Journal of Crohn's and Colitis (2013) 7, e1–e10

Available online at www.sciencedirect.com

Increased titers of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients with reduced H-ficolin levels but normal MASP-2 activity Thomas Schaffer, Beatrice Flogerzi, Alain M. Schoepfer, Frank Seibold 1 , Stefan Müller ⁎,1 Department of Clinical Research, Division of Gastroenterology, University Hospital Bern, University of Bern, Bern, Switzerland

Received 24 June 2011; received in revised form 23 January 2012; accepted 14 February 2012

KEYWORDS Anti-Saccharomyces cerevisiae antibodies; Crohn's disease; Ficolin; MASP-2; MBL

Abstract Background and aims: Mannan-binding lectin (MBL) and ficolins are microbial pattern recognition molecules that activate the lectin pathway of complement. We previously reported the association of MBL deficiency with anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease (CD). However, ASCA are also frequently found in MBL-proficient CD patients. Here we addressed expression/function of ficolins and MBL-associated serine protease2 (MASP-2) regarding potential association with ASCA. Methods: ASCA titers and MBL, ficolin and MASP-2 concentrations were determined by ELISA in the serum of patients with CD, ulcerative colitis (UC), and in healthy controls. MASP-2 activity was determined by measuring complement C4b-fixation. Anti-MBL autoantibodies were detected by ELISA. Results: In CD and UC patients, L-ficolin concentrations were significantly higher compared to healthy controls (p b 0.001 and p = 0.029). In contrast, H-ficolin concentrations were slightly reduced in CD and UC compared to healthy controls (p = 0.037 for UC vs. hc). CD patients with high ASCA titers had significantly lower H-ficolin concentrations compared to ASCA-low/negative CD patients (p = 0.009). However, MASP-2 activity was not different in ASCA-negative and ASCApositive CD patients upon both, ficolin- or MBL-mediated MASP-2 activation. Finally, anti-MBL autoantibodies were not over-represented in MBL-proficient ASCA-positive CD patients.

Abbreviations: CD, Crohn's disease; IBD, inflammatory bowel disease; UC, ulcerative colitis; ASCA, anti-Saccharomyces cerevisiae antibodies; MBL, mannan-binding lectin; MASP, MBL-associated serine protease; GlcNAc, N-acetyl glucosamine; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus. ⁎ Corresponding author at: Dept. of Clinical Research, Div. of Gastroenterology, University of Bern, Murtenstrasse 35, CH-3010, Bern, Switzerland. Tel.: + 41 31 632 8492; fax: + 41 31 632 3297. E-mail address: [email protected] (S. Müller). 1 These authors equally contributed to this work. 1873-9946/$ - see front matter © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.crohns.2012.02.013

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T. Schaffer et al. Conclusions: Our results suggest that low expression of H-ficolin may promote elevated ASCA titers in the ASCA-positive subgroup of CD patients. However, unlike MBL deficiency, we found no evidence for low expression of serum ficolins or reduced MASP-2 activity that may predispose to ASCA development. © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

1. Introduction Crohn's disease (CD) is one of the two major clinical forms of the inflammatory bowel diseases (IBD). The etiology of CD is incompletely understood but recent findings led to the widely accepted hypothesis that inappropriate immune responses to the commensal gut flora in genetically susceptible individuals may play an important role. 1–3 Antibodies against Saccharomyces cerevisiae mannan (ASCA) are found in about 60% of CD patients. 4 The major epitope of the yeast cell wall mannan recognized by ASCA has been well characterized. 5,6 In contrast, the triggering event leading to ASCA generation is still a matter of debate. We and others have shown that ASCA cross-react with many other yeast species and with some mycobacteria. 7–9 We have previously observed that deficiency for mannan-binding lectin (MBL) in CD patients, as well as their healthy first degree relatives, correlated with increased ASCA prevalence, compared to CD patients and their healthy first degree relatives with normal MBL expression levels. 10,11 MBL is a serum lectin recognizing mannosylated cell wall compounds of yeasts, bacteria and viruses. It serves as an opsonin by enhancing phagocytic clearance of mannose (and/or fucose) containing organisms from the blood stream, or activates the lectin pathway of complement in a complex with mannan-binding lectin-associated serineproteases (MASPs). 12–15 Deficiency for functional MBL is due to genetic polymorphisms in both the structural and the promoter region of the human mbl2 gene and is associated with a higher risk for certain infectious diseases particularly in childhood or in immunocompromised individuals. 16–19 Even though MBL-deficiency seems to facilitate the formation of ASCA, a considerable proportion of ASCA-positive CD patients have normal MBL serum levels. In addition a recent study in a large Hungarian cohort could not detect this association with patients MBL serum levels of less than 500 ng/ml. 20 The present study therefore aims to address other potential mechanisms within the lectin pathway of complement, which might be involved in the predisposition to develop this unusual antibody response. In addition to MBL, the ficolins can also form complexes with MASPs to initiate the lectin pathway of complement. 21 L-ficolin and H-ficolin are soluble pattern recognition receptors mainly binding to N-acetyl glucosamine (GlcNAc), which is present in bacterial and fungal cell walls, and in mannoproteins of the yeast cell wall. L-ficolin also binds to yeast mannan-Sepharose, 22 frequently used for absorption of MBL. Furthermore, L-ficolin as well as MBL is capable of activating MASP-2 upon binding to whole Mycobacterium bovis BCG. 23 Despite the fact that ficolins mainly recognize acetylated compounds and have a high affinity for GlcNAc, they show individual preferences in the recognition of different microorganisms. 24,25 Taken together, in addition to MBL,

