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INTRODUCTION Biologics targeting TNF are the mainstay of therapy for children with Crohn’s Disease (CD). However, a subset of patients do not respond, progressing to intestinal fibrosis requiring surgical resection. Prior studies have defined an ileal gene expression module linked to future strictures, and identified small molecules which may reverse this gene signature and suppress fibroblast activation. In the current study we developed a pre-clinical model system and tested a lead candidate, eicosatetraynoic acid (ETYA), a Peroxisome Proliferator-Activated Receptor (PPAR) agonist and arachidonic acid metabolism inhibitor. METHODS Peripheral blood samples were collected from pediatric CD patients and induced pluripotent stem cell (iPSC) lines were generated. iPSC were differentiated into human intestinal organoids (HIOs) and macrophage-like cells. Macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without eicosatetraynoic acid (ETYA) pre-treatment. Flow cytometry and cytospin characterized macrophage activation markers and morphology. Co-culture populations were harvested, and RNA and conditioned media were isolated for downstream analysis. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to quantify measures of inflammation and fibrosis, and to test whether introduction of ETYA abated any of these inflammatory or fibrotic changes. RESULTS iPSC-derived macrophages exhibited morphology similar to primary macrophages, and expressed inflammatory macrophage cell surface markers including CD68 (Fig. 1A). LPS-stimulated iPSC-derived macrophages expressed a global pattern of gene expression by RNA sequencing enriched in CD ileal inflammatory macrophages (ToppCell Atlas, p=4.397E-117), and produced cytokines and chemokines implicated in refractory disease (Fig. 1B). Co-culture of LPS-primed macrophages with HIO led to up-regulation of the fibroblast activation genes ACTA2 and COL1A1 (Fig. 2). Under these conditions, HIO collagen content measured by sirius red staining and polarized light microscopy was increased (Fig. 2). ETYA pre-treatment prevented these pro-fibrotic effects of LPS-primed macrophages upon HIO gene expression and collagen content. However, LPS induction of macrophage IL1B, TNF, and OSM production was not suppressed by ETYA, suggesting an alternative mechanism of action upon HIO fibroblast activation and collagen content in the co-culture system. CONCLUSIONS ETYA inhibits effects of LPS-primed macrophages upon HIO pro-fibrotic gene expression and collagen production. This was not associated with an effect of ETYA upon macrophage inflammatory cytokine production. Future studies will test alternate pathways including PPAR activation and arachidonic acid metabolism which may mediate this response.

BACKGROUND Mutations in the DUOX2 intestinal epithelial cell NADPH oxidase are associated with risk for Crohn’s Disease (CD), and may influence wound healing and the development of strictures. Patients who develop strictures exhibit reduced expression of genes encoding mitochondrial sub-units, and increased expression of genes encoding extra-cellular matrix proteins, in the ileum at diagnosis. We conducted a perturbagen bioinformatics analysis which predicted that the cyclooxygenase and lipoxygenase inhibitor eicosatetraynoic-acid (ETYA) would reverse the ileal gene signature associated with stricture development. In the current study we tested effects of ETYA in a human intestinal organoid model (HIO) system. METHODS HIO were derived from wild type (WT) and DUOX2 mutant (DUOX2var) induced pluripotent stem cells (iPSCs) prepared from CD patients. WT and DUOX2var HIO were examined under basal conditions, and following exposure to ETYA for 72 hours or 12 days. Superoxide production in EPCAM+ epithelial cells and CD90+ stromal cells was measured by flow cytometry. RNA was prepared and expression of mitochondrial and extra-cellular matrix genes linked to strictures in patients was determined using RNA sequencing and a TaqMan Low Density Array (TLDA) card. HIO tissue stiffness was measured using atomic force microscopy (AFM). RESULTS ETYA reduced superoxide production by HIO EPCAM+ epithelial cells only in WT organoids; no effect was observed in DUOX2var HIO. This was specific, as no effect of ETYA upon superoxide production was detected in CD90+ stromal cells. Under basal conditions, RNA sequencing demonstrated that WT HIO expressed core mitochondrial and extra-cellular matrix genes and enriched biologic functions implicated in CD strictures. Expression of extra-cellular matrix and wound healing genes was increased in DUOX2var HIO under basal conditions. ETYA up-regulated expression of the COX5B, POLG2, and SLC25A27 mitochondrial genes associated with lower rates of strictures, while reducing expression of the ACTA2, VIM, and COL1A1 extra-cellular matrix genes associated with higher rates of strictures, independent of DUOX2 genotype. WT and DUOX2var HIO exhibited tissue stiffness comparable to normal human ileum under basal conditions; this was significantly reduced by ETYA exposure only in DUOX2var HIO. CONCLUSIONS ETYA regulates mitochondrial and extra-cellular matrix genes implicated in stricture formation in an HIO model system. DUOX2var HIO exhibit increased extra-cellular matrix gene expression under basal conditions, which is reduced by ETYA exposure in conjunction with a reduction in tissue stiffness. Collectively, these data confirm that HIO provide a relevant model system to study mechanisms regulating stricture formation in CD, including screening of small molecules prioritized by perturbagen bioinformatics analysis.

A 4-year-old boy presented with perianal abscess and granulomatous colitis, which led the diagnosis of Crohn’s disease. He became refractory to all available therapies and required colectomy. Targeted sequencing revealed a deleterious variant in NCF4, causing severe neutrophil dysfunction. He underwent hematopoietic stem cell transplantation (HSCT) with an excellent outcome.

Author(s): Denson, Lee A; Jurickova, Ingrid; Karns, Rebekah; Shaw, Kelly A; Cutler, David J; Okou, David; Valencia, C Alexander; Dodd, Anne; Mondal, Kajari; Aronow, Bruce J; Haberman, Yael; Linn, Aaron; Price, Adam; Bezold, Ramona; Lake, Kathleen; Jackson, Kimberly; Walters, Thomas D; Griffiths, Anne; Baldassano, Robert N; Noe, Joshua D; Hyams, Jeffrey S; Crandall, Wallace V; Kirschner, Barbara S; Heyman, Melvin B; Snapper, Scott; Guthery, Stephen L; Dubinsky, Marla C; Leleiko, Neal S; Otley, Anthony R; Xavier, Ramnik J; Stevens, Christine; Daly, Mark J; Zwick, Michael E; Kugathasan, Subra | Abstract: BACKGROUND:Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's 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). Crohn's 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 l 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.

Background & aimsIndividuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients.MethodsWe performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production.ResultsWe identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P= .0008) and stricturing complications (P= .002) than children with CD without these mutations. Among patients withCD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P=.0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P= .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P= .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses.ConclusionsWe identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

BACKGROUND Human gastrointestinal (GI) organoid technologies have transformed GI disease research. By recapitulating the essential steps that occur during embryonic organ development, we could generate in vitro human colonic organoids (HCOs) (Munera et al, Cell Stem Cell. 2017) and human intestinal organoids (HIOs) (Spence et al, Nature. 2011) from iPSCs. Interestingly, HCOs contain both epithelial and surrounding mesenchymal derivatives, including myofibroblasts and tissue-resident immune cells. Our preliminary data demonstrate that HCOs co-develop CD163 positive macrophages derived from the hemogenic endothelium that develops within the colonic mesenchyme. Conversely, HIOs do not spontaneously develop tissue-resident immune cells. OBJECTIVE To incorporate tissue-resident immune cells into HIOs and use this new model to interrogate diseases such as IBD and Crohn’s Disease. RESULTS HIOs did not form endothelial tubes and immune cell floaters as seen in HCOs under the microscope 20 days after the start of differentiation. Moreover, HIO supernatants yielded few to no CFUs in Methocult assays compared to HCOs. Therefore, we tried two approaches to generate a novel HIO model system containing resident macrophages. First, we tried to transfer mesenchymal cells innately present in HCOs. We dissociate mesenchyme from HCOs and recombine them with HIO epithelium around day 20 from the start of differentiation (Figure 1A). We could successfully dissociate HCO-mesenchymal components from their epithelium and recombined them with HIO epithelium, but we could not detect CD163 positive macrophages in recombined HIOs. Second, we tried to transfer the supernatant of HCOs to HIOs around day 20 from the start of differentiation (Figure 1B). We confirmed the presence of CD163+ macrophages in day 35 HIOs using immunostaining and RNA sequencing (Figure 2A, B). We also confirmed the expression of cytokines such as IL10, IL12, and TGFβ via qPCR. Furthermore, we wanted to investigate whether these tissue resident macrophages were maintained after further maturation. As previously described, HIO transplantation into the mouse kidney capsule for 12 weeks allows for tissue maturation enabling crypt/villus formation and smooth muscle differentiation (Figure 2C) (Watson et al, Nat. Med. 2014). After recombined HIOs were transplanted, human CD163+ macrophages displayed expansion and were not displaced by bone marrow derived mouse macrophages (expressing F4/80). In the future, we also plan to differentiate monocytes directly from iPSCs and inject the monocytes into HIOs to see if they resident in the tissue as macrophages (Figure 1C). CONCLUSION We generated HIOs containing tissue-resident macrophages by transferring supernatant of HCOs. We expect to accelerate our understanding of the mechanisms of IBD such as CD because of the development of HIOs technology containing of immune cells.

