively functioned to suppress T cell proliferation. Through these acquired functions, our study determines a protective role for MDSC during the recovery phase of post-acute inflammation during persistent DSS-induced colitis.

BACKGROUND: Inflammatory bowel disease (IBD) is a group of conditions characterized by chronic inflammation of the gastrointestinal (GI) tract. A new diagnosis of IBD in children and adolescents can have significant psychosocial effects on both the patient and the family. Child and parental coping strategies play a crucial role in the adjustment to IBD, especially within the first year of the diagnosis. AIMS: The primary aim of the study was to assess the stability of coping measures over time in children and parents following a new pediatric IBD diagnosis. The study also aimed to assess the impact of parental coping on parental healthcare resource utilization for children with newly diagnosed IBD, as well as the impact of parental coping on anxiety, depression, and quality of life in children with newly diagnosed IBD. METHODS: This was a prospective, longitudinal cohort study at Boston Children’s Hospital (BCH) that focused on children and adolescents with newly diagnosed IBD, as well as their parents. Patients and their parents were approached at the time they enrolled in the study and then again about 12 months later as part of a one-year follow-up. At both time points, they were asked to fill out various questionnaires about psychological functioning and answer other questions about medical care. RESULTS: The study identified and encountered 465 IBD patients, of which 126 were eligible for recruitment. There were 70 patients and families who signed a consent form for enrollment, 55 who fully or partially completed the questionnaires at baseline, and only 5 who also completed the questionnaires at follow-up. Due to the limited number of participants who completed the questionnaires at follow-up, no definitive conclusions could be drawn about the stability of coping measures over time. Parental anxiety, parental depression, frequent parental stress, and difficult parental stress were all found to be positively correlated with healthcare utilization and negatively correlated with the child’s quality of life. Parental anxiety, frequent parental stress, and difficult parental stress were all found to be positively correlated with the child’s anxiety. Parental depression, frequent parental stress, and difficult parental stress were all found to be positively correlated with the child’s depression. CONCLUSION: Preliminary findings suggest that poor parental coping leads to decreased child quality of life and increased healthcare utilization, child anxiety, and child depression. A larger sample size is needed to accurately evaluate the stability of coping measures over time. The next steps for this study involve further examination of the impact of parental coping and enrollment of more patients and families.

Intestinal immune cells remain tolerant to the trillions of commensal bacteria present in the gut, with perturbations of this process implicated in development of inflammatory bowel disease (IBD). The cytokine TGF-β is a key factor promoting intestinal immune tolerance, but is secreted in a latent state that requires activation to function. Binding of TGF-β to the integrin αvβ8 is a principal mechanism of TGF-β activation, with mouse models demonstrating a crucial role for αvβ8 expression by dendritic cells and regulatory T cells in intestinal immune regulation. Despite this evidence, very little is known regarding the importance of this activating integrin in human intestinal homeostasis. Utilising flow cytometry here we find that integrin αvβ8 is highly expressed on peripheral blood monocytes with highest levels on intermediate CD14++CD16+ monocytes. Upon monocyte to macrophage differentiation high β8 expression is observed on anti-inflammatory M-CSF differentiated macrophages versus pro-inflammatory GM-CSF macrophages. In monocytes, expression of β8 is upregulated by specific bacterial TLR ligands. Utilising a TGF-β reporter cell line both monocytes and M-CSF MDM display an enhanced ability to activate TGF-β in an αvβ8-dependent manner. Data presented here indicate that macrophage αvβ8-dependent TGF-β activation does not alter expression of surface markers associated with a tolerogenic macrophage phenotype, phagocytosis, or production of the anti-inflammatory cytokine IL-10; nor does TGF-β appear to influence the metabolic profile of macrophages, key differences of which are associated with pro- or anti-inflammatory phenotype. However, the previously undescribed finding of integrin αvβ8 expression on human monocytes and macrophages, which was subsequently confirmed in intestinal populations and found to be downregulated in inflamed IBD mucosa, may highlight an important functional pathway in intestinal immune homeostasis and represent a potential future therapeutic target in IBD.

No description available.

