Functional Toll-Like Receptor (TLR)2 polymorphisms in the susceptibility to inflammatory bowel disease.

BACKGROUND: The recent genome-wide association studies (GWAS) in inflammatory bowel disease (IBD) suggest significant genetic overlap with complex mycobacterial diseases like tuberculosis or leprosy. TLR variants have previously been linked to susceptibility for mycobacterial diseases. Here we investigated the contribution to IBD risk of two TLR2 polymorphisms, the low-prevalence variant Arg753Gln and the GTn microsatellite repeat polymorphism in intron 2. We studied association with disease, possible correlations with phenotype and gene-gene interactions. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a large study in 843 patients with Crohn's disease, 426 patients with ulcerative colitis and 805 healthy, unrelated controls, all of European origin. Overall, the frequency for carriers of shorter GTn repeats in intron 2 of the TLR2 gene, which have previously been associated with low TLR2 expression and high IL-10 production, was slightly elevated in Crohn's disease and ulcerative colitis compared to healthy controls (16.0% resp. 16.7% vs. 12.8%). The highest frequency of short GTn carriers was noted among IBD patients on anti TNF-alpha therapy. However, none of these differences was significant in the multivariate analysis. The Arg753Gln polymorphism showed no association with any clinical subtype of IBD, including extensive colitis, for which such an association was previously described. We found no association with specific phenotypic disease subgroups. Also, epistasis analysis revealed no significant interactions between the two TLR2 variants and confirmed IBD susceptibility genes. CONCLUSIONS: The two functional relevant polymorphisms in TLR2, the GTn microsatellite repeat polymorphism in intron 2 and the Arg753Gln variant do not seem to play a role in the susceptibility to Crohn's disease or ulcerative colitis.

Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine-protective regulatory T cells.

The gut is home to a large number of Treg, with both CD4(+) CD25(+) Treg and bacterial antigen-specific Tr1 cells present in normal mouse intestinal lamina propria. It has been shown recently that intestinal mucosal DC are able to induce Foxp3(+) Treg through production of TGF-beta plus retinoic acid (RA). However, the factors instructing DC toward this mucosal phenotype are currently unknown. Curcumin has been shown to possess a number of biologic activities including the inhibition of NF-kappaB signaling. We asked whether curcumin could modulate DC to be tolerogenic whose function could mimic mucosal DC. We report here that curcumin modulated BM-derived DC to express ALDH1a and IL-10. These curcumin-treated DC induced differentiation of naïve CD4(+) T cells into Treg resembling Treg in the intestine, including both CD4(+)CD25(+) Foxp3(+) Treg and IL-10-producing Tr1 cells. Such Treg induction required IL-10, TGF-beta and retinoic acid produced by curcumin-modulated DC. Cell contact as well as IL-10 and TGF-beta production were involved in the function of such induced Treg. More importantly, these Treg inhibited antigen-specific T-cell activation in vitro and inhibited colitis due to antigen-specific pathogenic T cells in vivo.

rs224136 on chromosome 10q21.1 and variants in PHOX2B, NCF4, and FAM92B are not major genetic risk factors for susceptibility to Crohn's disease in the German population.