Neutrophil dysfunction and GM-CSF auto-antibodies are observed in pediatric and adult patients with Crohn’s disease (CD). We associated damaging coding variants with low GM-CSF induced STAT5 stimulation index (GMSI) in pediatric CD patients and implicated variation of neutrophil GM-CSF signaling in cell function and disease complications. Because many CD patients with low GMSI do not carry damaging coding mutations, we sought to test the hypothesis that non-coding variants contribute to this phenotype. We enrolled, performed whole genome sequencing, and measured the GMSI in 77 CD and ulcerative colitis (UC) patients (24 low and 53 normal GMSI). We identified 4 non-coding variants (rs3808851, rs10974787, rs10974788 and rs10974789) in RCL1 significantly associated with variation of GMSI level (p JAK2 (p 0.005 to 0.013), RCL1 (p 8.17E-13 to 2.98E-11) and AK3 (p 2.00E-68 to 3.03E-55) genes. Additionally, they influence proteins involved in differentiation of gut epithelium, inflammation, and immune system regulation. In summary, our study outlines the contribution of non-coding variants in neutrophil GM-CSF signaling and the potential importance of RCL1 and AK3 in CD pathogenesis.

Introduction The DUOX2 intestinal epithelial NADPH oxidase is upregulated in Crohn’s Disease (CD), and DUOX2 mutations are associated with increased CD risk. Oxidative stress and loss of mitochondrial function disrupt the intestinal barrier promoting inflammatory responses to commensals. The relative impact of DUOX2 mutations and microbial products in this regard is poorly understood. Hypothesis We hypothesized that DUOX2 genetic variation would be associated with differences in cellular reactive oxygen species (ROS) production and mitochondrial function in a Human Intestinal Organoid (HIO) model system. Methods Induced pluripotent stem cell lines derived from pediatric CD patients with and without combined DUOX2 missense mutations(R701Q, P982A, and H678R) were used to generate wild type (WT) and DUOX2mut HIOs. Reactive oxygen species (ROS) production was measured using the two-color ROS-ID® Total ROS/Superoxide detection kit, and the mitochondrial membrane potential (MMP) was measured using JC1 staining by flow cytometry in HIO EpCAM+ epithelial cells and CD90+ stromal cells. Expression of inflammatory and mitochondrial genes which varied with DUOX2 mutation carriage in CD patent ileal biopsies was measured by RT-PCR. HIO mitochondrial complex I and II activity was measured using an Oroboros respirometer. Results Epithelial ROS production was reduced in DUOX2mut HIO under basal conditions; this difference was not observed following pyocyanin stimulation (Fig. 1A). A profound suppression of epithelial ROS production was observed following butyrate treatment. Butyrate did not alter stromal cell ROS production. Under these conditions, induction of ROS by pyocyanin was abrogated in WT, but not DUOX2mut HIO epithelial cells (Fig. 1B). Butyrate increased expression of core genes regulating the mitochondrial respiratory chain and DNA synthesis (COX5B, NDUFA1, POLG2, SLC25A27) and HIF1A implicated in barrier function, independent of genotype (p Conclusions We confirmed epithelial effects of DUOX2 genotype and butyrate exposure on ROS production in the HIO model system. Genotype dependent effects on basal ROS production were largely abrogated by the microbial products pyocyanin and butyrate, although butyrate inhibition of pyocyanin induced ROS production was dependent on intact DUOX2 function. Data suggest a previously unanticipated effect of DUOX2 genetic variation on the epithelial and stromal cell MMP and cellular respiration. This may have implications for mechanisms by which DUOX2 regulates barrier function and inflammatory responses to commensals in CD.

