Erin Bonkowski

Loci on 20q13 and 21q22 are associated with pediatric-onset inflammatory bowel disease

Inflammatory bowel disease (IBD) is a common inflammatory disorder with complex etiology that involves both genetic and environmental triggers, including but not limited to defects in bacterial clearance, defective mucosal barrier and persistent dysregulation of the immune response to commensal intestinal bacteria. IBD is characterized by two distinct phenotypes: Crohns disease (CD) and ulcerative colitis (UC). Previously reported GWA studies have identified genetic variation accounting for a small portion of the overall genetic susceptibility to CD and an even smaller contribution to UC pathogenesis. We hypothesized that stratification of IBD by age of onset might identify additional genes associated with IBD. To that end, we carried out a GWA analysis in a cohort of 1,011 individuals with pediatric-onset IBD and 4,250 matched controls. We identified and replicated significantly associated, previously unreported loci on chromosomes 20q13 (rs2315008[T] and rs4809330[A]; P = 6.30 × 10−8 and 6.95 × 10−8, respectively; odds ratio (OR) = 0.74 for both) and 21q22 (rs2836878[A]; P = 6.01 × 10−8; OR = 0.73), located close to the TNFRSF6B and PSMG1 genes, respectively.

Activation of an IL-6:STAT3-dependent Transcriptome in Pediatric-onset Inflammatory Bowel Disease

Background: While activation of the IL‐6‐dependent transcription factor signal transducer and activator of transcription 3 (STAT3) has been implicated in the pathogenesis of inflammatory bowel disease (IBD), a direct effect on mucosal gene expression and inflammation has not been shown. We hypothesized that a proinflammatory IL‐6:STAT3‐dependent biological network would be up regulated in pediatric‐onset IBD patients, and would be associated with the severity of mucosal inflammation. Methods: Patients with pediatric‐onset IBD were enrolled at diagnosis and during therapy. Serum cytokine analysis was performed using Bioplex. STAT3 phosphorylation (pSTAT3) in peripheral blood leukocytes (PBLs) was assessed by flow cytometry. Immunohistochemistry of colonic mucosa was used to localize pSTAT3 and STAT3 target genes. Microarray analysis was used to determine RNA expression profiles from colon biopsies. Results: Circulating IL‐6 was upregulated in active IBD patients at diagnosis and during therapy. STAT3 activation was increased in PB granulocytes, IL‐6‐stimulated CD3+/CD4+ lymphocytes, and affected colon biopsies of IBD patients. The frequency of pSTAT3+ PB granulocytes and colon epithelial and lamina propria cells was highly correlated with the degree of mucosal inflammation. Microarray and Ingenuity Systems bioinformatics analysis identified IL‐6:STAT3‐dependent biological networks upregulated in IBD patients which control leukocyte recruitment, HLA expression, angiogenesis, and tissue remodeling. Conclusions: A proinflammatory IL6:STAT3 biologic network is upregulated in active pediatric IBD patients at diagnosis and during therapy. Specific targeting of this network may be effective in reducing mucosal inflammation.

Lipopolysaccharide exposure is linked to activation of the acute phase response and growth failure in pediatric Crohn's disease and murine colitis.

Background:Systemic exposure to lipopolysaccharide (LPS) has been linked to clinical disease activity in adults with inflammatory bowel disease (IBD). We hypothesized that markers of LPS exposure and the acute phase response (APR) would be increased in pediatric IBD patients with growth failure, and that LPS signaling would be required for induction of the APR in murine colitis. Methods:Serum markers of LPS exposure, endotoxin core IgA antibody (EndoCAb), and the APR, LPS binding protein (LBP) were quantified in pediatric IBD patients and controls. LBP and cytokine production were determined after administration of trinitrobenzene sulfonic acid (TNBS) enemas to mice with genetic deletion of Toll‐Like receptor 4 (TLR4), and wildtype (WT) controls. Results:Serum EndoCAb and LBP were significantly elevated in patients with Crohns disease (CD), compared to disease controls with ulcerative colitis (UC) and healthy controls (P < 0.001). This was independent of disease activity or location. CD patients with elevated serum EndoCAb and LBP exhibited linear growth failure which persisted during therapy. Serum LBP increased in WT mice following TNBS administration, in conjunction with increased serum TNF‐&agr;, IL‐6, and IL‐10, and expansion of regulatory T‐cell numbers. Both the APR and expansion of foxp3+ T cells were abrogated in TLR4‐deficient mice, in conjunction with a reduction in acute weight loss. Conclusions:LPS exposure and a persistent APR are associated with growth failure in pediatric CD. LPS signaling is required for the APR in murine colitis. Therapies targeting this pathway may benefit the subset of patients with refractory growth failure. (Inflamm Bowel Dis 2010)

Granulocyte-macrophage colony-stimulating factor autoantibodies: a marker of aggressive Crohn's disease.

Background: Neutralizing autoantibodies (Abs) against granulocyte–macrophage colony-stimulating factor (GM-CSF Ab) have been associated with stricturing ileal Crohns disease (CD) in a largely pediatric patient cohort (total 394, adult CD 57). The aim of this study was to examine this association in 2 independent predominantly adult inflammatory bowel disease patient cohorts. Methods: Serum samples from 742 subjects from the NIDDK IBD Genetics Consortium and 736 subjects from Australia were analyzed for GM-CSF Ab and genetic markers. We conducted multiple regression analysis with backward elimination to assess the contribution of GM-CSF Ab levels and established CD risk alleles and smoking on ileal disease location in the 477 combined CD subjects from both cohorts. We also determined associations of GM-CSF Ab levels with complications requiring surgical intervention in combined CD subjects in both cohorts. Results: Serum samples from patients with CD expressed significantly higher concentrations of GM-CSF Ab when compared with ulcerative colitis or controls in each cohort. Nonsmokers with ileal CD expressed significantly higher GM-CSF Ab concentrations in the Australian cohort (P = 0.002). Elevated GM-CSF Ab, ileal disease location, and disease duration more than 3 years were independently associated with stricturing/penetrating behavior and intestinal resection for CD. Conclusions: The expression of high GM-CSF Ab is a risk marker for aggressive CD behavior and complications including surgery. Modifying factors include environmental exposure to smoking and genetic risk markers.

A randomized controlled trial of growth hormone in active pediatric Crohn disease.

Objectives: Growth hormone (GH) may reduce symptoms and improve growth in Crohn disease (CD). The effect on mucosal inflammation is not known. We hypothesized that GH would improve both clinical and mucosal disease activity and stimulate linear growth in pediatric CD. Patients and Methods: Twenty patients ages 7 to 18 receiving corticosteroids (CTX) for active CD were randomized to begin GH, 0.075 mg · kg−1 · day−1 (group A), or continue CTX alone (group B). Clinical and endoscopic disease activities were assessed after 12 weeks. Group B began GH at 12 weeks, and clinical disease activity was assessed at 24 weeks. Subjects who experienced a clinical response after 12 weeks of GH therapy continued treatment for an additional 52 weeks, and linear growth was assessed. Results: Sixty-five percent of patients receiving GH achieved clinical remission, compared with 20% treated with CTX alone (P = 0.03). Although endoscopic disease activity trended toward an improvement at week 12 in group A, this did not differ between the groups. Sixty-one percent of week 12 GH responders maintained their clinical response through week 64. Mean (95th confidence interval) height z score on GH increased from −1.1 (−1.6, −0.6) to −0.4 (−1, 0.2), P = 0.004 during this 52-week extension phase. GH was well tolerated with no unexpected safety signals. Conclusions: The addition of GH to CTX therapy did not induce a reduction in mucosal inflammation, relative to CTX alone. However, GH was safe and effective as an adjunct to CTX for treatment of clinical disease activity and growth failure in pediatric CD.

Granulocyte macrophage colony-stimulating factor auto-antibodies and disease relapse in inflammatory bowel disease.

OBJECTIVES:Along with others, we have reported that neutralization of granulocyte macrophage colony-stimulating factor (GM-CSF) increases intestinal permeability and bacterial translocation, and reduces neutrophil bacterial killing and anti-microbial seroreactivity. The objective was to investigate the utility of serum GM-CSF auto-antibody (Ab) as a marker for confirmation of stable remission and prediction of relapses in patients with inflammatory bowel disease (IBD).METHODS:We consecutively included 181 adults and children with Crohns disease (CD, n=61) or ulcerative colitis (UC, n=120). Over a 3-year period, we collected 861 serum samples and 610 stool samples during regular follow-up visits. GM-CSF Abs and fecal S100 proteins were measured by an enzyme-linked immunoassay.RESULTS:Serum GM-CSF Ab levels correlated with disease activity, location, and extent. Time course analysis before and after relapse showed a clear increase of GM-CSF Ab concentrations up to 6 months before clinical relapse. At 1.7 μg/ml (CD) and 0.5 μg/ml (UC), the sensitivity and specificity of GM-CSF Ab for predicting relapse already 2–6 months earlier were 88% and 95% in CD and 62% and 68% in UC, respectively. A baseline GM-CSF Ab level of >1.7 μg/ml was significantly associated with relapse of CD within 18 months.CONCLUSIONS:As GM-CSF is required for myeloid cell antimicrobial functions and homeostatic responses to tissue injury, serum GM-CSF Ab levels might reflect the degree of bowel permeability and bacterial translocation. Therefore, GM-CSF Ab might identify IBD patients at risk of disease relapse at an early stage, which makes the test a potential tool for monitoring disease activity and optimizing therapy.