the ficolins are also involved in innate immunity against bacteria and very likely also yeast via the lectin pathway of complement. 26–28 To date, only one study addressed serum concentrations of H-ficolin in CD. 29 In this study, no differential expression was detected in CD patients compared to controls in a Danish cohort. L-ficolin has not been investigated in that respect, so far. MASP-2 is essential for complement activation by both MBL and ficolins, and therefore may be involved in determining the strength/success of the resulting anti-microbial innate immune response. MASP-2 polymorphisms have been described that lead to inherited MASP-2 deficiency or impaired functionality 30,31 while relevance for disease has not been thoroughly investigated. Anti-MBL autoantibodies have been described in other inflammatory disorders such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). 32–34 Such autoantibodies could have similar functional consequences as MBL deficiency. In order to obtain a complete overview of initial events in the recognition of yeast and the subsequent initiation of the lectin pathway of complement in view of a possible role in the generation of ASCA in CD, we measured H-ficolin, Lficolin, MASP-2 concentration and activity, and anti-MBL antibodies in samples from CD as compared to UC and healthy controls.

2. Methods 2.1. Patients and sera 316 patients treated in Gastroenterology at the University Hospital of Bern were included. Informed consent was obtained from all patients and the study was performed with the approval of the local ethics committees. Patients' characteristics are provided in Table 1. The diagnosis of CD and UC was based on standard endoscopic, histological, and radiographic features. All healthy volunteers were free of abdominal symptoms, had no 1st degree relatives with diagnosed IBD and were not on any regular medication. Blood was collected in serum-gel S-Monovettes (Sarstedt, Sevelen, Switzerland) and harvested sera were stored at − 20 °C until use. Sera were always tested in duplicates. ASCA titers were determined by our own accredited routine diagnostic test as described. 8

2.2. L-ficolin and H-ficolin serum concentrations Serum concentrations of L-ficolin and H-ficolin were determined by ELISA in Nunc-Immuno Maxisorp® 96-well plates (Nunc, Roskilde, Denmark). Plates were coated with 1 μg/ml monoclonal anti-human L-ficolin (clone GN4) or H-ficolin

MBL, filcolin, MASP-2 and ASCA in CD Table 1

Patients' characteristics.

Number of patients Female Age: mean ± SD, range Median age at onset (yrs) Disease duration (yrs) Age at diagnosis Disease phenotype a

Disease location (%) b

Disease activity Small bowel disease (%) Small bowel surgery (%) a b

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Crohn's disease

Ulcerative colitis

Healthy controls

182 79 41 ± 18 (18–85) 25 11 ± 13 (1–44) b 40 yrs: 118 (65%) ≥ 40 yrs: 64 (35%) B1: 72 (40%) B2: 64 (35%) B3: 46 (25%) L1: 49 (27%) L2: 46 (25%) L3: 80 (44%) L4: 7 (4%)

67 30 40 ± 12 (21–70) 27 10 ± 11 (2–33)

67 32 37 ± 8 (22–51) – – – –

Proctitis 11 Proctosigmoiditis 20 Left sided 12 Extensive 7 Pancolitis 17 Mayo score: 7 ± 3 (3–10) – –

CDAI: 137 ± 49 (13–226) 130 (71%) 51 (28%)

–

– – –

B1, non-stricturing, non-penetrating; B2, stricturing; B3, penetrating. L1, ileal; L2, colonic; L3, ileocolonic; L4, isolated upper disease.

(clone 4H5) antibodies (Hycult Biotechnology, Uden, The Netherlands) in carbonate–bicarbonate buffer pH 9.6, overnight at 4 °C. Plates were blocked 1 h at RT with phosphatebuffered saline (PBS) + 0.5% bovine serum albumin (BSA, Sigma, Buchs, Switzerland) (PBS-BSA). Sera were diluted 1:1000 for L-ficolin or 1:10,000 for H-ficolin in PBS-BSA. Recombinant human L-ficolin and H-ficolin (R&D Systems, Minneapolis, MN) were used as standards. Plates were incubated overnight at 4 °C and washed with PBS-BSA + 0.05% Tween 20 (PBST-BSA). Plates were then incubated for 2.5 h at room temperature (RT) with biotinylated polyclonal anti-human Lficolin or H-ficolin antibodies (R&D Systems) at 0.1 μg/ml in PBST-BSA. After washing, plates were further incubated for 2.5 h at RT with avidin-HRP (BD Biosciences, San Diego, CA) diluted 1:1000 in PBST-BSA. After washing, reaction was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate tablets (Sigma) dissolved in phosphate–citrate buffer pH 5 for 15 min in the dark. Color-development reaction was stopped with 0.5 M sulfuric acid and absorption read at 450 nm on a microplate reader (BioTek Instruments, Winooski, VT).

2.3. MBL and MASP-2 serum concentrations Serum concentrations of MBL were determined after 1/100 dilution using a commercial ELISA kit (BioPorto diagnostics, Gentofte, Denmark) according to the manufacturer's instructions. For the determination of MASP-2 concentrations, a commercial ELISA kit for human MASP-2 (Hycult Biotechnology) was used according to the manufacturer's protocol and with sera diluted 1/10.

2.4. Quantification of MBL/MASP-2 activation Sera were diluted 1:25 and mannan-bound MBL/MASP-2 activation quantified with the commercial human MBL/MASP-2-

dependent complement C4b deposition assay (Hycult Biotechnology) according to the manufacturer's protocol. Briefly, plate-bound mannan leads to activation of the MBL/ MASP-2 complex, leading to cleavage of exogenously added C4 into C4a and C4b. C4b remains attached to the MBL/ MASP-2 complex and is quantified using a specific antibody against its C4c subunit by ELISA. The calculated values (U/ ml) correspond to the extent of C4 cleavage products generated with a certain amount of a fully functional MBL under the assumption that MASP-2 is not rate limiting (using the assay kit's MBL/MASP-2 complex standard).

2.5. Quantification of ficolin-dependent MASP-2 activation The complement C4 fixation (Hycult Biotechnology) assay was adapted to measure ficolin-, instead of MBL-mediated MASP-2 activation. Briefly, Nunc-Immuno Maxisorp® 96well plates were coated with 1 mg/ml BSA (Sigma) in PBS overnight at 4 °C. Plates were then incubated with 100 μl 96% ethanol containing 0.1% (v/v) acetic anhydride per well at RT for 1 h for acetylation of the plate-bound BSA. Plates were washed and sera added at 1:25 dilution. The assay kit's MBL/MASP-2 standard was used as negative control to confirm specificity for ficolin-mediated MASP-2 activation via recognition of the plate-bound acetyl groups. Subsequent steps were identical to those of the MBL/MASP2-mediated C4 fixation according to the protocol of the assay.

2.6. Anti-MBL autoantibody ELISA The anti-MBL autoantibody ELISA was adapted from Seelen et al. 34 and performed in Nunc-Immuno Maxisorp® 96-well plates. Plates were coated with 0.5 μg/ml recombinant MBL (kindly provided by Enzon Pharmaceuticals, Bridgewater, NJ)

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T. Schaffer et al.

in carbonate–bicarbonate buffer pH 9.6, overnight at 4 °C. Plates were blocked 1 h at RT with PBS + 1% BSA. Plates were incubated for 2 h at RT with patients' sera diluted 1:100 in PBS-BSA. Plates were washed with PBST-BSA and incubated for 1 h at RT with peroxidase-coupled anti-human IgG (Sigma) diluted 1:5000 in PBST-BSA. After washing, a color reaction was developed using TMB substrate tablets (Sigma) dissolved in phosphate–citrate buffer pH 5 for 30 min. in the dark. The reaction was stopped with 0.5 M sulfuric acid and the absorption read at 450 nm on a microplate reader (BioTek Instruments). Elevated anti-MBL autoantibody titers were defined as OD at 450 nm higher than the mean OD450 of all healthy controls plus two times the standard deviation.

2.7. Statistics Statistical analyses were performed using a statistical package program (STATA Vs 9.0, Texas) and InStat software (GraphPad Software, San Diego, CA). The statistical significance was determined using the Mann–Whitney test for comparison of 2 independent groups with numerical data and the Fisher's exact or chi-square test for comparison of two independent groups with categorical data. Correlations between two variables were calculated with the Spearman rank correlation coefficient. A p b 0.05 was considered statistically significant.

3. Results 3.1. Serum concentrations of L-ficolin and H-ficolin Concentrations of L-ficolin and H-ficolin were determined in the serum of patients with CD, UC and in healthy controls. Table 1 shows the clinical and serological characteristics of the study population. Patients with IBD were found to have higher L-ficolin titers (1.78 ± 0.88 μg/ml for CD and 1.48 ± 0.84 μg/ml for UC) compared to healthy controls (1.17 ± 0.77 μg/ml). This difference was statistically highly significant for CD (p b 0.001) but not for UC (p = 0.059). L-ficolin levels were not significantly different between CD and UC patients (p = 0.098) (Fig. 1A). Regarding H-ficolin, no significant differences were observed between CD patients (22.7 ± 10.6 μg/ml), UC patients (21.3 ± 6.8 μg/ml; p = 0.662), and healthy controls (24.3 ± 9.7 μg/ml; p = 0.645) (Fig. 1B).

Figure 1 L-ficolin (A) and H-ficolin (B) concentrations were measured by ELISA in serum samples from 182 patients with Crohn's disease (CD), 67 patients with ulcerative colitis (UC) and 67 healthy controls (HC) and are displayed as box plots for each category. p-Values were determined with the Mann–Whitney U (MWU) test.

controls (p b 0.001) (Fig. 2). In contrast, L-ficolin expression levels were not different in the ASCA-negative/low compared to the ASCA-high expressers (p = 0.085).

3.3. MASP-2 serum concentrations MBL or ficolins form multimeric complexes with the MASPs to initiate the lectin pathway of complement. In a first step, we investigated serum expression levels of MASP-2 in a cohort of 21 ASCA-negative and 55 ASCA-positive CD patients, where MBL cannot be the limiting factor for the activation

3.2. Association of ASCA with serum levels of ficolin Table 2

To detect a possible association between ASCA positivity and ficolin titers, categorical groups were defined according to the 95% Confidence Intervals, as described before for Lficolin 35 (Tables 2 and 3). The chi-squared test did not reveal any association of low, normal or high L- or H-ficolin expressers with the presence or absence of ASCA, even when low expressers were directly compared with high expressers, i.e. omitting all those patients with expression levels in between. When CD patients were grouped according to their levels of ASCA expression, we found that CD patients with high ASCA titers had significantly lower H-ficolin levels compared to those with no or low ASCA expression (p b 0.001) as well as compared to UC patients (p b 0.001) and healthy

Correlation of L-ficolin with ASCA.

Categories of L-ficolin expression

ASCAnegative

ASCApositive

Low Normal High

46 32 25

47 40 30

Chi-square test

g

p = 0.522

g

p = 0.637

CD patients' sera were categorized according to low, normal or high L-ficolin expression. “Low” expression was defined as below the 95% confidence interval (CI) which was 1.55–1.86 μg/ml for our cohort of CD patients. Similarly, “High” was defined as above the 95% CI, while “Normal” was defined as everything above the lower value of the 95% CI.

MBL, filcolin, MASP-2 and ASCA in CD Table 3

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Correlation of H-ficolin with ASCA.

Categories of H-ficolin expression

ASCAnegative

ASCApositive

Low Normal High

32 46 30

45 42 33

Chi-square test

g

p = 0.169

g

p = 0.473

CD patients' sera were categorized according to low, normal or high H-ficolin expression. “Low” expression was defined as below the 95% confidence interval (CI) which was 21.96–26.36 μg/ml for our cohort of CD patients. Similarly, “High” was defined as above the 95% CI, while “Normal” was defined as everything above the lower value of the 95% CI.

of the lectin pathway of complement (MBL N 500 ng/ml). There was no significant difference in the median of MASP2 concentrations between the two groups (265.2 ng/ml vs. 310.0 ng/ml, respectively; p = 0.508) (Fig. 3A). In addition, no correlation between individual MASP-2 and ASCA titers was found (r = 0.098, p = 0.293) (Fig. 3B). According to a Danish study normal MASP-2 concentrations range from 170 to 1196 ng/ml. 36 Thus, we compared b 170 ng/ml with N 170 ng/ ml MASP-2 expressers regarding their ASCA status. We found that CD patients with low MASP-2 concentrations showed the same prevalence for ASCA-positivity than normal MASP-2 expressers (Fisher's exact test p = 0.496).

3.4. Mannan-induced MBL/MASP-2 activation Since the presence of ELISA-reactive MASP-2 does not necessarily correlate with the functional activity of MASP-2, the

Figure 3 MASP-2 concentrations were measured by ELISA in randomly selected MBL-positive sera from ASCA-negative (n = 21) or ASCA-positive (n = 55) CD patients. (A) Dot plot with medians showing the distribution of the individual serum concentrations of MASP-2. (B) Arbitrary values between 0 (ASCA −) and 6 (ASCA+++) were determined for ASCA expression according to the grading used in our diagnostic unit. Values for serum MASP-2 concentrations were plotted against these arbitrary units of the 76 tested CD patients. Correlation was assessed using the Spearman rank correlation coefficient.

level of MBL/MASP-2 complex activity upon interaction of MBL with mannan was assessed by measuring cleavage of complement C4 into C4a and C4b. A spot check of 12 ASCAnegative and 17 ASCA-positive CD patients, 10 UC patients and 6 healthy controls, all with MBL oligomer serum levels above 500 ng/ml, were analyzed (Table 4). No significant differences were observed between all tested groups.

Table 4

Figure 2 ASCA and H-ficolin concentrations were determined by ELISA and results are shown in a dot plot with medians. ASCAnegative/weak positive (ASCA ±) and highly ASCA-positive (ASCA++/+++) categories were defined according to the number of standard deviations the respective OD450 was above the mean of 10 healthy control sera, as described in. 7 ASCA± and ASCA++/ +++ CD patients were compared to UC patients and healthy controls (HC). p-Values were determined using the MWU test.

MBL/MASP-2 activity.

Subgroups

MASP-2 activity [U/ml]: median

MASP-2 activity [U/ml]: range

Chi-square test

CD ASCA− (n = 12) CD ASCA+ (n = 17) UC (n = 10) HC (n = 6)

338.7

118–502

n.s.

257.4

74–509

n.s.

254.0 308.1

127–462 68–502

n.s. n.s.

CD patients were split into ASCA-positive and ASCA-negative subgroups, and compared with UC patients and healthy controls (HC) for mannan− bound MBL − dependent MASP − 2 activity (MBL/MASP-2 activity), measured as complement C4b deposition by ELISA. The median and range of MBL/MASP-2 activity are shown as units per ml (U/ml) according to the standard provided with the ELISA kit.

e6 (Three MBL-negative sera were assessed to confirm MBLdependency of the assay, not shown). Thresholds for low MBL/MASP-2 activity were set at the lower 95% CI (248.5 U/ ml) or at 160 U/ml, the latter based on a clearly distinguishable group of CD sera with an MBL/MASP-2 activity below 160 U/ml despite a mean MBL concentration of around 1500 ng/ml. ASCA-positive patients were not more frequently within the low MBL/MASP-2 activity groups than ASCA-negative patients (Fisher's exact test, p= 1.000 for the 248.5 U/ml as well as the 160 U/ml threshold). There was no significant correlation between MASP-2 concentrations and mannan-bound MBL-mediated MASP-2 activity (r= 0.105, p = 0.480) (Fig. 4A). In contrast, MBL concentrations strongly correlated with MASP-2 activity (r = 0.808, p b 0.001) (Fig. 4B). Thus, in the context of mannan as initial trigger, it is mostly the large variations in individual MBL serum titers that define mannan-bound MBL-mediated MASP-2 activity.

Figure 4 Mannan-bound MBL-mediated MASP-2 activity (MBL/ MASP-2 activity) was measured by the C4b deposition assay in randomly selected MBL-positive sera from ASCA-negative (n = 12) and ASCA-positive (n = 17) CD patients, as well as an unbiased spot check with 10 UC patients and 6 healthy controls (HC). (A) Correlation of MASP-2 concentration (x-axis) with MBL/MASP-2 activity (y-axis) across all groups. (B) Correlation of MBL concentration (x-axis) with MBL/MASP-2 activity (yaxis) across all groups. Two MBL-deficient healthy control sera were also included. Correlations were assessed using the Spearman rank correlation coefficient.

