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Background The gut and oral microbiome have independently been shown to be associated with inflammatory bowel disease (IBD). However, it is not known to what extent gut and oral microbial disease markers converge in terms of their composition in IBD. Further, the spatial and temporal variation within the oral microenvironments of IBD remain to be elucidated. Patients and Methods We used a prospectively recruited cohort of patients with IBD (n = 47) and unrelated healthy control patients (n = 18) to examine the spatial and temporal distribution of microbiota within the various oral microenvironments, represented by saliva, tongue, buccal mucosa, and plaque, and compared them with stool. Microbiome characterization was performed using 16S rRNA gene sequencing. Results The oral microbiome displayed IBD-associated dysbiosis, in a site- and taxa-specific manner. Plaque samples depicted a relatively severe degree of dysbiosis, and the disease-associated dysbiotic bacterial groups were predominantly the members of the phylum Firmicutes. Our 16S rRNA gene analyses show that oral microbiota can distinguish patients with IBD from healthy control patients, with salivary microbiota performing the best, closely matched by stool and other oral sites. Longitudinal profiles of microbial composition suggest that some taxa are more consistently perturbed than others, preferentially in a site-dependent fashion. Conclusions Collectively, these data indicate the potential of using oral microbial profiles in screening and monitoring patients with IBD. Furthermore, these results support the importance of spatial and longitudinal microbiome sampling to interpret disease-associated dysbiotic states and eventually to gain insights into disease pathogenesis.

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.