Background: Caveolin-1 (Cav-1), the major component of caveolae, is a multifunctional scaffolding protein that serves as a platform for the cell’s signal-transduction and plays a role in inflammation. However, its role in inflammatory bowel disease (IBD), a chronic inflammatory condition in the gastrointestinal tract, is not clear. A recent study shows that Cav-1 mediates angiogenesis in dextran sodium sulphate (DSS)-induced colitis. These results contradict our data, in which Cav-1 levels decreased significantly in 2,4,6-trinitrobenzene sulphonic acid (TNBS)–induced colitis. Methods: To test whether Cav-1 is involved in IBD pathogenesis, various models representing different dominant Th subtype responses and mimicking the immune pathologic mechanisms of different clinical IBD setting were employed: acute colitis was induced by intra-rectal administration of a single dose of TNBS in BALB/c and C57BL/6J mice, or by drinking 3% DSS water for 6 days in C57BL/6J mice. Chronic colitis was induced by administration of TNBS once a week for 7 weeks in BALB/c mice. To assess the effects of complete loss of Cav-1, Cav-1 knock-out (Cav-1-/-) and control wild-type C57BL/6J mice received a single TNBS administration. To further test the possible role of Cav-1, one of two peptides (that either mimicked (Caveolin scaffolding domain; CSD) or antagonized (Caveolin-1 binding domain; CBD1) Cav-1)) was administered intraperitoneally to mice receiving TNBS. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified by ELISA. Inflammation was evaluated through histological analysis. Results: Cav-1 levels in mouse colon tissue were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation and cytokine levels. Furthermore, a loss of Cav-1 (Cav-1-/-) showed increased clinical and inflammatory scores and increased body weight loss. Mice receiving peptides to alter Cav-1 levels, showed surprising effects. The mimicking peptide (CSD) showed decreased Cav-1 levels, while the antagonizing peptide (CBD1) showed increased Cav-l levels. These changes in levels were associated with clinical and inflammatory scores and body weight loss that supported the TNBS-induced data. DSS-induced colitis mice showed increased disease activity index, however no significant difference in Cav-1 levels was found between colitis and normal mice. Conclusions: Cav-1 plays an important role in the protection of TNBS-induced colitis, but not in DSS-induced colitis, an entirely different result from a previous report, suggesting that enhancement of Cav-1 expression and functions may be beneficial to IBD treatment in some specific clinical settings. Further studies are warranted.

ABSTRACT Background Calprotectin is a protein found in the cytoplasm of neutrophile granulocytes. In the course of inflammatory bowel disease (IBD), calprotectin is released during chronic inflammation in the gut. Activation of neutrophils during the inflammation is followed by activation and secretion of pro-inflammatory molecules such as calprotectin. Calprotectin is stable in stool up to 7 days and can therefore be used as a non-invasive marker for diagnosis, treatment and measurement of the disease activity in patients with IBD. The most common method for analysis of calprotectin concentration is ELISA. This method is time-consuming and many manufactures have therefore developed rapid tests as a faster alternative for quantification of calprotectin in stool. Aim The aim of the study was to evaluate one ELISA and one rapid test from the same manufacture compare the data with the existing ELISA-method used in the laboratory for routine analysis. Methods A rapid test (CalFast) and an ELISA method (CalPrest) from Eurospital, were used for analysis of calprotectin in stool. These two methods were compared with known concentrations of calprotectin obtained by the ELISA method from Bühlmann used in the routine work.  Results The results showed poor correlation between the rapid test and the ELISA method. Furthermore, the comparison between the two ELISA-methods showed a poor correlation. Conclusion Evaluation of the two new methods showed poor correlation with the existing ELISA method from Bühlmann. Evaluation of the rapid test did not show any correlation with the two ELISA methods and the data cannot be trusted. It is difficult to conclude which of the two ELISA methods gives accurate results due to the absence of an international standard.

Background Calprotectin is a protein expressed in the cytoplasm inside the neutrophile granulocytes. During inflammatory bowel disease (IBD), the neutrophile granulocytes are involved in a complex interaction at the inflammatory area where they die and release their content into the intestinal lumen. Therefore, calprotectin in stool is a suitable marker for diagnosis and measurement of the disease-activity in patients with IBD. The most commonly used method to detect calprotectin in stool is ELISA, but the process of manual preparation of stool samples is time-consuming. Aim The objective of the study was to evaluate an extraction method that could replace manual preparation of fecal samples and to compare different methods for measuring Calprotectin in stool using two ELISA-methods from two manufacturers and one rapidtest. Methods For extraction of calprotectin from stool samples we used sample collector tubes from Epitope Diagnostics and fecal preparation kits from Roche. Two different ELISA-kits for measuring calprotectin concentration in stool were compared. Measurements of calprotectin with rapid-test from Epitope Diagnostics were also performed and were compared with the two ELISA kits. Results The results indicate a poor correlation between two extraction methods with Sample Collector Tube and Roche preparation kit. The comparison between the two ELISA-kits showed poor correlation. Evaluation of rapid test showed 33% false negative results with a cut-off value at 50 mg/kg. Conclusion Evaluation of products from Epitope Diagnostics showed poor correlation with the Bühlmann ELISA and an unreliable rapid test. Therefore, none of evaluated products from Epitope Diagnostics is accurate enough to be used for clinical diagnosis in the laboratory.

Background: Caveolin-1 (Cav-1), the major component of caveolae, is a multifunctional scaffolding protein that serves as a platform for the cell’s signal-transduction and plays a role in inflammation. However, its role in inflammatory bowel disease (IBD), a chronic inflammatory condition in the gastrointestinal tract, is not clear. A recent study shows that Cav-1 mediates angiogenesis in dextran sodium sulphate (DSS)-induced colitis. These results contradict our data, in which Cav-1 levels decreased significantly in 2,4,6-trinitrobenzene sulphonic acid (TNBS)–induced colitis. Methods: To test whether Cav-1 is involved in IBD pathogenesis, various models representing different dominant Th subtype responses and mimicking the immune pathologic mechanisms of different clinical IBD setting were employed: acute colitis was induced by intra-rectal administration of a single dose of TNBS in BALB/c and C57BL/6J mice, or by drinking 3% DSS water for 6 days in C57BL/6J mice. Chronic colitis was induced by administration of TNBS once a week for 7 weeks in BALB/c mice. To assess the effects of complete loss of Cav-1, Cav-1 knock-out (Cav-1-/-) and control wild-type C57BL/6J mice received a single TNBS administration. To further test the possible role of Cav-1, one of two peptides (that either mimicked (Caveolin scaffolding domain; CSD) or antagonized (Caveolin-1 binding domain; CBD1) Cav-1)) was administered intraperitoneally to mice receiving TNBS. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified by ELISA. Inflammation was evaluated through histological analysis. Results: Cav-1 levels in mouse colon tissue were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation and cytokine levels. Furthermore, a loss of Cav-1 (Cav-1-/-) showed increased clinical and inflammatory scores and increased body weight loss. Mice receiving peptides to alter Cav-1 levels, showed surprising effects. The mimicking peptide (CSD) showed decreased Cav-1 levels, while the antagonizing peptide (CBD1) showed increased Cav-l levels. These changes in levels were associated with clinical and inflammatory scores and body weight loss that supported the TNBS-induced data. DSS-induced colitis mice showed increased disease activity index, however no significant difference in Cav-1 levels was found between colitis and normal mice. Conclusions: Cav-1 plays an important role in the protection of TNBS-induced colitis, but not in DSS-induced colitis, an entirely different result from a previous report, suggesting that enhancement of Cav-1 expression and functions may be beneficial to IBD treatment in some specific clinical settings. Further studies are warranted.

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

Inflammatory bowel disease (IBD) is associated with an inappropriate immune response to the gut microbiota and disruption of intestinal homeostasis. IBD patients and experimental animal models have consistently shown alterations in the gut microbiota composition. However, these studies have mainly focused on faecal microbiota samples taken after the onset of inflammation and IBD establishment. The colonic microbiota inhabits both the gut lumen and the mucus layer covering the intestinal epithelium. Thus, information about mucus-resident microbiota is not necessarily conveyed in the routine microbiota analyses of faecal samples. To address potential changes in microbial composition and function before the onset of IBD, we compared both mucus and faecal microbiota in the mdr1a-/- spontaneous model of colitis over times that we histologically defined as before onset of colitis, during and after colitis onset. We showed that alterations in microbiota composition preceded the onset of intestinal inflammation and that these changes were evident in the mucus, but not in faeces. This altered microbiota composition was coupled with a reduced inner mucus layer, indicating a compromised mucus barrier prior to colitis development. Upon emergence of inflammation, compositional differences were found in both mucus and faecal microbial communities. Spatial segregation of microbiota with intestinal mucosa was also disrupted on disease onset which we hypothesise contributes to a more severe intestinal pathology. Therefore, our data indicate that microbial changes start locally in the mucus and then proceed to the faecal matter concomitantly with colitis development. Next, we examined whether microbial gene functional potential and endogenous metabolite profiles followed alterations in gut microbiota taxonomic composition. Our findings showed that the microbial gene content was similar between mdr1a-/- mice and wild-type littermate controls, demonstrating stability of the gut microbiome at the face of ensuing gut inflammation. In further support of these findings, urinary metabolite analysis revealed that metabolite profiles were unaffected by intestinal inflammation. Metabolites previously reported to change in IBD were similar between mdr1a-/- and wild-type mice at stages preceding and during inflammation. We also found that changes in metabolite profiles did not correlate with colitis scores. However, metabolite changes could discriminate mdr1a-/- mice from wild-type controls, suggesting they could have value in predicting risk of IBD with a potential clinical use in at least a subset of individuals with MDR1A polymorphisms. To assess whether changes in antimicrobial proteins (AMPs) accounted for observed differences in mucus microbiota composition, we also investigated the expression of regenerating islet-derived protein 3 γ (Reg3γ), angiogenin 4 (Ang4), β-defensin 1 and resistin-like molecule beta (Relm-β) in the colon. We found similar levels of these AMPs as well as IgA-producing plasma cells between mdr1a-/- and wild-type mice, suggesting that other factors contribute to alterations in microbiota composition. Overall, our data indicate that the mdr1a-/- is a good model of colitis, as it enables us to look at pre-clinical changes in the gut microbiota. This work suggests the importance of mucus sampling for sensitive detection of microbiota changes. Furthermore, metabolite profiling may be a helpful way to discriminate genetic susceptibility to disease.