Granulocyte macrophage-colony-stimulating factor autoantibodies and increased intestinal permeability in Crohn disease.

Background:Alterations in intestinal permeability have been implicated in the pathogenesis of Crohn disease (CD). We have reported that granulocyte macrophage-colony-stimulating factor (GM-CSF) is required for mucosal barrier function in mice, and elevated neutralizing GM-CSF autoantibodies (Ab) are associated with stricturing ileal disease and surgery in patients with CD. We hypothesized that children with CD with elevated GM-CSF Ab would exhibit increased intestinal permeability. Patients and Methods:Subjects were divided into 3 groups: 15 with CD and high GM-CSF Ab (≥1.6 μg/mL, GM-CSF Ab Hi), 12 with CD and low GM-CSF Ab (<1.6 μg/mL, GM-CSF Ab Lo), and 15 healthy controls. Subjects ingested a lactulose:mannitol (L:M) solution, and urinary excretion of LM was measured by high-performance liquid chromatography. Serum GM-CSF Ab, endotoxin core Ab (EndoCAb), and lipopolysaccharide-binding protein (LBP), and fecal S100A12 were determined by enzyme-linked immunosorbent assay. Results:The CD groups did not vary by age, sex, disease location, or activity. Neither systemic (serum LBP) nor mucosal (fecal S100A12) inflammation differed between the CD groups. Intestinal permeability as measured by the urine L:M ratio and endotoxin exposure as measured by serum EndoCAb were increased in the GM-CSF Ab Hi group compared to the GM-CSF Ab Lo group and controls. Conclusions:Patients with CD with elevated GM-CSF Ab exhibit an increase in bowel permeability relative to patients with CD with lower levels of GM-CSF Ab in the absence of differences in systemic or intestinal inflammation. Therapies that target the mucosal barrier may be of particular benefit in this subgroup of patients with CD.

Lipopolysaccharide (LPS) directly suppresses growth hormone receptor (GHR) expression through MyD88-dependent and -independent Toll-like receptor-4/MD2 complex signaling pathways

INTRODUCTION Sepsis is associated with growth hormone (GH) insensitivity and in the intact animal the major surface component of the bacterial cell wall, lipopolysaccharide (LPS), inhibits GH receptor (GHR) gene expression. The prevailing explanation for LPS-induced effects on the GHR promoter is that this effect is indirect via generation of cytokines. Our recent studies demonstrate that saturated free fatty acids (FFAs) inhibit the activity of the murine GHR promoter. Saturated FFAs are an essential component of the lipid A moiety of LPS required for biological activity of LPS. HYPOTHESIS LPS directly modulates the activity of the dominant GHR promoter via interaction with Toll-like receptor(s) (TLR)/MD2 complex and activation of cognate signaling pathway(s). RESULTS In transient transfection experiments with RAW 264.7 cells which express endogenous TLR4 and MD2, LPS treatment inhibited GHR promoter activity. Co-transfection of dominant negative TLR4 abrogated this effect on GHR promoter activity. In HEK 293T cells, which are devoid of endogenous TLR4 or MD2, ectopic expression of TLR4 and MD2 resulted in LPS-induced inhibition of the GHR promoter activity. The inhibition of GHR promoter activity was demonstrable by 5-6h after exposure to LPS and persisted at 24h. Fatty-acid free LPS failed to elicit a similar effect on the GHR promoter and the effect of LPS was abrogated by Polymyxin B. The essential role of the cofactor MD2 on the effect of LPS on the GHR promoter was established in experiments using ectopic expression of wild type and mutant MD2. Cotransfection of CD14 in these cells failed to alter the effect of LPS on the activity of the GHR promoter. Analysis of cell culture supernatant excluded the possibility that the effect of LPS was secondary to release of cytokines from the transfected cells. The effect of LPS on the endogenous GHR promoter activity and protein expression was confirmed in F442A preadipocyte cells. In HEK 293T cells, ectopic expression of mutant MyD88 or mutant TRIF abrogated the effect of LPS on the GHR promoter, suggesting that the effect of LPS on the GHR promoter was via both MyD88-dependent and -independent pathways. CONCLUSIONS LPS acts through both MyD88-dependent and -independent TLR4 signaling pathways to directly inhibit GHR gene expression. Our results establish a novel cytokine-independent mechanism for decrease in GHR expression in bacterial sepsis.

