Caroline T. Brown

High-throughput multi-analyte Luminex profiling implicates eotaxin-1 in ulcerative colitis.

Accurate and high-throughput technologies are needed for identification of new therapeutic targets and for optimizing therapy in inflammatory bowel disease. Our aim was to assess multi-analyte protein-based assays of cytokines/chemokines using Luminex technology. We have reported that Luminex-based profiling was useful in assessing response to L-arginine therapy in the mouse model of dextran sulfate sodium colitis. Therefore, we studied prospectively collected samples from ulcerative colitis (UC) patients and control subjects. Serum, colon biopsies, and clinical information were obtained from subjects undergoing colonoscopy for evaluation of UC or for non-UC indications. In total, 38 normal controls and 137 UC cases completed the study. Histologic disease severity and the Mayo Disease Activity Index (DAI) were assessed. Serum and colonic tissue cytokine/chemokine profiles were measured by Luminex-based multiplex testing of 42 analytes. Only eotaxin-1 and G-CSF were increased in serum of patients with histologically active UC vs. controls. While 13 cytokines/chemokines were increased in active UC vs. controls in tissues, only eotaxin-1 was increased in all levels of active disease in both serum and tissue. In tissues, eotaxin-1 correlated with the DAI and with eosinophil counts. Increased eotaxin-1 levels were confirmed by real-time PCR. Tissue eotaxin-1 levels were also increased in experimental murine colitis induced by dextran sulfate sodium, oxazolone, or Citrobacter rodentium, but not in murine Helicobacter pylori infection. Our data implicate eotaxin-1 as an etiologic factor and therapeutic target in UC, and indicate that Luminex-based assays may be useful to assess IBD pathogenesis and to select patients for anti-cytokine/chemokine therapies.

MTG16 contributes to colonic epithelial integrity in experimental colitis

Objective The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8−/− and Mtgr1−/− mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity. Methods Baseline and stress induced colonic phenotypes were examined in Mtg16−/− mice. To unmask phenotypes, we treated Mtg16−/− mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation. Results Mtg16−/− mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16−/− mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1+, F4/80+, CD11c+ and MHCII+; CD11c+) and Th1 adaptive (CD4) immune cells in Mtg16−/− colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16−/− colons. Compared with wild-type mice, Mtg16−/− mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16−/− recipients did not rescue the Mtg16−/− injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16−/− mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16−/− mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues. Conclusions These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.

Serum Fatty Acids Are Correlated with Inflammatory Cytokines in Ulcerative Colitis

Background and Aims Ulcerative colitis (UC) is associated with increased dietary intake of fat and n-6 polyunsaturated fatty acids (PUFA). Modification of fat metabolism may alter inflammation and disease severity. Our aim was to assess differences in dietary and serum fatty acid levels between control and UC subjects and associations with disease activity and inflammatory cytokines. Methods Dietary histories, serum, and colonic tissue samples were prospectively collected from 137 UC subjects and 38 controls. Both histologic injury and the Mayo Disease Activity Index were assessed. Serum and tissue cytokines were measured by Luminex assay. Serum fatty acids were obtained by gas chromatography. Results UC subjects had increased total fat and oleic acid (OA) intake, but decreased arachidonic acid (AA) intake vs controls. In serum, there was less percent saturated fatty acid (SFA) and AA, with higher monounsaturated fatty acids (MUFA), linoleic acid, OA, eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA) in UC. Tissue cytokine levels were directly correlated with SFA and inversely correlated with PUFA, EPA, and DPA in UC subjects, but not controls. 5-aminosalicylic acid therapy blunted these associations. Conclusions In summary, we found differences in serum fatty acids in UC subjects that correlated with pro-inflammatory tissue cytokines. We propose that fatty acids may affect cytokine production and thus be immunomodulatory in UC.

L-Arginine Availability and Metabolism Is Altered in Ulcerative Colitis.

