Search

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.

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.

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.

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.