OBJECTIVES: Recently, a North American genome-wide association study identified three novel gene variants in PHOX2B, NCF4, and FAM92B as well as one single nucleotide polymorphisms (SNP; rs224136) in the intergenic region on chromosome 10q21.1 as being associated with Crohn's disease (CD). However, their influence on European CD patients as well as ulcerative colitis (UC) is unknown. Therefore we aimed to replicate these novel CD susceptibility variants in a large European cohort with inflammatory bowel disease and analyzed potential gene-gene interactions with variants in the NOD2/CARD15, IL23R, and ATG16L1 genes. METHODS: Genomic DNA from 2,833 Caucasian individuals including 854 patients with CD, 476 patients with UC, and 1,503 healthy unrelated controls was analyzed for SNPs in PHOX2B (rs16853571), NCF4 (rs4821544), and FAM92B (rs8050910), including rs224136 on chromosome 10q21.1. RESULTS: In our study population, no association of PHOX2B (P=0.563), NCF4 (P=0.506), FAM92B (P=0.401), and rs224136 (P=0.363) with CD was found. Similarly, none of these SNPs was associated with UC. In contrast, all analyzed SNPs in NOD2/CARD15, IL23R, and ATG16L1 were strongly associated with CD with P values ranging from 5.0x10(-3) to 1.6x10(-22), but there was no epistasis with polymorphisms in PHOX2B, NCF4, FAM92B, and rs224136. CONCLUSIONS: In contrast to the North American population, PHOX2B, NCF4, FAM92B, and rs224136 are not associated with CD in the European population, whereas NOD2/CARD15, IL23R, and ATG16L1 are strongly associated with CD in both the North American and European populations, confirming these three genes as major CD susceptibility genes in Caucasian populations.

CXCL16 is a surrogate marker of inflammatory bowel disease.

OBJECTIVE: Impaired barrier function of the gut and inadequate immunological response to intestinal pathogens are the cornerstones in the pathogenesis of inflammatory bowel disease (IBD). CXCL16 is a protein which shares pattern recognition receptor functions, relevant for adhesion and phagocytosis of bacterial products, with the properties of an adhesion molecule and inflammatory chemokine. The relevance of CXCL16 in IBD has so far been elusive. This objective of this study was to determine the association between CXCL16 and IBD. MATERIAL AND METHODS: Soluble CXCL16 (sol-CXCL16) serum levels in a cohort of 239 patients with Crohn's disease were measured, 114 patients with ulcerative colitis and 144 controls. RESULTS: In a univariate analysis, sol-CXCL16 was found to be markedly increased in patients with Crohn's disease or ulcerative colitis compared with that in controls (p < 0.001). This was significantly associated with an increase of the inflammatory marker C-reactive protein (CRP) (p < 0.01). In the multivariate analysis (adjusted for age, gender, body mass index (BMI), white blood cell (WBC) count, resistin and CRP) sol-CXCL16 was associated with Crohn's disease above versus below the median (OR 10.53 (3.97-27.78) p < 0.001) and ulcerative colitis (OR 3.46 (1.40-8.55) p < 0.01). CONCLUSION: Our findings suggest that CXCL16 may play a pro-inflammatory role in IBD, particularly Crohn's disease.

Pharmacogenetics of Crohn's disease.

The considerable interindividual differences in efficacy and side effects of commonly used medications in Crohn's disease are partly owing to genetic polymorphisms. Many genetic variants have been studied in genes possibly involved in the metabolism or mechanism of action of therapeutic agents such as glucocorticosteroids, azathioprine/6-mercaptopurine, methotrexate, calcineurin inhibitors or anti-TNF agents. However, the only test translated into clinical practice is thiopurine S-methyltransferase (TPMT) genotyping for hematological toxicity of thiopurine treatment. To date, there are no other meaningful applications for pharmacogenomics in clinical practice of Crohn's disease. In the future, designed therapeutic trials should possibly permit the development of predictive models including genotypic markers, such as that proposed for the clinical outcome after infliximab therapy, which includes an apoptotic pharmacogenetic index. The recent identification of new susceptibility genes provides additional candidate markers that have possible effects on the outcomes of therapies, and prioritizes new therapeutic targets, such as the IL-23 pathway. Further innovative approaches might be relevant for the pharmacogenetic investigation of gene variants implied in innate immune pattern recognition and autophagy.

The ATG16L1 gene variants rs2241879 and rs2241880 (T300A) are strongly associated with susceptibility to Crohn's disease in the German population.