T. Schaffer et al.

3.5. Ficolin-dependent MASP-2 activity Plate-bound BSA was acetylated to provide a high-affinity binding partner for both, L- and H-ficolin. An unbiased selection of our cohort of CD patients (n= 50) showed significantly higher ficolin/MASP-2 activity compared to randomly chosen healthy controls (n= 13) (p = 0.001). The ficolin/MASP-2 activity was slightly but not significantly (p= 0.123) higher in the ASCA-positive compared to the ASCA-negative subgroup of CD patients (Fig. 5A). L-ficolin concentrations were normalized

Figure 5 Acetylated BSA-bound ficolin-mediated MASP-2 activity (ficolin/MASP-2 activity) was measured by a C4b deposition assay with specific adaptations as described in the Methods section. C4b deposition was assessed in a randomly selected cohort of 26 ASCA-positive and 24 ASCA-negative CD patients, as well as 14 healthy controls (HC). (A) Dot plot with medians showing individual OD450 values of the assay for the different groups with the means indicated by horizontal bars. p-Values indicate significant differences between groups using the non-parametric Mann–Whitney U-test. (B) L-ficolin concentrations were normalized to the range obtained for H-ficolin and the resulting values added to the corresponding Hficolin concentrations (x-axis). The resulting arbitrary units were correlated with ficolin/MASP-2 activity (y-axis). The correlation was assessed using the Spearman rank correlation coefficient.

MBL, filcolin, MASP-2 and ASCA in CD

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to the numerical range of H-ficolin and both values added together for each individual. The arbitrary values obtained by this, served as a means to represent total soluble serum ficolin concentrations, and were plotted against ficolin/MASP-2 activity (Fig. 5B). There was no correlation between combined ficolin concentrations and the ficolin-dependent MASP-2 activity (r= 0.017, p = 0.903), indicating that the combined action of L- and H-ficolins was not rate limiting in ficolin/MASP-2 activation. Dependency of MASP-2 activity on acetylated BSA in this approach was confirmed using a number of MBL-deficient sera with normal MASP-2 levels (data not shown).

3.6. Anti-MBL autoantibodies Another possibility, how MBL/MASP-2-mediated activation of the lectin pathway of complement could be impaired, is the presence of anti-MBL autoantibodies as described for SLE or RA. Anti-MBL IgG were measured by ELISA in sera of 72 MBLpositive (N 500 ng/ml) and 10 MBL-negative (b 100 ng/ml) CD patients, 21 MBL-positive and 2 MBL-negative UC patients, and 25 MBL-positive and 5 MBL-negative healthy controls. On average, anti-MBL autoantibody titers were significantly lower in CD compared with healthy controls (p = 0.038), and slightly but non-significantly lower compared to UC patients (Fig. 6A). A total of nine sera (6%) showed elevated levels of anti-MBL autoantibodies above the rather conservative cutoff defined by the mean + 2 standard deviations of all healthy controls [Fig. 6A dotted line]. Among these anti-MBL-positive sera, 5 were from CD patients (6%), 1 from UC patients (4%) and 3 from healthy controls (10%). While all anti-MBLpositive sera were from MBL-positive individuals, none of them were from ASCA-positive CD patients. This suggests that anti-MBL antibody expression above the cut-off is “not independent” of ASCA-negativity (Fisher's exact test, p = 0.009). ASCA-positive CD patients showed a significantly lower mean of OD450 values in the anti-MBL IgG ELISA than ASCA-negative CD patients, which was more pronounced in the MBL-positive subgroup of ASCA-positive CD patients (Fig. 6B). The levels of anti-MBL autoantibodies in healthy controls were comparable to those of ASCA-negative CD patients significantly increased above those of the ASCA-positive/MBL-positive subgroup (p b 0.001). In UC patients, average anti-MBL IgG titers were between those of ASCA-negative/MBL-positive CD patients (p = 0.026), and those of ASCA-positive/MBL-positive CD patients (p = 0.036) (Fig. 6B).

4. Discussion ASCA are a hallmark of a large subgroup of CD patients but why and how this unusual antibody response against yeast oligomannan develops is not clear. We systematically addressed the initial steps of the lectin pathway of complement, which is a major innate immunity pathway recognizing the same oligomannan structures as ASCA. We have previously shown that MBL-deficiency was associated with ASCA in CD. 10,37 Here we focused on the soluble ficolins, which also activate the lectin pathway of complement via MASPs. Furthermore, MASP-2 activity was of special interest, as loss of function variants had been described for this critical component of the lectin pathway of complement. 38–40

