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 Crohn's 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.

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.

Single nucleotide polymorphisms in C-type lectin genes, clustered in the IBD2 and IBD6 susceptibility loci, may play a role in the pathogenesis of inflammatory bowel diseases.

BACKGROUND AND AIM: The balance between microbes and host defence mechanisms at the mucosal frontier plays an important, yet unclarified role in the pathogenesis of inflammatory bowel disease (IBD). The importance of microorganisms in IBD is supported by the association of IBD with mutations in pattern recognition receptors (PRRs) such as NOD2 and TLR4. We aimed to examine whether polymorphisms in another type of PRRs, the so-called C-type lectin receptors (CLRs), are associated with IBD. Growing insights into the pathogenetic role of NOD2 mutations in Crohn's disease (CD) and the fact that the majority of CLR-encoding genes are located in IBD susceptibility loci provide strong arguments for further exploration of the role of CLRs in IBD. METHODS: In this study, we selected four single nucleotide polymorphisms (SNPs) in different CLRs to determine whether there could be a role for these CLRs in IBD. Functional SNPs in the genes coding for the candidate CLRs DC-SIGN, LLT1, DCIR and MGL were examined. Genotyping of all SNPs was performed at the Academic Medical Center. In this study, around 1572 samples were included from a maximum of 621 CD patients, 457 ulcerative colitis (UC) patients and 586 healthy controls (HCs). RESULTS AND CONCLUSION: No association was found between our IBD cohort and the candidate SNPs for DC-SIGN (CD/HC: P=0.25 and UC/HC: P=0.36), DCIR (CD/HC: P=0.22 and UC/HC: P=0.41) and MGL (CD/HC: P=0.37 and UC/HC: P=0.25). However, one polymorphism in LLT1 was found to be associated with our CD population (P<0.034). Our UC cohort was not associated with the variation in LLT1 (P=0.33). LLT1 is a ligand for the recently discovered CD161. CD161 is a new surface marker for human interleukin (IL)-17-producing Th17 cells. The Th17 phenotype has been linked to CD by the fact that IL-22, IL-17 and IL-23 receptor levels are increased in CD. The signal transduction pathways involving LLT1 and CD161 are not completely clarified and are currently under investigation in our laboratory.

In Vivo Inflammation Does Not Impair ABCA1-Mediated Cholesterol Efflux Capacity of HDL.

HDL provides atheroprotection by facilitating cholesterol efflex from lipid-laden macrophages in the vessel wall. In vitro studies have suggested impaired efflux capacity of HDL following inflammatory changes. We assessed the impact of acute severe sepsis and mild chronic inflammatory disease on the efflux capacity of HDL. We hypothesize that a more severe inflammatory state leads to stronger impaired cholesterol efflux capacity. Using lipid-laden THP1 cells and fibroblasts we were able to show that efflux capacity of HDL from both patients with severe sepsis or with Crohn's disease (active or in remission), either isolated using density gradient ultracentrifugation or using apoB precipitation, was not impaired. Yet plasma levels of HDL cholesterol and apoA-I were markedly lower in patients with sepsis. Based on the current observations we conclude that inflammatory disease does not interfere with the capacity of HDL to mediate cholesterol efflux. Our findings do not lend support to the biological relevance of HDL function changes in vitro.

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 Crohn's 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 Crohn's 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 Crohn's 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 Crohn's disease who carry ATG16L1 risk alleles.

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 Crohn's 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.

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 Crohn's 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 Crohn's 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 Crohn's 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 Crohn's 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 Crohn's disease patient cohort showed no association of the variants in ATG16L1 or IRGM and the presence of granulomas.

Confirmation of multiple Crohn's disease susceptibility loci in a large Dutch-Belgian cohort.

OBJECTIVES: Inflammatory bowel diseases (IBD)-Crohn's 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.

Hypnotherapy for children with functional abdominal pain or irritable bowel syndrome: a randomized controlled trial.

BACKGROUND & AIMS: Functional abdominal pain (FAP) and irritable bowel syndrome (IBS) are highly prevalent in childhood. A substantial proportion of patients continues to experience long-lasting symptoms. Gut-directed hypnotherapy (HT) has been shown to be highly effective in the treatment of adult IBS patients. We undertook a randomized controlled trial and compared clinical effectiveness of HT with standard medical therapy (SMT) in children with FAP or IBS. METHODS: Fifty-three pediatric patients, age 8-18 years, with FAP (n = 31) or IBS (n = 22), were randomized to either HT or SMT. Hypnotherapy consisted of 6 sessions over a 3-month period. Patients in the SMT group received standard medical care and 6 sessions of supportive therapy. Pain intensity, pain frequency, and associated symptoms were scored in weekly standardized abdominal pain diaries at baseline, during therapy, and 6 and 12 months after therapy. RESULTS: Pain scores decreased significantly in both groups: from baseline to 1 year follow-up, pain intensity scores decreased in the HT group from 13.5 to 1.3 and in the SMT group from 14.1 to 8.0. Pain frequency scores decreased from 13.5 to 1.1 in the HT group and from 14.4 to 9.3 in the SMT group. Hypnotherapy was highly superior, with a significantly greater reduction in pain scores compared with SMT (P < .001). At 1 year follow-up, successful treatment was accomplished in 85% of the HT group and 25% of the SMT group (P < .001). CONCLUSIONS: Gut-directed HT is highly effective in the treatment of children with longstanding FAP or IBS.