Simone C. Wolfkamp

Confirmation of Multiple Crohn's Disease Susceptibility Loci in a Large Dutch–Belgian Cohort

OBJECTIVES:Inflammatory bowel diseases (IBD)—Crohns disease (CD) and ulcerative colitis (UC)—are chronic gastrointestinal inflammatory disorders with a complex genetic background. A genome-wide association scan by the Wellcome Trust Case Control Consortium (WTCCC) recently identified several novel susceptibility loci.METHODS:We performed a large replication study in 2,731 Dutch and Belgian IBD patients (1,656 CD and 1,075 UC) and 1,086 controls. In total, 40 single nucleotide polymorphisms (SNPs) that showed moderate or strong association in the WTCCC study, along with SNPs in the previously identified genes IL23R, ATG16L1, and NELL1, were studied.RESULTS:We confirmed the associations with IL23R (rs11209026, P=2.69E-12), ATG16L1 (rs2241880, P=4.82E-07), IRGM (rs4958847, P=2.26E-05), NKX2-3 (rs10883365, P=5.91E-06), 1q24 (rs12035082, P=1.51E-05), 5p13 (rs17234657, P=2.62E-05), and 10q21 (rs10761659, P=8.95E-04). We also identified associations with cyclin Y (CCNY; rs3936503, P=2.09 E-04) and Hect domain and RCC1-like domain 2 (HERC2; rs916977, P=1.12E-04). Pooling our data with the original WTCCC data substantiated these associations. Several SNPs were also moderately associated with UC. Two genetic risk profiles based on the number of risk alleles and based on a weighted score were created. On the basis of these results, we calculated sensitivities, specificities, positive and negative predictive values, and likelihood ratios for CD.CONCLUSIONS:We replicated genetic associations for CD with IL23R, ATG16L1, IRGM, NKX2-3, 1q24, 10q21, 5p13, and PTPN2 and report evidence for associations with HERC2 and CCNY. Pooling our data with the results of the WTCCC strengthened the results, suggesting genuine genetic associations. We show that a genetic risk profile can be constructed that is clinically useful and that can aid in making treatment decisions.

Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease

Objective Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). Design In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. Results In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. Conclusions The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Autophagy Attenuates the Adaptive Immune Response by Destabilizing the Immunologic Synapse

BACKGROUND & AIMS Variants in the genes ATG16L1 and IRGM affect autophagy and are associated with the development of Crohns disease. It is not clear how autophagy is linked to loss of immune tolerance in the intestine. We investigated the involvement of the immunologic synapse-the site of contact between dendritic cells (DCs) and T cells, which contains molecules involved in antigen recognition and regulates immune response. METHODS DC autophagy was reduced using small interfering RNAs or pharmacologic inhibitors. DC phenotype and function were analyzed by confocal microscopy, time-lapse microscopy, and flow cytometry. We also examined DCs isolated from patients with Crohns disease who carried the ATG16L1 risk allele. RESULTS Immunologic synapse formation induced formation of autophagosomes in DCs; the autophagosomes were oriented toward the immunologic synapse and contained synaptic components. Knockdown of ATG16L1 and IRGM with small interfering RNAs in DCs resulted in hyperstable interactions between DCs and T cells, increased activation of T cells, and activation of a T-helper 17 cell response. LKB1 was recruited to the immunologic synapse, and induction of autophagy in DC required inhibition of mammalian target of rapamycine signaling by the LKB1-AMP activated protein kinase (AMPK) pathway. DCs from patients with Crohns disease who had an ATG16L1 risk allele had a similar hyperstability of the immunologic synapse. CONCLUSIONS Autophagy is induced upon formation of the immunologic synapse and negatively regulates T-cell activation. This mechanism might increase adaptive immunity in patients with Crohns disease who carry ATG16L1 risk alleles.

ATG16L1 and NOD2 polymorphisms enhance phagocytosis in monocytes of Crohn's disease patients

AIM To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohns disease (CD). METHODS In this study, we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls. As both autophagy related like 1 (ATG16L1) and immunity-related guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleotide-binding ligomerization domain-containing protein 2 (NOD2) has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction. The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo™ Escherichia coli Bioparticles Phagocytosis kit for flowcytometry. RESULTS In this study, we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity (ratio of mean fluorescence intensity) between the patient groups and the healthy controls. CD patients show a significantly higher phagocytic capacity (ratio mean percentage of phagocytic cells) compared to healthy controls (51.91% ± 2.85% vs 37.67% ± 7.06%, P = 0.05). The extend of disease was not of influence. However, variants of ATG16L1 (WT: 2.03 ± 0.19 vs homozygoot variant: 4.38 ± 0.37, P < 0.009) as well as NOD2 (C-ins) (heterozygous variant: 42.08 ± 2.94 vs homozygous variant: 75.58 ± 4.34 (P = 0.05) are associated with the phagocytic activity in patients with CD. CONCLUSION Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants. This could be part of the pathophysiological mechanism resulting in the disease.

Association of FcgR2a, but not FcgR3a, with inflammatory bowel diseases across three Caucasian populations.