Figure 6 Anti-MBL IgG autoantibodies were measured in sera from 82 CD patients, 23 UC patients and 30 healthy controls (HC) by ELISA using recombinant human MBL for coating the plates. Antibody titers are shown as OD at 450 nm (OD450). (A) Comparison of anti-MBL IgG between CD, UC and HC. The dashed line indicates the cut off at an OD450 of 0.55 between anti-MBL IgG-negative and positive sera (mean of all HC sera with the exception of two outliers plus two standard deviations). (B) CD patients were separated into ASCA-negative/MBL-positive, ASCA-positive/MBL-positive and ASCA-positive/MBL-negative subgroups and OD450 values for anti-MBL IgG compared between the subgroups and with UC and HC. MBL-positivity and -negativity were defined as MBL oligomer serum concentrations of N 500 ng/ml, and b 100 ng/ml, respectively. Medians are indicated by horizontal bars. p-Values indicate statistically significant differences for mean OD450 values between the respective groups using the nonparametric Mann–Whitney U-test.

First, we analyzed the serum levels of L- and H-ficolins. The increased titers of L-ficolin in both, CD and UC, compared to controls suggest an acute-phase-like regulation. By definition, plasma concentrations of acute-phase proteins increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) by at least 25% during inflammation. 41 In our study population this criterion was fulfilled

e8 for L-ficolin (CD = + 44% and UC = +26.5% compared to healthy controls). Furthermore, as for other classical acute-phase proteins L-ficolin is produced mainly by hepatocytes. Indeed, we observed a positive correlation between L-ficolin levels and the corresponding CRP values as determined by routine diagnostics from 25 randomly chosen sera of CD patients (r = 0.496, p = 0.022). In contrast to L-ficolin, we observed similar H-ficolin concentrations in CD and healthy controls, which is in accordance with a Danish study population. 29 Regulation of ficolin expression has not been extensively studied so far and examples have been shown for reduced 42 as well as elevated 43 H-ficolin concentrations in inflammatory disorders. Besides hepatocytes, H-ficolin is also expressed in the lung and bile duct epithelium and from the latter is secreted into the bile duct. 44 Therefore, we cannot conclusively state that it is not regulated under inflammatory conditions in IBD as we only determined its serum levels. Regarding ASCA, we could not detect an association of differential ficolin expression in ASCA-positive vs. ASCAnegative CD patients. However, H-ficolin titers were statistically significantly decreased in sera from a subgroup of CD patients characterized by very high ASCA titers as clearly shown in Fig. 2. Theoretically, presence of anti-H-ficolin autoantibodies might mask crucial antigens of the H-ficolin ELISA as one of the matched ELISA antibodies is monoclonal. However, testing a number of low- and high H-ficolin serum samples for the presence anti-H-ficolin autoantibodies did not support this hypothesis (data not shown). Thus, low Hficolin levels, which are most likely genetically determined, 45 may predispose ASCA-positive CD patients for enhanced production of these antibodies. We have been able to show that MBL-deficiency facilitates dissemination of the opportunistic pathogenic yeast Candida albicans in mice. 46 Similarly, neonatal gram-positive sepsis is more frequent in low H-ficolin expressers, reflecting an in most cases asymptomatic innate immune deficiency due to low H-ficolin serum levels. 47 Therefore, our results could let us speculate that H-ficolin might also be involved in the acute control of fungal infections and low levels would facilitate enhanced antibody production upon repeated exposure to fungal antigens. However, speaking against this hypothesis is our finding, that there is no similar inverse correlation of MBL with ASCA serum levels, and complete lack of MBL correlates with ASCA-positivity but not with the level of ASCA expression. 10 Downstream of MBL and the ficolins are the MASPs. In our cohort of patients and controls we did not find functional MASP-2 deficiency. There were a few individuals with MASP-2 titers below 170 ng/ml, which were considered as MASP-2 low expressers. 36 However, we found no difference in the over-all distribution of MASP-2 concentrations in ASCAnegative vs. ASCA-positive CD patients, further reflected by the lack of correlation between MASP-2 concentrations and ASCA expression (Fig. 3). The MASP-2 concentrations we measured corresponded well with data on MASP-2 found in a cohort of Danish Caucasians 40 (Danish Caucasians: 416 ng/ml, 125–1152; our cohort: 415 ng/ml, 78–1255). When MASP-2 activity was measured using the complement C4b fixation assay, a large and comparable range of mannan-bound MBL-dependent MASP-2 activity was found in all groups tested in this study. This indicates that in addition to varying serum levels, variability in MASP-2 activity is