Innate dysfunction promotes linear growth failure in pediatric Crohn's disease and growth hormone resistance in murine ileitis

Background: Growth failure remains a common complication of pediatric Crohns disease (CD) and has been associated with small bowel involvement and need for surgery. We have reported that patients with elevated (≥1.6 &mgr;g/mL) granulocyte macrophage colony stimulating factor autoantibodies (GM‐CSF Ab) are more likely to experience complicated ileal disease requiring surgery. We hypothesized that concurrent GM‐CSF Ab and CARD15 risk allele carriage (C15+GMAb+) would be associated with growth failure in CD and growth hormone (GH) resistance in murine ileitis. Methods: We enrolled 229 pediatric CD patients at two sites and determined CARD15 genotype, serum GM‐CSF Ab, and GH binding protein (GHBP), and height (HTz) and weight (WTz) z‐scores at diagnosis. Ileitis was induced in card15‐deficient mice by GM‐CSF neutralization and nonsteroidal antiinflammatory drug (NSAID) exposure. Hepatic GH receptor (GHR) abundance and GH‐dependent Stat5 activation were determined by western blot and Igf‐I mRNA expression by real‐time polymerase chain reaction (PCR). Results: Mean (95% confidence interval [CI]) HTz at diagnosis was reduced to −0.48 (−4.2, 2.3) in C15+GMAb+patients, compared to −0.07 (−4.9, 3.4) in disease controls (P ≤ 0.05). Circulating GHBP, as a marker for tissue GHR abundance, was reduced in C15+GMAb+ patients. Hepatic GHR abundance, GH induction of Stat5 tyrosine phosphorylation, and Igf‐I mRNA expression were reduced in male card15‐deficient mice with ileitis due to GM‐CSF neutralization and NSAID exposure. Conclusions: Innate dysfunction due to concurrent genetic variation in CARD15 and neutralizing GM‐CSF Ab is associated with linear growth failure in pediatric CD, and hepatic GH resistance in murine ileitis. (Inflamm Bowel Dis 2011;)

Granulocyte‐macrophage colony stimulating factor blockade promotes ccr9+ lymphocyte expansion in Nod2 deficient mice

Background: Ileal involvement in Crohns disease (CD) is associated with NOD2 mutations and granulocyte‐macrophage colony stimulating factor autoantibodies (GM‐CSF Ab), and GM‐CSF blockade promotes ileitis in Nod2/Card15‐deficient (C15KO) mice. RALDH2‐expressing dendritic cells (DC) and IL‐4 promote CCR9 imprinting and small bowel homing of T lymphocytes, in conjunction with CCL25 expression by ileal epithelial cells (IEC). We hypothesized that GM‐CSF neutralization promotes ileal disease by modulating expression of CCL25 by IEC and CCR9 by T lymphocytes via Nod2‐dependent and independent pathways. Methods: CCL25 and CCR9 expression were determined in pediatric CD patients stratified by GM‐CSF Ab. Ileitis was induced in C15KO mice via GM‐CSF Ab administration followed by nonsteroidal antiinflammatory drug (NSAID) exposure, and expression of CCL25, CCR9, FOXP3, intracellular cytokines, and RALDH2 was determined in IEC and immune cell populations. Results: The frequency of CCL25+ IEC and CCR9+ T lymphocytes was increased in CD patients with elevated GM‐CSF Ab. In the murine model, GM‐CSF blockade alone induced IEC CCL25 expression, and reduced the frequency of mesenteric lymph node (MLN) CD4+FOXP3+ cells, while Card15 deficiency alone enhanced MLN DC RALDH2 expression. Both GM‐CSF neutralization and Card15 deficiency were required for downregulation of MLN DC IL‐10 expression; under these conditions NSAID exposure led to an expansion of IL‐4+ and IL‐17+ CCR9+ lymphocytes in the ileum. Conclusions: GM‐CSF prevents ileal expansion of CCR9+ lymphocytes via Nod2‐dependent and independent pathways. CCR9 blockade may be beneficial in CD patients with elevated GM‐CSF Ab.