OBJECTIVES: We analyzed ATG16L1, a recently identified Crohn's disease (CD) susceptibility gene, in a large cohort with inflammatory bowel disease (IBD) including potential interactions with other IBD genes as well as factors regulating its gene expression. METHODS: Genomic DNA from 2,890 Caucasians including 768 patients with CD, 507 patients with ulcerative colitis (UC), and 1,615 healthy controls was analyzed for 9 different ATG16L1 single nucleotide polymorphisms (SNPs). Genotyping included CARD15/NOD2 variants p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008 and polymorphisms in SLC22A4/OCTN1 (1672 C-->T) and SLC22A5/OCTN2 (-207 G-->C) as well as 10 CD-associated IL23R variants. The transcriptional regulation of ATG16L1 was studied in intestinal epithelial cells following stimulation with Toll-like receptor (TLR) ligands and proinflammatory cytokines and in a murine ileitis model and CD biopsies. RESULTS: All nine ATG16L1 gene variants analyzed displayed highly significant associations with CD demonstrating a CD-protective effect for the minor allele. The strongest associations were found for rs2241879 and the coding SNP rs2241880 (T300A); P= 3.6 x 10(-6) and 3.7 x 10(-6), respectively (OR 0.74, 95% CI 0.65-0.84 for both variants). The genotype-phenotype analysis revealed no significant associations. In UC, only rs6431660 was weakly disease-associated. There was no evidence for epistasis between the ATG16L1 gene and other susceptibility genes (IL23R, CARD15, SLC22A4/5). ATG16L1 mRNA expression was not upregulated in CD and murine ileitis, and was less than threefold increased in cells stimulated with proinflammatory cytokines and TLR ligands. CONCLUSION: ATG16L1 is a CD susceptibility gene without epistatic interaction with other CD susceptibility genes and is not upregulated in intestinal inflammation.

Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD.

BACKGROUND: Interleukin (IL)-17F, produced in IL-23R-expressing Th17 cells, is a novel member of the IL-17 cytokine family. Given the association of IL23R with inflammatory bowel disease (IBD), we characterized the role of IL-17F in IBD including its intestinal gene expression and the effect of the IL17F p.His161Arg polymorphism on disease susceptibility and phenotype of Crohn's disease (CD) and ulcerative colitis (UC). In addition, we analyzed the IL17F p.His161Arg polymorphism for potential epistasis with IL23R and NOD2/CARD15 variants. METHODS: Intestinal IL-17F mRNA expression was measured by quantitative polymerase chain reaction (PCR). Genomic DNA from 1682 individuals (CD: n = 499; UC: n = 216; controls: n = 967) was analyzed for the presence of the IL17F p.His161Arg polymorphism, the 3 NOD2 variants, p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008, and 10 CD-associated IL23R variants. RESULTS: Intestinal IL-17F mRNA expression was 4.4-fold increased in inflamed colonic lesions compared to uninflamed biopsies in CD (P = 0.016) but not in UC. However, the mean intestinal IL-17F mRNA expression was higher in UC than in CD (P < 0.0001). The IL17F p.His161Arg substitution was observed with similar frequencies in IBD patients and controls and was not associated with a certain disease phenotype, but weakly associated with a low body mass index (BMI; P = 0.009) and an earlier age of disease onset (P = 0.039) in UC. There was no evidence for epistasis between the IL17F p.His161Arg polymorphism and IBD-associated single nucleotide polymorphisms within the IL23R gene. CONCLUSIONS: Intestinal IL17F gene expression is increased in active CD. The IL17F p.His161Arg polymorphism is not associated with IBD susceptibility and has no epistatic interaction with CD-associated IL23R variants.

Linking genetic susceptibility to Crohn's disease with Th17 cell function: IL-22 serum levels are increased in Crohn's disease and correlate with disease activity and IL23R genotype status.

BACKGROUND: We analyzed the influence of Crohn's disease (CD)-associated IL23R gene variants on IL-22 that is expressed in IL-23R+ Th17 cells. METHODS: IL-22 serum levels were measured in 242 CD patients and in 31 healthy controls. Subanalyses included serum levels of IL-6, TNF-alpha, IL-17A, IL-17F, C-reactive protein (CRP), and leukocyte count. In all patients, genotyping for 10 CD-associated IL23R single nucleotide polymorphisms (SNPs) and the 3 main CD-associated CARD15 variants was performed. RESULTS: There was a highly significant increase in IL-22 serum expression in CD patients compared to healthy controls (P = 2.53 x 10(-9)). IL-22 serum levels correlated with disease activity: IL-22 levels in patients with a Crohn's disease activity index (CDAI) >150 were significantly higher than in patients with a CDAI <150 (P = 0.001), while TNF-alpha and IL-6 were not significantly different between these 2 groups. Analyzing the effect of 10 IL23R variants on IL-22 serum levels, we demonstrated that the quotients of mean IL-22 serum levels of carriers of the minor allele to the mean serum IL-22 in wildtype carriers correlated highly with the corresponding CD susceptibility risk for each gene variant (r = 0.807). The IL-22 levels in carriers of CD risk-increasing IL23R variants were significantly higher than in carriers of CD risk-decreasing IL23R variants (P = 0.008). CONCLUSIONS: The Th17 cytokine IL-22 is expressed at high levels in CD and correlates with disease activity, offering a better separation between active and inactive CD than IL-6 and TNF-alpha. IL23R genotypes influence IL-22 serum expression, linking genetic CD susceptibility to Th17 cell function for the first time.

Resistin is an inflammatory marker of inflammatory bowel disease in humans.

OBJECTIVES: Resistin, a recently discovered adipokine, has been shown to be associated with inflammatory conditions such as insulin resistance, obesity, atherosclerosis and rheumatoid arthritis. We therefore hypothesized that (i) resistin levels may be elevated in patients with inflammatory bowel disease (IBD) and (ii) resistin levels may be associated with disease activity in IBD. METHODS: We addressed these questions by testing for associations between resistin plasma levels, inflammatory parameters and clinical disease activity in a case-control study with 235 patients with Crohn's disease (CD), 112 patients with ulcerative colitis (UC) and 144 healthy controls. RESULTS: Patients with IBD showed significantly higher resistin levels compared with controls (P<0.0001). In both, patients with CD and UC, resistin concentrations were significantly associated with elevated white blood cell count (P<0.0001), C-reactive protein (CRP) (P<0.0001) and disease activity (P< or =0.0001). In multivariate logistic regression analysis, resistin levels were identified as an independent predictor of active disease (odds ratio 1.014, 95% confidence interval 1.002-1.027, P=0.02) in patients with CD after adjusting for sex, age, body mass index, white blood cell count and CRP. In UC patients, resistin was associated with active disease in multivariate regression analysis after control for sex, age, body mass index and white blood cell count (odds ratio 1.015, 95% confidence interval 1.002-1.029, P=0.02). Addition of CRP, however, abolished this association. CONCLUSION: Resistin levels are an independent predictor of disease activity in patients with CD. Resistin may represent a novel link between inflammation and IBD.

rs1004819 is the main disease-associated IL23R variant in German Crohn's disease patients: combined analysis of IL23R, CARD15, and OCTN1/2 variants.

BACKGROUND: The IL23R gene has been identified as a susceptibility gene for inflammatory bowel disease (IBD) in the North American population. The aim of our study was to test this association in a large German IBD cohort and to elucidate potential interactions with other IBD genes as well as phenotypic consequences of IL23R variants. METHODS: Genomic DNA from 2670 Caucasian individuals including 833 patients with Crohn's disease (CD), 456 patients with ulcerative colitis (UC), and 1381 healthy unrelated controls was analyzed for 10 IL23R SNPs. Genotyping included the NOD2 variants p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008 and polymorphisms in SLC22A4/OCTN1 (1672 C-->T) and SLC22A5/OCTN2 (-207 G-->C). RESULTS: All IL23R gene variants analyzed displayed highly significant associations with CD. The strongest association was found for the SNP rs1004819 [P = 1.92x10(-11); OR 1.56; 95 % CI (1.37-1.78)]. 93.2% of the rs1004819 TT homozygous carriers as compared to 78% of CC wildtype carriers had ileal involvement [P = 0.004; OR 4.24; CI (1.46-12.34)]. The coding SNP rs11209026 (p.Arg381Gln) was protective for CD [P = 8.04x10(-8); OR 0.43; CI (0.31-0.59)]. Similar, but weaker associations were found in UC. There was no evidence for epistasis between the IL23R gene and the CD susceptibility genes CARD15 and SLC22A4/5. CONCLUSION: IL23R is an IBD susceptibility gene, but has no epistatic interaction with CARD15 and SLC22A4/5. rs1004819 is the major IL23R variant associated with CD in the German population, while the p.Arg381Gln IL23R variant is a protective marker for CD and UC.

Macrophage migration inhibitory factor (MIF) -173G/C promoter polymorphism influences upper gastrointestinal tract involvement and disease activity in patients with Crohn's disease.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with increased expression in inflammatory bowel disease. The aim of the study was to analyze the role of the MIF -173G/C single nucleotide polymorphism in Crohn's disease (CD). METHODS: Using restriction fragment length polymorphism analysis, genomic DNA of 198 patients with CD and 159 unrelated controls was analyzed for the -173G/C SNP in the MIF promoter region. Colonic MIF mRNA expression was measured by quantitative polymerase chain reaction (PCR), serum MIF levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-six of the 146 G/G wildtype carriers (24.7%) but only 3 of the 45 G/C heterozygotes (6.7%) and only 1 of the C/C homozygotes (14.3%) were diagnosed with upper gastrointestinal tract involvement (P = 0.009, odds ratio [OR] = 0.22, 95% confidence interval [CI], 0.06-0.75 for wildtype versus hetero- and homozygous carriers). This result was confirmed in a second prospective study, in which all patients diagnosed with upper gastrointestinal involvement (n = 13) were G/G wildtype carriers (P = 0.01 versus controls). All patients (n = 12; 100%) with a Crohn's disease activity index (CDAI) > 300 were G/G wildtype carriers compared to only 65.6% wildtype carriers in the group with a CDAI < 150 (P = 0.016). MIF is expressed in the colonic mucosa of CD patients and intestinal epithelial cells but its mRNA expression does not correlate with the degree of inflammation and is not upregulated by proinflammatory cytokines. In CD, MIF serum levels are not influenced by the MIF -173G/C polymorphism. CONCLUSIONS: The MIF -173G/C polymorphism appears to be a factor contributing to a particular CD phenotype characterized by protection against upper gastrointestinal tract involvement and severe disease activity.

Tight mucosal compartmentation of the murine immune response to antigens of the enteric microbiota.

BACKGROUND & AIMS: The normal host immune response to antigens of the enteric microbiota is poorly defined. In this study, we isolated recombinant microbial antigens from commensal bacteria and used them to probe the normal murine immune response. METHODS: A plasmid DNA expression library was generated from cecal bacteria of C3H/HeJ mice and used to express 20 recombinant intestinal bacterial proteins (rIBs). Antibody responses in serum and secretions were measured by an enzyme-linked immunosorbent assay, and CD4+ T-cell responses were measured by [3H]-thymidine incorporation. Two immunodominant commensal flagellins were also tested. RESULTS: No baseline serum immunoglobulin (Ig)G antibody or splenic CD4+ T-cell systemic response to any rIB or to either flagellin was detected in normal C3H/HeJ mice. However, there were strong systemic responses to all 20 rIBs after parenteral immunization, which were equivalent to the responses to ovalbumin. Substantial levels of intestinal IgA were detected to half the rIBs and to both commensal flagellins. Mucosal immunization with flagellin plus ovalbumin stimulated an intestinal IgA but not a serum IgG response. Antigen-pulsed dendritic cells (DCs) stimulated production of specific IgA in the absence of T-cell help via costimulation by BAFF and/or APRIL, members of the TNF family. CONCLUSIONS: The host immune response to enteric bacteria is tightly compartmentalized to the mucosa in normal mice, with systemic B cells and CD4+ T cells remaining naive rather than tolerant. We postulate that mucosal DCs play a crucial role in this compartmentation.

Increased expression of the chemokine fractalkine in Crohn's disease and association of the fractalkine receptor T280M polymorphism with a fibrostenosing disease Phenotype.

BACKGROUND: The fractalkine receptor CX3CR1 has been shown to be involved in inflammation and immune response. Recently, two polymorphisms of CX3CR1 (V249I and T280M) were reported. AIMS: Our aim was to analyze fractalkine expression and the role of CX3CR1 polymorphisms in Crohn's disease (CD). METHODS: We determined fractalkine mRNA expression in the intestinal epithelial cell (IEC) line SW480 after stimulation with proinflammatory cytokines as well as in inflamed (n = 14) and noninflamed (n = 14) CD lesions by quantitative PCR. By restriction fragment length polymorphism analysis, genomic DNA from 206 patients with CD and 211 unrelated controls was analyzed for the two single nucleotide polymorphisms in the CX3CR1 gene, which result in the V249I and T280M substitutions. RESULTS: All proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly increased fractalkine mRNA expression in IEC. There was also a significant increase in fractalkine mRNA expression in inflamed lesions of CD patients when compared to noninflamed colonic mucosa (p = 0.02). Intestinal fractalkine mRNA levels correlated highly with IL-8 mRNA expression levels (r = 0.931). However, there was no difference in the V249I and T280M genotype frequencies between CD patients and the control group. In the CD group, 33.0% were heterozygous and 8.3% homozygous for the V249I polymorphism, while 23.3% were heterozygous and 4.4% homozygous for the T280M polymorphism. All T280M homozygotes were diagnosed of intestinal stenosis (p = 0.03 vs wildtype and heterozygous carriers) and had significantly more often ileocolonic involvement more often than patients with wildtype and heterozygous genotypes (p = 0.01). These associations were independent of the CARD15 genotype status. CONCLUSIONS: The expression of the chemokine fractalkine is upregulated by proinflammatory cytokines and enhanced in inflamed CD lesions. The CX3CR1 T280M polymorphism appears to influence CD phenotype and localization.

The role of Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and CARD15/NOD2 mutations in the susceptibility and phenotype of Crohn's disease.

BACKGROUND: We investigated the influence of 2 common Toll-like receptor 4 (TLR4) polymorphisms on susceptibility and disease characteristics of Crohn's disease (CD). METHODS: Genomic DNA from 204 patients with CD and 199 unrelated controls was analyzed for the presence of 2 single nucleotide polymorphisms in the TLR4 gene, resulting in the amino acid substitutions Asp299Gly and Thr399Ile. In addition, the carrier status for the 3 common CD-associated CARD15/NOD2 gene mutations, Arg702Trp, Gly908Arg, and 1007fs, was determined. The frequency of the different genotypes was compared, and a detailed genotype-phenotype correlation was performed. RESULTS: An almost 2-fold increase in the frequency of the TLR4 Asp299Gly phenotype was observed in patients with CD (14.2%) compared with healthy controls (7.5%, P = 0.038, odds ratio = 2.03). The prevalence of a stricturing phenotype was increased in patients heterozygous for 1 of the TLR4 polymorphisms studied (Asp299Gly, 34.5%; Thr399Ile, 36.7%) compared with patients with wild-type TLR4 (17.1% and 16.7%; P = 0.04 and 0.02, respectively). The presence of the Asp299Gly polymorphism in the absence of CARD15/NOD2 mutations was a particularly strong predictor of the stricturing disease phenotype that was present in 47.4% of the patients with Asp299Gly+/NOD2- compared with 10.1% of the patients with the Asp299Gly-/NOD2+ status (P = 0.0009; P = 0.0004 for Thr399Ile+/NOD2- versus Thr399Ile-/NOD2+). In contrast, there was a trend toward a higher prevalence of the penetrating phenotype in the TLR4-/NOD2+ group (71.6%) compared with the TLR4+/NOD2- group (47.4%, P = 0.059). CONCLUSIONS: The TLR4 Asp299Gly polymorphism is a risk factor for CD. TLR4 and CARD15/NOD2 mutations may contribute to distinct disease phenotypes.

Generation of antigen-specific, Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function.

We tested the hypothesis that immature APC, whose NF-kappaB-signaling pathway and thus maturation was blocked by the proteosome inhibitor benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal (PSI), could be a source of Ag-specific regulatory T (Treg) cells. DO11.10 CD4(+) T cells that were incubated with Ag- and PSI-pulsed APC proliferated poorly, produced less IL-2, IFN-gamma, and IL-10 in secondary cultures, and inhibited the response of both naive and memory CD4(+) T cells stimulated by Ag-pulsed APC. The generation of PSI-APC Treg cells required IL-10 production by APC. PSI-APC Treg cell inhibition required cell-cell contact but not IL-10 or TGF-beta. Addition of IL-2 did not reverse, but Ab to CTLA-4 did reverse partially the inhibitory effect. Depletion of CD25(+) T cells before initial culture with PSI-APC did not affect Treg generation. PSI-APC Treg cells expressed high levels of Foxp3, inhibited proliferation of naive DO11.10 T cells in vivo, and abrogated colitis driven by a memory Th1 response to bacterial-associated Ag. We conclude that NF-kappaB-blocked, immature APC are able to induce the differentiation of Treg cells that can function in vitro and in vivo in an Ag-specific manner.

Anti-Saccharomyces cerevisiae antibody titers are stable over time in Crohn's patients and are not inducible in murine models of colitis.

AIM: To investigate ASCA production over time in CD and murine colitis in order to further our understanding of their etiology. MATERIALS AND METHODS: Sixty-six CD patients were compared to ulcerative colitis (UC) and irritable bowel syndrome patients with respect to ASCA production as measured by ELISA. ASCA IgG or IgA positivity as well as change in titers over a period of up to 3 years (Delta IgG/A) was correlated with clinical parameters such as CD activity index (CDAI) and C-reactive protein levels (CRP). Moreover, two murine models of colitis (DSS and IL-10 knock out) were compared to control animals with respect to ASCA titers after oral yeast exposure. RESULTS: ASCA IgG and IgA titers are stable over time in CD and non-CD patients. Fistular disease was associated with a higher rate of ASCA IgA positivity (P = 0.014). Ileal disease was found to have a significant influence on the Delta IgG of ASCA (P = 0.032). There was no correlation found between ASCA positivity or Delta IgG/A and clinical parameters of CD: CDAI and CRP. In mice, neither healthy animals nor animals with DSS-induced or spontaneous colitis exhibited a marked increase in ASCA titers after high-dose yeast exposure. On the other hand, mice immunized intraperitoneally with mannan plus adjuvant showed a marked and significant increase in ASCA titers compared to adjuvant-only immunized controls (P = 0.014). CONCLUSION: The propensity to produce ASCA in a subgroup of CD patients is largely genetically predetermined as evidenced by their stability and lack of correlation with clinical disease activity parameters. Furthermore, in animal models of colitis, mere oral exposure of mice to yeast does not lead to the induction of marked ASCA titers irrespective of concomitant colonic inflammation. Hence, environment may play only a minor role in inducing ASCA.

Ameliorative effect of IDS 30, a stinging nettle leaf extract, on chronic colitis.

BACKGROUND AND AIMS: Anti-TNF-alpha antibodies are very effective in the treatment of acute Crohn's disease, but are limited by the decline of their effectiveness after repeated applications. The stinging nettle leaf extract, IDS 30, is an adjuvant remedy in rheumatic diseases dependent on a cytokine suppressive effect. We investigated the effect of IDS 30 on disease activity of murine colitis in different models. METHODS: C3H.IL-10-/- and BALB/c mice with colitis induced by dextran sodium sulphate (DSS) were treated with either IDS 30 or water. Mice were monitored for clinical signs of colitis. Inflammation was scored histologically, and faecal IL-1beta and mucosal cytokines were measured by ELISA. Mononuclear cell proliferation of spleen and Peyer's patches were quantified by 3H-thymidine. RESULTS: Mice with chronic DSS colitis or IL-10-/- mice treated with IDS 30 clinically and histologically revealed significantly (p < 0.05) fewer signs of colitis than untreated animals. Furthermore, faecal IL-1beta and mucosal TNF-alpha concentrations were significantly lower (p < 0.05) in treated mice. Mononuclear cell proliferation after stimulation with lipopolysaccharide was significantly (p < 0.001) reduced in mice treated with IDS 30. CONCLUSIONS: The long-term use of IDS 30 is effective in the prevention of chronic murine colitis. This effect seems to be due to a decrease in the Th1 response and may be a new therapeutic option for prolonging remission in inflammatory bowel disease.

Genetic variants of the mannan-binding lectin are associated with immune reactivity to mannans in Crohn's disease.

BACKGROUND & AIMS: Some patients with Crohn's disease (CD) develop antibodies against mannan, a component of the yeast Saccharomyces cerevisiae cell wall. Mannan-binding lectin (MBL), a component of the innate immune system, can bind to S. cerevisiae . MBL concentration depends on genetic polymorphisms. The aim of this study was to evaluate whether low MBL contributes to anti-S. cerevisiae antibody (ASCA) production. METHODS: ASCA and MBL concentrations in sera from patients with CD (n = 74), ulcerative colitis (UC) (n = 22), and healthy controls (n = 32) were measured by an enzyme-linked immunosorbent assay (ELISA). Genetic MBL variants were determined from 58 CD patients, 18 UC patients, and 47 controls by DNA sequencing. Lymphocytes were tested for proliferative response after stimulation with mannan. RESULTS: ASCA were found in 47% of the patients with CD and in 0% of the controls. More ASCA-positive patients (52%) had low serum MBL concentrations compared with ASCA-negative patients (4%) (P < 0.0001). T-cell proliferation in response to mannan stimulation was observed in ASCA-positive patients and could be inhibited by the addition of MBL. These patients had significantly lower MBL serum concentrations than patients whose lymphocytes did not proliferate on mannan stimulation (P < 0.0001). Homozygous or compound heterozygous MBL mutations in the exon 1 and promoter occurred in 12 patients with cellular or humoral immune reactivity to mannan as compared with only 1 nonreactive patient (P < 0.0001). CONCLUSIONS: A subgroup of CD patients is characterized by ASCA positivity, T-cell proliferation on mannan stimulation, and mutations in the MBL gene that result in MBL deficiency. Thus, we propose that enhanced mannan exposure stimulates specific immune responses in a subgroup of CD patients with genetically determined low MBL concentrations. This enhanced exposure contributes to the generation of ASCA.

Immune sensitization to yeast antigens in ASCA-positive patients with Crohn's disease.

BACKGROUND: Alimentary antigens may play a role in the perpetuation of inflammation in Crohn's disease (CD). Yeast antigens are widespread components of food. A proportion of CD patients develop antibodies against the yeast Saccharomyces cerevisiae (ASCA), but little is known about the cellular immune reactivity against food antigens in antibody-positive and -negative patients. METHODS: Lymphocytes from patients with CD, ulcerative colitis, and healthy controls were tested for their proliferative response after stimulation with the yeast antigen mannan and ovalbumin. The cellular phenotypes and activation markers were analyzed via FACS. Cytokine concentrations and antibody titers were determined by ELISA. RESULTS: Only lymphocytes of ASCA-positive patients with CD proliferated after stimulation with mannan. These lymphocytes expressed increased activation markers (CD25, CD69). Activation of T cells was mediated by antigen-presenting cells and was associated with increased tumor necrosis factor-alpha (TNF-alpha) levels. The immune reactivity to ovalbumin was predominantly found in CD patients. It was weaker compared with mannan, independent of ASCA status, and also present in healthy controls. CONCLUSIONS: A disturbed humoral and cellular response to the yeast antigen mannan is specifically seen in a subgroup of CD patients. This phenomenon may be due to a loss of tolerance toward yeast and is possibly genetically determined.