T. Schaffer et al. frequent and cannot be considered to play a role in ASCA generation. In this setup, MASP-2 activity was mainly determined by the MBL, rather than by the MASP-2 serum concentrations, indicating that functional MASP-2 deficiency may only rarely occur as a result of defective MASP-2. Using acetylated BSA as binding partner for the ficolins to activate MASP-2 further indicated that most sera have normal MASP-2 activity irrespective of the type of MASP-2 complex, i.e. MBL/MASP-2 or ficolin/MASP-2. Ficolin/MASP-2 activity was even significantly higher in CD compared to healthy controls, most likely due to the elevated L-ficolin concentrations. However, ASCA-negative and ASCA-positive CD sera showed comparable ficolin/MASP-activities, again speaking against a role of inefficient MASP-2 activation in the generation of ASCA in this context. Collectively, our results regarding MASP-2 concentrations and MBL/MASP-2 or ficolin/MASP-2 activities suggest that we do not have individuals homozygous for the known MASP-2 missense polymorphisms 38–40 in our cohort, and that neither MASP-2 nor L- or H-ficolin play a major role in the predisposition of CD patients to develop ASCA. The third aspect in this study was to test whether anti-MBL autoantibodies may be more frequently present in ASCApositive CD patients. Anti-MBL antibodies are frequently detected in SLE patients 34 and in rheumatoid arthritis (RA), 32 and have been shown to interfere with correct MBL function. However, we rarely found anti-MBL IgG in all groups tested and the levels were even lower in CD than in healthy controls. Most strikingly, none of the ASCA-positive sera reached anti-MBL IgG-specific signals above a cut-off that was defined to separate negative from clearly positive events. On the contrary, ASCA-positive sera, in particular those with normal MBL levels, showed significantly reduced signals for anti-MBL IgG compared to ASCA-negative or healthy control sera. Therefore, absence rather than presence of anti-MBL autoantibodies seems to associate with ASCA-positivity, although this is only true when absolute OD450 values and not positive/negative discrimination upon a cut-off level are considered with the present size of our cohort. Reduced absorption values in the anti-MBL autoantibody ELISA for ASCApositive CD patients is counter-intuitive, as IBD patients are generally known for their predisposition to develop many kinds of autoantibodies. 48 Seelen et al. 34 speculate that increased anti-MBL autoantibodies in SLE patients may be due to decreased target-bound MBL. Accordingly, one could speculate that ASCA-positive CD patients may have less targetbound MBL, which would consequently result in lower absorption values in the anti-MBL autoantibody ELISA. In addition, the concept of reduced target-bound MBL (which does not have to be restricted to purified mannan) could hypothetically explain a predisposition for ASCA development. In conclusion, while not predisposing for ASCA generation per se, low H-ficolin plasma concentrations associate with particularly high anti-yeast oligomannan antibody expression within the ASCA-positive subgroup of CD patients. With this background, determination of potential binding activities of H-ficolin to yeast cells or cell wall compounds may be of particular interest. In contrast, MBL so far remains the main determinant for ASCA seroconversion in the lectin pathway of complement. Finally, it is possible that the newly discovered phylogenetically conserved protein MAp44, which competes with MASP-2 for binding to MBL and the ficolins, may influence predisposition for ASCA generation. 49 However, since MASP-2

MBL, filcolin, MASP-2 and ASCA in CD

e9

activity in the context of mannan and MBL was comparable in ASCA-positive and ASCA-negative CD patients' sera, such an influence would most likely not occur within the circulation and consequently may be difficult to address experimentally.

10.

Conflict of interest statement

11.

On behalf of all authors, the corresponding author would like to state that none of the listed authors of the present study has any financial conflict of interest to declare.

12.

Acknowledgments

13.

The present study was supported by the Swiss National Science Foundation grants SNSF 3247BO-118112/1 to F.S., and SNSF 3347C01089792/1 of the Swiss IBD cohort study group, and by an unrestricted research grant from Abbott AG, Switzerland to A.M.S.

14.

The authors would like to thank Anders Krarup for providing us his protocol for binding and activation of ficolin to ELISA plates via acetylation of plate-bound BSA, Enzon pharmaceuticals for generously providing recombinant human MBL, and Emma Slack for carefully reading the manuscript. Author contributions: TS performed most experiments and wrote the initial version of the manuscript. BF coordinated blood samples and performed experiments. AMS recruited patients and controls, did clinical work-up and performed statistics. SM contributed to the original idea, designed and coordinated experiments, and wrote the final version of the manuscript. FS had the original idea, contributed to design and coordination of the experiments and acquired funding.

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