Ingrid Jurickova

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)

Regulation of intestinal barrier function by signal transducer and activator of transcription 5b

Background: Colon epithelial cell (CEC) apoptosis and nuclear factor-κB (NF-κB) activation may compromise barrier function, and it has been reported that signal transducer and activator of transcription 5b (STAT5b)-deficient mice exhibit increased susceptibility to colitis. It is hypothesised that the growth hormone (GH) target STAT5b maintains mucosal barrier integrity by promoting CEC survival and inhibiting NF-κB activation. Methods: The GH effect upon mucosal injury due to 2,4,6-trinitro-benzenesulfonic acid (TNBS) administration was determined in STAT5b-deficient mice and wild-type (WT) controls. The effect of STAT5b deficiency upon CEC survival and NF-κB activation was determined and related to differences in intestinal permeability and bacterial translocation. RNA interference (RNAi) was used to knock down STAT5b expression in the T84 CEC line, and the effect upon basal and GH-dependent regulation of proapoptotic and inflammatory pathways induced by tumour necrosis factor α (TNFα) was determined. Results: GH suppression of mucosal inflammation in TNBS colitis was abrogated in STAT5b-deficient mice. STAT5b deficiency led to activation of a proapoptotic pattern of gene expression in the colon, and increased mucosal permeability. The frequency of apoptotic CECs was increased in STAT5b-deficient mice while tight junction protein abundance was reduced. This was associated with upregulation of CEC Toll-like receptor 2 expression and NF-κB activation. STAT5b knockdown in T84 CEC increased TNFα-dependent NF-κB and caspase-3 activation. GH inhibition of TNFα signalling was prevented by STAT5b knockdown. Conclusion: STAT5b maintains colonic barrier integrity by modulating CEC survival and NF-κB activation. STAT5b activation may therefore represent a novel therapeutic target in inflammatory bowel disease.

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.

Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury

Background Administration of granulocyte-macrophage colony stimulating factor (GM-CSF) relieves symptoms in Crohns disease (CD). It has been reported that reduced GM-CSF bioactivity is associated with more aggressive ileal behaviour and that GM-CSF-null mice exhibit ileal barrier dysfunction and develop a transmural ileitis following exposure to non-steroidal anti-inflammatory drugs (NSAIDs). STAT5 signalling is central to GM-CSF action. It was therefore hypothesised that GM-CSF signalling in non-haematopoietic cells is required for ileal homeostasis. Methods Bone marrow (BM) chimeras were generated by reconstituting irradiated GM-CSF receptor (gm-csfr) β chain or GM-CSF (gm-csf) deficient mice with wild type BM (WTBM→GMRKO and WTBM→GMKO). Intestinal barrier function and the response to NSAID-induced ileal injury were examined. Expression of gm-csf, gm-csfr or stat5 in Caco-2 and HT-29 intestinal epithelial cell (IEC) lines was knocked down and the effect of GM-CSF signalling on IEC survival and proliferation was determined. Results Elevated levels of GM-CSF autoantibodies in ileal CD were found to be associated with dysregulation of IEC survival and proliferation. GM-CSF receptor-deficient mice and WTBM→GMRKO chimeras exhibited ileal hyperpermeability. NSAID exposure induced a transmural ileitis in GM-CSF receptor-deficient mice and WTBM→GMRKO chimeras. Transplantation of wild type BM into GM-CSF-deficient mice prevented NSAID ileal injury and restored ileal barrier function. Ileal crypt IEC proliferation was reduced in WTBM→GMRKO chimeras, while STAT5 activation in ileal IEC following NSAID exposure was abrogated in WTBM→GMRKO chimeras. Following knock down of gm-csf, gm-csfr α or β chain or stat5a/b expression in Caco-2 cells, basal proliferation was suppressed. GM-CSF normalised proliferation of Caco-2 cells exposed to NSAID, which was blocked by stat5a/b RNA interference. Conclusions Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury; furthermore, GM-CSF signalling in non-haematopoietic cells regulates ileal epithelial homeostasis via the STAT5 pathway. The therapeutic use of GM-CSF may therefore be beneficial in chronic ileitis associated with CD.

Utility of neutrophil Fcγ receptor I (CD64) index as a biomarker for mucosal inflammation in pediatric Crohn's disease.

Background:Neutrophil expression of the Fc&ggr; receptor I (CD64) is upregulated in adult patients with clinically active inflammatory bowel disease (IBD). We tested the relationship of CD64 with mucosal inflammation and clinical relapse in pediatric Crohns disease (CD). Methods:In a cohort of 208 newly diagnosed CD and 43 non-IBD controls, ileal expression of Fc&ggr;RI/S100A9 was determined by RNA sequencing from biopsies obtained at ileocolonoscopy. In a second cohort, we tested for the peripheral blood polymorphonuclear neutrophil (PMN) CD64 index from 26 newly diagnosed CD, 30 non-IBD controls, and 83 children with established CD. Results:Ileal Fc&ggr;RIA mRNA expression was significantly elevated in CD at diagnosis compared with non-IBD controls (P < 0.001), and correlated with ileal S100A9 (calprotectin) expression (r = 0.83, P < 0.001). The median (range) PMN CD64 index for newly diagnosed CD was 2.3 (0.74–9.3) compared with 0.76 (0.39–1.2) for non-IBD controls (P < 0.001) with 96% sensitivity and 90% specificity at the cut point of 1.0. The PMN CD64 index significantly correlated with mucosal injury as measured by the simple endoscopic score for CD (r = 0.62, P < 0.001). Patients with CD in clinical remission receiving maintenance therapy with a PMN CD64 index <1.0 had a sustained remission rate of 95% over the following 12 months compared with 56% in those with a PMN CD64 index >1.0 (P < 0.01). Conclusions:An elevated PMN CD64 index is associated with both mucosal inflammation and an increased risk for clinical relapse in pediatric CD. The PMN CD64 index is a reliable marker for sustained remission in patients with CD receiving maintenance therapy.

Paediatric Crohn disease patients with stricturing behaviour exhibit ileal granulocyte–macrophage colony-stimulating factor (GM-CSF) autoantibody production and reduced neutrophil bacterial killing and GM-CSF bioactivity

Granulocyte–macrophage colony‐stimulating factor (GM‐CSF) autoantibodies are associated with stricturing behaviour in Crohn disease (CD). We hypothesized that CD ileal lamina propria mononuclear cells (LPMC) would produce GM‐CSF autoantibodies and peripheral blood (PB) samples would contain GM‐CSF neutralizing capacity (NC). Paediatric CD and control PBMC and ileal biopsies or LPMC were isolated and cultured and GM‐CSF, immunoglobulin (Ig)G and GM‐CSF autoantibodies production were measured by enzyme‐linked immunosorbent assay (ELISA). Basal and GM‐CSF‐primed neutrophil bacterial killing and signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation (pSTAT5) were measured by flow cytometry. GM‐CSF autoantibodies were enriched within total IgG for LPMC isolated from CD ileal strictures and proximal margins compared to control ileum. Neutrophil bacterial killing was reduced in CD patients compared to controls. Within CD, neutrophil GM‐CSF‐dependent STAT5 activation and bacterial killing were reduced as GM‐CSF autoantibodies increased. GM‐CSF stimulation of pSTAT5 did not vary between controls and CD patients in washed PB granulocytes in which serum was removed. However, GM‐CSF stimulation of pSTAT5 was reduced in whole PB samples from CD patients. These data were used to calculate the GM‐CSF NC. CD patients with GM‐CSF NC greater than 25% exhibited a fourfold higher rate of stricturing behaviour and surgery. The likelihood ratio (95% confidence interval) for stricturing behaviour for patients with elevation in both GM‐CSF autoantibodies and GM‐CSF NC was equal to 5 (2, 11). GM‐CSF autoantibodies are produced by LPMC isolated from CD ileal resection specimens and are associated with reduced neutrophil bacterial killing. CD peripheral blood contains GM‐CSF NC, which is associated with increased rates of stricturing behaviour.

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.

Genetic and Transcriptomic Variation Linked to Neutrophil Granulocyte–Macrophage Colony-Stimulating Factor Signaling in Pediatric Crohn’s Disease

BACKGROUND Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohns disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling. METHODS Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined. RESULTS We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohns disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P < 0.0001). CONCLUSIONS Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD.

Granulocyte-Macrophage Colony Stimulating Factor Auto-Antibodies Are Produced by Lamina Propria Mononuclear Cells Isolated From Ileal Strictures of Pediatric Patients With Crohn Disease Undergoing Ileo-Cecal Resection

G A A b st ra ct s that the STAT3 risk allele would be associated with increased cellular responsiveness to IL6 stimulation as measured by STAT3 tyrosine phosphorylation. Methods: B cells were isolated by magnetic bead columns from peripheral blood samples obtained from 69 pediatric IBD patients and transfected with EBV to create immortalized EBV-transformed lymphoblastoid cell lines (EBLs). Patients were genotyped for the STAT3 rs744166 and JAK2 rs10758669 risk alleles. EBLs selected for experimentation included STAT3 AA or GG (A=risk) while including only those which were also JAK2 AA (homozygote for the non-risk allele). EBLs were cultured overnight in serum-free media with 1x106 cells/mL and then stimulated with 100 ng/mL IL-6 for 30 minutes prior to analysis. Analyses included flow cytometry for cell surface IL-6 receptor and Glycoprotein130 (GP130) and intracellular pSTAT3 and pSTAT1; Real Time Polymerase Chain Reaction (RT-PCR) for STAT3, JAK2, and GP130; and western blot for nuclear pSTAT3 and pSTAT1, cytosolic STAT3, STAT1, JAK2, GP130, and IL6 receptor, and membrane fraction JAK2. Protein bands were quantified by normalized chemoluminescent arbitrary units (AU) via LAS Image Reader and MultiGauge Software (Fujifilm®). Differences were tested using unpaired and paired t-tests in GraphPad Prism Software®. Results: Pediatric IBD patient demographics did not vary by STAT3 genotype (age 15.6 vs 14.3 years, percent male 50 vs 40, CD patients 2 vs 2, UC patients 8 vs 6). Neither cell surface IL-6 receptor or GP130; cytosolic abundance of STAT3, STAT1, JAK2, GP130, IL-6 receptor; nor messenger RNA expression of STAT3, JAK2, or GP130 varied by STAT3 genotype. The mean nuclear protein pSTAT3 AU following IL-6 stimulation was 31.5 AU in EBLs from patients homozygous for the STAT3 risk allele compared to 19.1 AU in those lacking the STAT3 risk allele (p=0.04). The mean nuclear protein pSTAT1 AU following IL-6 stimulation was inversely 2.8 AU in EBLs from patients homozygous for the STAT3 risk allele compared to 4.3 AU in those lacking the STAT3 risk allele (p=0.001). The mean membrane fraction JAK2 AU was 80.2 AU in EBLs from patients homozygous for the STAT3 risk allele compared to 24.9 AU in those lacking the STAT3 risk allele (p= 0.008). Conclusions: The STAT3 genetic variant which increases risk for IBD is associated with increased JAK2 membrane fraction abundance and increased IL-6 dependent STAT3 tyrosine phosophorylation in EBV-transformed lymphocytes. These differences in cell signaling may promote IBD risk via STAT3 dependent effects upon T-lymphocyte differentiation and survival.