Background:L-arginine (L-Arg) is the substrate for both inducible nitric oxide (NO) synthase (NOS2) and arginase (ARG) enzymes. L-Arg is actively transported into cells by means of cationic amino acid transporter (SLC7) proteins. We have linked L-Arg and arginase 1 activity to epithelial restitution. Our aim was to determine if L-Arg, related amino acids, and metabolic enzymes are altered in ulcerative colitis (UC). Methods:Serum and colonic tissues were prospectively collected from 38 control subjects and 137 UC patients. Dietary intake, histologic injury, and clinical disease activity were assessed. Amino acid levels were measured by high-performance liquid chromatography. Messenger RNA (mRNA) levels were measured by real-time PCR. Colon tissue samples from 12 Crohns disease patients were obtained for comparison. Results:Dietary intake of arginine and serum L-Arg levels were not different in UC patients versus control subjects. In active UC, tissue L-Arg was decreased, whereas L-citrulline (L-Cit) and the L-Cit/L-Arg ratio were increased. This pattern was also seen when paired involved (left) versus uninvolved (right) colon tissues in UC were assessed. In active UC, SLC7A2 and ARG1 mRNA levels were decreased, whereas ARG2 and NOS2 were increased. Similar alterations in mRNA expression occurred in tissues from Crohns disease patients. In involved UC, SLC7A2 and ARG1 mRNA levels were decreased, and NOS2 and ARG2 increased, when compared with uninvolved tissues. Conclusions:Patients with UC exhibit diminished tissue L-Arg, likely attributable to decreased cellular uptake and increased consumption by NOS2. These findings combined with decreased ARG1 expression indicate a pattern of dysregulated L-Arg availability and metabolism in UC.

Alterations in lipid, amino acid, and energy metabolism distinguish Crohn’s disease from ulcerative colitis and control subjects by serum metabolomic profiling

IntroductionBiomarkers are needed in inflammatory bowel disease (IBD) to help define disease activity and identify underlying pathogenic mechanisms. We hypothesized that serum metabolomics, which produces unique metabolite profiles, can aid in this search.ObjectivesThe aim of this study was to characterize serum metabolomic profiles in patients with IBD, and to assess for differences between patients with ulcerative colitis (UC), Crohn’s disease (CD), and non-IBD subjects.MethodsSerum samples from 20 UC, 20 CD, and 20 non-IBD control subjects were obtained along with patient characteristics, including medication use and clinical disease activity. Non-targeted metabolomic profiling was performed using ultra-high performance liquid chromatography/mass spectrometry (UPLC-MS/MS) optimized for basic or acidic species and hydrophilic interaction liquid chromatography (HILIC/UPLC-MS/MS).ResultsIn total, 671 metabolites were identified. Comparing IBD and control subjects revealed 173 significantly altered metabolites (27 increased and 146 decreased). The majority of the alterations occurred in lipid-, amino acid-, and energy-related metabolites. Comparing only CD and control subjects revealed 286 significantly altered metabolites (54 increased and 232 decreased), whereas comparing UC and control subjects revealed only five significantly altered metabolites (all decreased). Hierarchal clustering using significant metabolites separated CD from UC and control subjects.ConclusionsWe demonstrate that a number of lipid-, amino acid-, and tricarboxylic acid cycle-related metabolites were significantly altered in IBD patients, more specifically in CD. Therefore, alterations in lipid and amino acid metabolism and energy homeostasis may play a key role in the pathogenesis of CD.

Mo1734 Serum Fatty Acids Are Correlated With Tissue Cytokine Levels in Ulcerative Colitis

until used for cytokine determination. The concentration of intestine mucosa cytokines involved in the Th1 (IL-2, IL-12p70, IFN-γ), Th2 (IL-4), Th17 (IL-6, IL-17A, IL-23) and Treg (IL-10, TGF-β) cell commitment and IL-1 β and TNF-α (cytokines of the innate immune response) was assayed in duplicate by ELISA (Biosource, Camarillo, CA) and the results were expressed as picogram of cytokine per milligram of protein. Data were analyzed with SPSS. Comparisons between the groups were done by the two-tailed Mann-Whitney U-test. The ileal and colonic concentration of all proinflammatory cytokines was significantly higher in the patients with CD and also UC than in control group (p<0.001 for all). Otherwise, ileal IL-10 (369.7±47.9 vs. 1569.3±81.7) and TGF-β (10534.9±391.2 vs. 13045.8±1005.8) and colonic IL-10 (225.2±47.1 vs. 1563.3±81.7) and TGF-β (7224.0±306.7 vs. 12962.8±991.4) concentration was lower in UC than in controls. Also ileal IL-10 (224.9±23.9) and TGF-β (7073.6±54.4) and colonic IL-10 (310.7±55.3) and TGF-β (9089.1±1850.3) concentration was lower in CD patients than in controls. Significant differences between CD and UC were also observed. Thus, IL-2, IL-6, IL-17, IL-23 ileal and colonic concentration was higher in DC than in UC. Otherwise, IL-10 ileal concentration was lower in CD and IL-10 colonic concentration was lower in UC. The concentration of other cytokines including those of the innate immunity and Th1 response was higher in the ileum of CD and higher in the colon of UC when the diseases where compared. Next CD with and without complications were compared. Although no significant differences were observed between patients with stenosis (n=17) and fistula (n=7), the colonic concentration of Th17-associated cytokines and TNF-α was significantly higher (p<0.03), either in patients with stenosis or with fistula than in those without complications .In conclusion, in CD predominates a Th17 shift, and an insuficient Treg cell activation to suppress the overwhelming proinflammatory milieu was observed in both diseases.

Tu1118 Non-Invasive Determination of Disease Activity in Ulcerative Colitis by Serum Luminex Profiling

Background: In a previous study utilizing multiplex Luminex technology in a cohort of control and ulcerative colitis (UC) patients, we identifiedmultiple proinflammatory cytokines/ chemokines that were increased in the serum and tissue of patients with active UC, determined by histology, compared to control patients. In order to better assess the potential for Luminex profiling for clinical trials, our aim was to investigate the relationship between a validated clinical disease activity tool, the Mayo Disease Activity Index (DAI) and the endoscopy score (ES) subset of the DAI, and the cytokine/chemokine profile in UC patients. Methods: Human serum and colon tissue samples were collected prospectively from 40 control subjects undergoing screening colonoscopy and 137 UC patients. The DAI (a combination of stool frequency, blood in the stool, physician global assessment, and ES) was assessed for UC patients at the time of colonoscopy. Serum and tissue cytokines/chemokines were measured by a 39-plex MILLIPLEX bead assay. We classified UC patients by DAI or ES alone into inactive (DAI 0-2; ES 0) and active (DAI 3-12; ES 1-3) disease. Cytokine/chemokine levels were analyzed based upon the DAI and ES classifications and were compared to control patients. We also assessed the correlation of DAI with the serum and tissue cytokine/ chemokine levels. Results: When active versus inactive UC was defined by a DAI cutoff of 3 or greater, patients with active UC had increased serum levels of eotaxin-1, G-CSF, GMCSF, IL-8, and TGF-α compared to control patients (all with at least p,0.05). None of the tissue or serum cytokine/chemokine levels resulted in a correlation greater than an r2 value of 0.18 with DAI as a continuous variable. When active disease was defined by an ES of 1 or greater, serum eotaxin-1, G-CSF, GM-CSF, IL-8, and IL-9 levels were increased in active UC (all p,0.05). Eotaxin-1, GM-CSF, and IL-8 were also increased in tissues when compared to controls when active UC was defined by either increased DAI or ES. In total, there were 21 of 39 cytokines/chemokines increased in tissues from patients with a DAI of 3 or greater vs. normal control subjects, and 20 were increased when ES was used. In active vs. inactive UC by DAI or ES, there were increases in serum levels of G-CSF, IFNγ, and IL-8; IL-15 and TGF-α were also increased when active UC was defined by DAI. In active vs. inactive UC defined by DAI, there were 17 analytes increased in tissues. When defined by ES, there were 22 analytes increased, including eotaxin-1, G-CSF, and IL-8. Conclusions: Luminex profiling was robust in identifying active disease defined by using either the DAI or the ES. Testing for serum cytokines/chemokines, especially G-CSF, IL-8, and eotaxin-1, may represent a non-invasive strategy for detecting active UC that compares favorably with current clinical tools.