Tara Willson

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.

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)

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.

Deletion of Intestinal Epithelial Cell STAT3 Promotes T-Lymphocyte STAT3 Activation and Chronic Colitis Following Acute Dextran Sodium Sulfate Injury in Mice

Background:Intestinal epithelial cell (IEC) STAT3 is required for wound healing following acute dextran sodium sulfate (DSS) injury. We hypothesized that loss of IEC STAT3 would promote the development of chronic colitis following acute DSS injury. Methods:Colitis was induced in IEC-specific STAT3-deficient mice (STAT3[INCREMENT]IEC) and littermate controls (STAT3Flx/Flx) with 4% DSS for 7 days, followed by water consumption for 21 days. Epithelial and immune mediators and severity of colitis were determined. Results:Survival, colon length, and histologic injury were significantly worse at day 28 in STAT3[INCREMENT]IEC mice. IEC proliferation and apoptosis did not vary by genotype at day 14 or day 28. The colonic lamina propria frequency of pSTAT3+ cells was increased at day 28 and correlated with histologic injury in STAT3[INCREMENT]IEC mice. The frequency of colonic F480+ pSTAT3+ macrophages and CD3+ pSTAT3+ T lymphocytes were increased in STAT3[INCREMENT]IEC mice as compared with STAT3Flx/Flx controls. In STAT3[INCREMENT]IEC mice, colonic expression of STAT3 target genes Reg3&bgr; and Reg3&ggr;, which mediate epithelial restitution, were significantly decreased, whereas expression of interleukin (IL)-17a, IFN&ggr;, CXCL2, CXCL10, and CCL2 were significantly increased and correlated with the increase in histologic severity at day 28(P < 0.05). IL-17a expression also correlated with the increased lamina propria frequency of CD3+ pSTAT3+ T lymphocytes. Conclusions:Loss of intestinal epithelial STAT3 leads to more severe chronic inflammation following acute injury, which is not accounted for by a sustained defect in epithelial proliferation or apoptosis 7 or 21 days after 1 cycle of DSS but rather defective REG3 expression and expansion of pSTAT3+ lymphocytes and IL-17A expression.

Paired Immunoglobulin-Like Receptor B (PIR-B) Negatively Regulates Macrophage Activation in Experimental Colitis

BACKGROUND & AIMS Innate and adaptive immune responses are regulated by cross talk between activation and inhibitory signals. Dysregulation of the inhibitory signal can lead to aberrant chronic inflammatory diseases such as the inflammatory bowel diseases (IBD). Little is known about negative regulation of innate intestinal immune activation. We examined the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of macrophage function in innate intestinal immunity. METHODS We examined the susceptibility of Pirb-/- and wild-type (WT) mice to dextran sodium sulfate (DSS)-induced colitis. We assessed proinflammatory cytokine release and mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) activation in Pirb-/- and WT macrophages following Escherichia coli stimulation. Macrophage transfer experiments were performed to define the role of PIR-B in the negative regulation of macrophage function in DSS-induced colitis. We also assessed expression of PIR-B human homologues (immunoglobulin-like transcript [ILT]-2 and ILT-3) in colon biopsy samples from healthy individuals (controls) and patients with IBD. RESULTS Pirb-/- mice had increased susceptibility to DSS-induced colitis. In vitro analysis showed increased production of proinflammatory cytokines (interleukin-6, interleukin-1beta, and tumor necrosis factor alpha) and activation of MAPK and NF-kappaB in Pirb-/- macrophages following bacterial activation. Adoptive transfer of bone marrow-derived Pirb-/- macrophages into WT mice was sufficient to increase disease susceptibility. ILT-2 and ILT-3 were expressed on CD68+ and CD68- mononuclear cells and intestinal epithelium in colon biopsy samples from patients and controls. CONCLUSIONS PIR-B negatively regulates macrophage functions in response to pathogenic bacteria and chronic intestinal inflammatory responses. Inhibitory receptors such as PIR-B might be used as therapeutic targets for treatment of patients with IBD.

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;)

Pretreatment with anti-VEGF therapy may exacerbate inflammation in experimental acute colitis

AIM Our previous investigations of angiogenesis in inflammatory bowel disease showed that vascular endothelial growth factor (VEGF) blockade reduced colonic neovascularization and inflammation. We hypothesized that pretreatment with bevacizumab, a monoclonal anti-VEGF antibody, would attenuate the severity of angiogenesis and inflammation in a murine model of colitis. METHODS C57BL/6 mice were treated with intraperitoneal injections of bevacizumab (250 μg/dose) before induction of colitis with dextran sulfate sodium (DSS). The colons were examined at predetermined time points. Colonic inflammation and microvessel density were assessed microscopically. RESULTS All mice acutely developed melena and weight loss (18.8% ± 1.1% control vs 20.2% ± 1.1% treated, P = .37) and regained a similar weight percentage after the recovery (26.5% ± 4.0% vs 20.9% ± 4.4%, P = .37). Microvessel density acutely increased in both groups in response to DSS, with a trend toward inhibited angiogenesis in the treated group at the conclusion of the acute phase (194,100 ± 14,240 vs 149,400 ± 17,590 μm(2), P = .11). Bevacizumab-treated mice exhibited significantly increased inflammation after the acute phase (8.3 ± 0.8 vs 13.0 ± 2.0, P = .05), but were similar to control after the recovery (7.3 ± 1.5 vs 5.5 ± 1.0, P = .27). CONCLUSIONS Preemptive VEGF inhibition does not significantly attenuate angiogenesis and, in fact, worsens inflammation in a model of acute colitis. Preventive VEGF blockade may disrupt healing and exacerbate injury via alternative angiogenic or inflammatory pathways.

498 Autocrine Intestinal Epithelial Cell GM-CSF Signaling Promotes Homeostatic Responses to Gut Injury

Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium ofmice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohns disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBαmRNA, a downstream target of NFκBwas substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship