Mannan-binding lectin deficiency results in unusual antibody production and excessive experimental colitis in response to mannose-expressing mild gut pathogens.

BACKGROUND: In Crohn's disease (CD) the deficiency of mannan-binding lectin (MBL) is associated with an increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of the disease. However, the role of MBL in intestinal inflammation is currently unclear. A study was undertaken to analyse local MBL expression in human intestine and the consequences of MBL deficiency in experimental colitis and yeast infection. METHODS: ASCA were measured by ELISA. MBL was assessed by ELISA and quantitative PCR. Wild type and MBL-deficient mice were administered dextran sulfate sodium (DSS) in the presence or absence of viable Candida albicans or adhesive invasive Escherichia coli (AIEC). Mice were infected with C albicans to assess generation of anti-yeast mannan antibodies. RESULTS: MBL expression was virtually undetectable in the intestinal mucosa of both healthy controls and patients with CD, irrespective of macroscopic inflammation, indicating that systemic MBL must be responsible for the reduced risk of complicated disease in MBL-competent patients with CD. MBL-deficient mice showed enhanced DSS colitis upon oral challenge with C albicans or AIEC. C albicans could be recovered from the kidneys of colitic/C albicans-fed MBL-deficient, but not wild type mice. Infection with C albicans induced high titres of anti-C albicans mannan IgM and IgG in MBL-deficient mice but only a modest and transient IgM response with no class switch to IgG in wild type mice. Cross-reactive ASCA IgM continuously increased in MBL-deficient mice but rapidly declined after transient induction in wild type mice. In MBL-deficient mice, increased C albicans dissemination correlated with reduced early retention in the circulation. CONCLUSIONS: These results suggest that systemic MBL helps to prevent excessive inflammation upon access of normally mild pathogens across the damaged intestinal epithelium. Lack of this innate defence promotes antibody responses with cross-reactive potential against common mannan epitopes. These interpretations are compatible with the increased prevalence of ASCA and complicated disease phenotypes in MBL-deficient patients with CD.

Low Mannan-binding lectin serum levels are associated with complicated Crohn's disease and reactivity to oligomannan (ASCA).

OBJECTIVES: Mannan-binding lectin (MBL) acts as a pattern-recognition molecule directed against oligomannan, which is part of the cell wall of yeasts and various bacteria. We have previously shown an association between MBL deficiency and anti-Saccharomyces cerevisiae mannan antibody (ASCA) positivity. This study aims at evaluating whether MBL deficiency is associated with distinct Crohn's disease (CD) phenotypes. METHODS: Serum concentrations of MBL and ASCA were measured using ELISA (enzyme-linked immunosorbent assay) in 427 patients with CD, 70 with ulcerative colitis, and 76 healthy controls. CD phenotypes were grouped according to the Montreal Classification as follows: non-stricturing, non-penetrating (B1, n=182), stricturing (B2, n=113), penetrating (B3, n=67), and perianal disease (p, n=65). MBL was classified as deficient (<100 ng/ml), low (100-500 ng/ml), and normal (500 ng/ml). RESULTS: Mean MBL was lower in B2 and B3 CD patients (1,503+/-1,358 ng/ml) compared with that in B1 phenotypes (1,909+/-1,392 ng/ml, P=0.013). B2 and B3 patients more frequently had low or deficient MBL and ASCA positivity compared with B1 patients (P=0.004 and P<0.001). Mean MBL was lower in ASCA-positive CD patients (1,562+/-1,319 ng/ml) compared with that in ASCA-negative CD patients (1,871+/-1,320 ng/ml, P=0.038). In multivariate logistic regression modeling, low or deficient MBL was associated significantly with B1 (negative association), complicated disease (B2+B3), and ASCA. MBL levels did not correlate with disease duration. CONCLUSIONS: Low or deficient MBL serum levels are significantly associated with complicated (stricturing and penetrating) CD phenotypes but are negatively associated with the non-stricturing, non-penetrating group. Furthermore, CD patients with low or deficient MBL are significantly more often ASCA positive, possibly reflecting delayed clearance of oligomannan-containing microorganisms by the innate immune system in the absence of MBL.

Phenotypic associations of Crohn's disease with antibodies to flagellins A4-Fla2 and Fla-X, ASCA, p-ANCA, PAB, and NOD2 mutations in a Swiss Cohort.

BACKGROUND: Distinct Crohn's disease (CD) phenotypes correlate with antibody reactivity to microbial antigens. We examined the association between antibody response to 2 new flagellins called A4-Fla2 and Fla-X, anti-Saccharomyces cerevisiae antibodies (ASCA), anti-neutrophil cytoplasmic antibodies (p-ANCA), anti-pancreas antibodies (PAB), NOD2 mutations (R702W, G908R, and L1007fsinsC), and clinical CD phenotypes (according to Vienna criteria). METHODS: All the above-mentioned antibodies as well as NOD2 mutations were determined in 252 CD patients, 53 with ulcerative colitis (UC), and 43 healthy controls (HC) and correlated with clinical data. RESULTS: A seroreactivity for A4-Fla2/Fla-X/ASCA/p-ANCA/PAB (in percent) was found in 59/57/62/12/22 of CD patients, 6/6/4/51/0 of UC patients, and 0/2/5/0/0 of healthy controls. CD behavior: 37% B1, 36% B2, and 27% B3. In multivariate logistic regression, antibodies to A4-Fla2, Fla-X, and ASCA were significantly associated with stricturing phenotype (P = 0.027, P = 0.041, P < 0.001), negative associations were found with inflammatory phenotype (P = 0.001, P = 0.005, P < 0.001). Antibodies to A4-Fla2, Fla-X, ASCA, and NOD2 mutations were significantly associated with small bowel disease (P = 0.013, P = 0.01, P < 0.001, P = 0.04), whereas ASCA was correlated with fistulizing disease (P = 0.007), and small bowel surgery (P = 0.009). Multiple antibody responses against microbial antigens were associated with stricturing (P < 0.001), fistulizing disease (P = 0.002), and small bowel surgery (P = 0.002). CONCLUSIONS: Anti-flagellin antibodies and ASCA are strongly associated with complicated CD phenotypes. CD patients with serum reactivity against multiple microbes have the greatest frequency of strictures, perforations, and small bowel surgery. Further prospective longitudinal studies are needed to show that antibody-based risk stratification improves the clinical outcome of CD patients.

Anti-Saccharomyces cerevisiae mannan antibodies (ASCA) of Crohn's patients crossreact with mannan from other yeast strains, and murine ASCA IgM can be experimentally induced with Candida albicans.

BACKGROUND: Anti-Saccharomyces cerevisiae antibodies (ASCA) present in a subgroup of Crohn's disease (CD) patients indicate loss of tolerance against commensal antigens. ASCA can be induced in Candida albicans-infected rabbits, suggesting their potential crossreactive nature. The present study aimed to determine crossreactivities of ASCA with cell wall mannans from other yeasts, including the opportunistic pathogen C. albicans, and to define the requirements for (crossreactive) ASCA in experimental mice. METHODS: ASCA were determined by enzyme-linked immunosorbent assay (ELISA). ASCA were neutralized by preincubating sera with purified mannans. Binding of ASCA was visualized by Western blot. Mice were immunized with live yeasts and experimental colitis was induced with dextran sodium sulfate (DSS). RESULTS: Seroreactivity of ASCA-positive CD patients against S. cerevisiae mannan significantly correlates with that against mannans from 5 other yeast species, including C. albicans. This correlation is due to crossreactive IgG, demonstrated by the loss of reactivity after preincubation of sera with mannans from the other yeasts. Immunization of mice with S. cerevisiae or C. albicans fails to induce (crossreactive) ASCA IgM or IgG antibodies. Subsequent chronic experimental colitis concomitant with feeding live yeasts promotes ASCA IgM but not IgG generation, while titers remain modest compared to those in ASCA-positive CD patients. CONCLUSIONS: Correlations of ASCA reactivities against mannans from different yeasts are due to crossreactive IgGs. The inability of mice to readily generate ASCA is in line with the current opinion that genetic predisposition is a prerequisite for the development of this and other unusual immune reactivities in CD.

Association of deficiency for mannan-binding lectin with anti-mannan antibodies in Crohn's disease: a family study.

BACKGROUND: Antibodies against mannan, a component of the yeast Saccharomyces cerevisiae cell wall, are more frequently found in Crohn's disease (CD) patients with low levels of mannan-binding lectin (MBL). MBL concentration depends on genetic polymorphisms. The aim of this study was to evaluate whether low MBL is related to ASCA production in healthy family members of CD patients. METHODS: ASCA and MBL concentrations in sera from patients (n=52), and their 158 healthy relatives were measured by enzyme-linked immunosorbent assay (ELISA). Genetic MBL variants were determined by DNA sequencing. RESULTS: Thirty-five (67%) patients were ASCA-positive. Twenty-six (74%) of the 35 ASCA-positive patients had low MBL levels (<500 ng/mL), whereas only 4 (24%) of the 17 ASCA-negative patients had low values for MBL (P=0.001). ASCA were found in 38 (24%) family members. Twenty-three (50%) of 46 family members with low values for MBL were ASCA-positive compared to 15 (13%) of 112 family members with normal values for MBL (P<0.0001). ASCA were found in 33 of 104 (32%) family members of ASCA-positive patients and in 5 family members (9%) of ASCA-negative patients (P=0.002). Relatives with mutations leading to MBL deficiency had significantly more frequent ASCA than relatives without these mutations (P=0.018). CONCLUSIONS: MBL deficiency is associated with ASCA positivity not only in patients with CD, but also in their relatives. An impaired innate immune system defined by low MBL serum concentrations may lead to an increased reactivity of the specific immune system to mannan antigens, and therefore facilitate the generation of ASCA.

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.

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.