Molecular mechanisms driving disease course and response to therapy in ulcerative colitis (UC) are not well understood. Here, we use RNAseq to define pre-treatment rectal gene expression, and fecal microbiota profiles, in 206 pediatric UC patients receiving standardised therapy. We validate our key findings in adult and paediatric UC cohorts of 408 participants. We observe a marked suppression of mitochondrial genes and function across cohorts in active UC, and that increasing disease severity is notable for enrichment of adenoma/adenocarcinoma and innate immune genes. A subset of severity genes improves prediction of corticosteroid-induced remission in the discovery cohort; this gene signature is also associated with response to anti-TNFα and anti-α4β7 integrin in adults. The severity and therapeutic response gene signatures were in turn associated with shifts in microbes previously implicated in mucosal homeostasis. Our data provide insights into UC pathogenesis, and may prioritise future therapies for nonresponders to current approaches., The severity of ulcerative colitis, and response to treatment, is highly variable. Here, the authors examine rectal gene expression signatures and faecal microbiomes of children and adults with the disease and provide new insights in to pathogenesis.

Summary 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.

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.Colitis was induced in IEC-specific STAT3-deficient mice (STAT3)[INCREMENT]IEC and littermate controls (STAT3 Flx/Flx) with 4% DSS for 7 days, followed by water consumption for 21 days. Epithelial and immune mediators and severity of colitis were determined.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 STAT3 Flx/Flx controls. In STAT3[INCREMENT]IEC mice, colonic expression of STAT3 target genes Reg3β and Reg3γ, which mediate epithelial restitution, were significantly decreased, whereas expression of interleukin (IL)-17a, IFNγ, CXCL2, CXCL10, and CCL2 were significantly increased and correlated with the increase in histologic severity at day 28(P0.05). IL-17a expression also correlated with the increased lamina propria frequency of CD3* pSTAT3* T lymphocytes.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.

The multi tumor suppressor genes MTS1 (CDKN2, p16INK4A) and MTS2 (CDKN1, p15INK4B) located at 9p21-22 are inactivated in some human cancers via several mechanisms including deletion and hypermethylation. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (TALL). We have investigated the deletion, methylation and p16 protein expression status of MTS1 in Tcell childhood acute lymphoblastic leukemia (19 cases) and cell lines[11]. On Southern blot homozygous deletions or hemizygous deletion with rearangement were detected in 4/19 T-ALL. The expression of p16 protein was not observed on Western blot in 4/15 T-ALL with intact p16 gene. The p16 gene was methylated 3/15 in T-ALL. Only one of three expressed p16 protein. The other 11/15 T-ALL had p16 protein expression but different level. Loss of MTS1 was observed in 3/11 cell lines. Cell line with MTS1 gene had p16 protein expression in 6/8. After treatment with the demethylating agent (5-AzoCyt) RD cell line showed p16 expression. This has not been observed with the other cell lines. Thus hypermethylation of MTS1 is rare in childhood T-ALL. Although inactivation of MTS1 by deletion is common in T-ALL and cell lines. Furthermore our data show that the p16 gene inactivation by hypermethylation and deletion may play a role in the leukemogenesis.

Genotypic variation in signal transducer and activator of transcription 3 (STAT3) increases risk for inflammatory bowel disease (IBD), and STAT3-dependent inflammatory networks are induced in the colon in these patients. We hypothesized that STAT3 "A" risk allele carriage would be associated with increased cellular STAT3 activation and colon leukocyte recruitment.Colonic expression of genes regulating STAT3 signaling and leukocyte recruitment and function was measured in pediatric patients with Crohn disease (CD) stratified by STAT3 genotype. The frequency of colonic pSTAT3* and CXCR2* neutrophils was determined using immunohistochemistry. STAT3 tyrosine phosphorylation (pSTAT3) was measured in circulating leukocytes by flow cytometry, and mechanisms regulating STAT3 activation were tested in IBD Epstein-Barr virus (EBV)-transformed lymphocytes (EBL).Colonic expression of interleukin 6 (IL-6), the STAT3 target gene SOCS3, the neutrophil chemoattractants IL-8, CXCL1, and CXCL3, and the neutrophil products S100A8, S100A9, and S100A12 were increased in patients carrying the STAT3 "A" risk allele. The frequency of neutrophils expressing the cognate receptor for IL-8, CXCR2, was increased in colonic biopsies from patients carrying the risk allele, and the frequency of pSTAT3* or CXCR2* neutrophils correlated with histologic severity. The frequency of CD4 lymphocytes and granulocytes expressing pSTAT3 was increased in patients carrying the STAT3 "A" risk allele. EBLs from patients carrying the STAT3 "A" risk allele exhibited increased basal and IL-6-stimulated STAT3 tyrosine phosphorylation, increased transcription of STAT3 and SOCS3 after IL-6 stimulation, and increased membrane localization of the IL-6 receptor, GP130, and Janus-associated kinase 2.The STAT3 "A" risk allele is associated with increased cellular STAT3 activation and upregulation of pathways that promote recruitment of CXCR2* neutrophils to the gut.

Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least 11 different genes. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intragenic variations, emphasizing the importance of identifying the complementation groups. In the present study we have employed bicistronic retrovirus vectors that coexpress FA-specific cDNAs for complementation groups A, C, F, and G, together with the enhanced green fluorescence protein (EGFP), allowing for specific analysis of transduced EGFP+ cells within bulk cultures by flow cytometry. In addition, the assay relies on the correction of the characteristic FA-associated G2/M arrest after treatment of cells with DNA-damaging agents, which is analyzed by flow cytometry. Results obtained with this assay matched the complementation groups known for 12 control lymphoblast cell lines tested. We report here the results obtained for 48 FA patients with unknown complementation groups using this new assay. Complementation groups were identified for 24 patients. We have identified mutations in the genes corresponding to the assigned complementation group in 23 samples. This assay has now been established in a standardized fashion for complementation assignments in FA patients and the subsequent directing of rapid mutation analysis in those patients.

Relapse is the major cause of death after allogeneic bone marrow transplantation (BMT). This study tested the hypothesis that the numbers of donor mononuclear cells, lymphocytes, and CD34+cells influence relapse and event-free survival (EFS) after BMT. The study population consisted of 113 consecutive patients with hematologic malignancies who underwent non–T-cell–depleted BMT from HLA-matched siblings. Sixty-four patients had low-risk diagnoses (ALL/AML CR1, MDS RA/RARS, and CML CP1); 49 patients had high-risk diagnoses (all others). CD34+ cells, T cells, B cells, natural killer cells, monocytes, and a rare population of CD3−, CD4bright cells in the allografts were measured by flow cytometry. The CD3−, CD4bright cells in bone marrow had the same frequency and phenotype as CD123brighttype 2 dendritic cell (DC) progenitors, and they differentiated into typical DCs after short-term culture. Cox regression analyses evaluated risk strata, age, gender, and the numbers of nucleated cells, CD3+ T cells, CD34+ hematopoietic cells, and CD4bright cells as covariates for EFS, relapse, and nonrelapse mortality. Recipients of larger numbers of CD4bright cells had significantly lower EFS, a lower incidence of chronic graft-versus-host disease (cGVHD), and an increased incidence of relapse. Recipients of larger numbers of CD34+ cells had improved EFS; recipients of fewer CD34+ cells had delayed hematopoietic engraftment and increased death from infections. In conclusion, the content of donor CD4bright cells was associated with decreased cGVHD and graft-versus-leukemia effects in recipients of allogeneic bone marrow transplantation, consistent with a role for donor DCs in determining immune responses after allogeneic BMT.

Background: Ileal involvement in Crohn's 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. (Inflamm Bowel Dis 2011;)

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.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.Serum EndoCAb and LBP were significantly elevated in patients with Crohn's disease (CD), compared to disease controls with ulcerative colitis (UC) and healthy controls (P0.001). 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-alpha, 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.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.

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.

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, Crohn's 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