Background: The Fc receptors II and III (FcgR2a, and FcgR3a) play a crucial role in the regulation of the immune response. The FcgR2a*519GG and FcgR3a*559CC genotypes have been associated with several autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, nephritis, and possibly to type I diabetes, and celiac disease. In a large multicenter, two‐stage study of 6570 people, we tested whether the FcgR2a and FcgR3a genes were also involved in inflammatory bowel disease (IBD), which includes Crohns disease (CD) and ulcerative colitis (UC). Methods: We genotyped the FcgR2a*A519G and FcgR3a*A559C functional variants in 4205 IBD patients in six well‐phenotyped Caucasian IBD cohorts and 2365 ethnically matched controls recruited from the Netherlands, Spain, and New Zealand. Results: In the initial Dutch study we found a significant association of FcgR2a genotypes with IBD (P‐genotype = 0.02); while the FcgR2a*519GG was more common in controls (23%) than in IBD patients (18%; odds ratio [OR] = 0.75; 95% confidence interval [CI] 0.61–0.92; P = 0.004). This association was corroborated by a combined analysis across all the study populations (Mantel–Haenszel [MH] OR = 0.84; 0.74–0.95; P = 0.005) in the next stage. The Fcgr2a*GG genotype was associated with both UC (MH‐OR = 0.84; 0.72–0.97; P = 0.01) and CD (MH‐OR = 0.84; 0.73–0.97; P = 0.01), suggesting that this genotype confers a protective effect against IBD. There was no association of FcgR3a*A559C genotypes with IBD, CD, or UC in any of the three studied populations. Conclusions: The FcgR2a*519G functional variant was associated with IBD and reduced susceptibility to UC and to CD in Caucasians. There was no association between FcgR3a*5A559C and IBD, CD or UC. (Inflamm Bowel Dis 2010)

The ATG16L1 risk allele associated with Crohn’s disease results in a Rac1-dependent defect in dendritic cell migration that is corrected by thiopurines

Thiopurines are commonly used drugs in the therapy of Crohn’s disease, but unfortunately only show a 30% response rate. The biological basis for the thiopurine response is unclear, thus hampering patient selection prior to treatment. A genetic risk factor associated specifically with Crohn’s disease is a variant in ATG16L1 that reduces autophagy. We have previously shown that autophagy is involved in dendritic cell (DC)-T-cell interactions and cytoskeletal regulation. Here we further investigated the role of autophagy in DC cytoskeletal modulation and cellular trafficking. Autophagy-deficient DC displayed loss of filopodia, altered podosome distribution, and increased membrane ruffling, all consistent with increased cellular adhesion. Consequently, autophagy-deficient DC showed reduced migration. The cytoskeletal aberrations were mediated through hyperactivation of Rac1, a known thiopurine target. Indeed thiopurines restored the migratory defects in autophagy-deficient DC. Clinically, the ATG16L1 risk variant associated with increased response to thiopurine treatment in patients with Crohn’s disease but not ulcerative colitis. These results suggest that the association between ATG16L1 and Crohn’s disease is mediated at least in part through Rac1 hyperactivation and subsequent defective DC migration. As this phenotype can be corrected using thiopurines, ATG16L1 genotyping may be useful in the identification of patients that will benefit most from thiopurine treatment.

Is there a role for Crohn's disease-associated autophagy genes ATG16L1 and IRGM in formation of granulomas?

Objective Autophagy-related 16-like 1 gene (ATG16L1) and immunity-related guanosine triphosphatase gene (IRGM) are associated with Crohns disease susceptibility and autophagy. Elimination of invading pathogens is a function of autophagy. Formation of granulomas can be attributed to impaired recognition of bacterial components by the innate immune system. This study was undertaken to elucidate whether disease-associated variants in ATG16L1 and IRGM by affecting autophagy pathways impair pathogen clearance in the cell and thereby cause increased prevalence of granulomas in Crohns disease patients. Methods Genotypes of the inflammatory bowel disease patient cohort consisting of 819 inflammatory bowel disease patients and over 1700 histology reports on intestinal biopsies obtained during ileocolonoscopy stating the presence or absence of granulomas were included in this case–control study. Results We confirm the association of the ATG16L1 variant and IRGM variants with Crohns disease. Comparison of the genotype frequency of the ATG16L1 SNP (rs2241880) and the presence or absence of granuloma in 179 cases showed a P value of 0.16. Both variants in IRGM (rs4958847 and rs13361189) showed P values of 0.7 after comparison with granuloma prevalence in 213 cases. A total of 169 Crohns disease patients were genotyped for both the genes, but no evidence for gene–gene interaction between ATG16L1 and IRGM and granuloma formation was found. Conclusion Our Crohns disease patient cohort showed no association of the variants in ATG16L1 or IRGM and the presence of granulomas.

Mo1751 Relating Genetic Variants in IBD to Aberrant Cytokine Profiles: A Focus on TNFSF15

A focus on TNFSF15 N.W. Duijvis1, F.H. van Dooren1, D. Oudejans1, S.C. Wolfkamp2, E.W. Vogels1, P. Henneman3, P.C. Stokkers4, G.R. D’Haens2, W. de Jonge1, A.A. te Velde1 1. Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands 2. Dept. of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands 3. Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands 4. Dept. of Gastroenterology and Hepatology, Sint Lucas Andreas Ziekenhuis, Amsterdam, the Netherlands