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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.

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.

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.

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.

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.

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