Genetic variations that affect innate immunity increase risk of ileal Crohn's disease (CD). However, the penetrance of susceptibility genes, including NOD2, is low, suggesting additional risk factors. Neutralizing autoantibodies (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF Ab) reduce neutrophil antimicrobial function in patients with primary alveolar proteinosis (PAP). We investigated whether GM-CSF Ab regulates neutrophil function in CD.Serum samples from 354 adult and pediatric patients with inflammatory bowel disease (IBD) were analyzed for GM-CSF Ab and IBD markers. Levels of GM-CSF Ab were compared with patients' CD features and neutrophil function. Intestinal barrier function and nonsteroidal anti-inflammatory drug (NSAID)-induced injury were assessed in GM-CSF-null and NOD2-null mice.Median GM-CSF Ab levels increased from 0.4 microg/mL in control serum to 2.4 microg/mL in pediatric CD and 11.7 microg/mL in adult CD serum and were associated with ileal involvement (P.001). Ileal location, duration of disease, and increased GM-CSF Ab levels were associated with stricturing/penetrating behavior (odds ratio, 2.2; P=.018). The positive and negative predictive values of GM-CSF Ab for stricturing/penetrating behavior were comparable with that of other IBD serum markers. CD patients with increased GM-CSF Ab had reduced neutrophil phagocytic capacity and increased accumulation of pSTAT3+ neutrophils in the affected ileum. GM-CSF-null mice and NOD2-null mice in which GM-CSF was neutralized had defects in mucosal barrier function and developed a transmural ileitis following NSAID exposure.GM-CSF regulates ileal homeostasis in CD and in mouse models. CD patients with increases in serum GM-CSF Ab might benefit from GM-CSF administration.

Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least eleven different genes (A, B, C, D1, D2, E, F, G, I, J, L). Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intra-genic variations, underlining the importance of subtyping complementation groups. Previously we have utilized retroviral mediated gene transfer of FA cDNAs and subsequent analysis for the correction of hypersensitivity to Mitomycin C, to develop a valid method for subtyping of groups (Hanenberg et al., 2002). However, for lymphoblastic cell lines (LCL), G418 selection was required to compensate for low transduction efficiencies achieved. In the present study we have utilized RD114/GALV psuedotyped (high titer stable producer clones) bicistronic retroviral vectors that coexpress EGFP. This results in higher transduction efficiencies in LCLs (20–70% for C, F, G and 10–20 % for A vector) and allows for specific analysis of transduced EGFP-positive cells within the bulk cultures, thus obviating the need for selection. In addition, the assay relies on the correction of G2/M arrest that is analyzed by FLOW, further reducing the assay time. After transduction with cDNA-containing vectors for groups A, C, F and G, cells are exposed for 48 hours to the alkylating agent Melphalan, stained with Hoechst 33342 dye and cell cycle analysis for FA-specific G2/M arrest is performed by flow cytometry. The percentage of EGFP-positive cells in the G2/M phase of cell cycle for each vector is calculated and compared using the MODFIT software. A complementation group is identified when correction in the G2/M arrest occurs. Results obtained with the assay matched the complementation group known for 12 control LCLs tested. We report here the results obtained for 39 FA patients with unknown complementation groups using this new assay. Of the 39 cell lines tested, complementation group was identified for 20 lines [FA-A(17), FA-C(1), FA-F(1), FA-G(1)], while 15 lines were typed as Non A/C/F/G. Cells from 4 patients did not undergo G2/M arrest. Mutation analysis by direct sequencing of genomic DNA after DHPLC completed for 11 patients thus far, has detected a mutation in one or both alleles confirming the results of the rapid complementation assay in every case (Table). For the 15 cell lines typed as Non A/C/F/G, additional studies including vectors for the FA genes FANCD2, FANCE, FANCL are underway. In summary, we have developed a rapid and efficient method for the identification of complementation groups in FA patients that also serves as a guide for quick mutational analysis. This methodology can also be applied to myeloid progenitor cells as well as fibroblasts, an advantage in patients with lymphoid mosaicism. Table 1. Mutational analysis of cell lines identified by cell cycle analysis | Cell Line Ref # | Cell cycle analysis | Mutation 1(paternal) | Mutation 2 (maternal) | |:--------------- | ------------------- | -------------------- | --------------------- | | RA2593 | A | c.1471-?_2151+?del | c.1471-?_2151+?del | | RA2483 | A | | c.1901-?_2014+?del | | RA2625 | A | | c.3239G>T(p.R1080L) | | RA2644 | A | c.2806G>A(p.E936K) | c.2806G>A(p.E936K) | | RA2685 | A | c.2678G>A(p.W893X) | | | RA2698 | A | c.337_338delTC | | | RA2704 | A | c.3514-?_3626+?del | c.3520_3522delTGG | | RA2724 | C | c.322delG | c.711+4A>T(IVS4+4A>T) | | RA2191 | A | c.3520_3522dellTGG | | | RA2709 | F | c.484-485delCT | c.219delG | | RA2727 | G | c.908T>C | c.908T>C