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TNFα genotype influences development of IgA-ASCA antibodies in Crohn's disease patients with CARD15 wild type
Clinical Immunology (2006) 121, 305 — 313
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TNFA genotype influences development of IgA-ASCA antibodies in Crohn’s disease patients with CARD15 wild type Patricia Castro-Santos a, Lourdes Mozo a, Carmen Gutie ´rrez a,b, Ana Sua ´rez b,* a
Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain Department of Functional Biology, Immunology Area, University of Oviedo, C/Julia´n Claverı´a s/n, 33006 Oviedo, Spain
b
Received 11 April 2006; accepted with revision 18 July 2006 Available online 6 September 2006
KEYWORDS Inflammatory bowel disease; CD; Cytokine polymorphisms; ASCA; PAB; TNFa; IL-10; CARD15
Abstract A typical feature of Crohn’s disease (CD) patients is the development of antibodies against self- (PAB) or exogenous (ASCA) antigens, a process in which mucosal cytokine expression pattern might be involved. On the other hand, mutations in CARD15, a genetic risk factor for CD, alter cytokine production in response to bacterial infection. In the present study, we evaluated the role of functionally relevant IL-10 and TNFa gene polymorphisms in the synthesis of these antibodies and their relationship with CARD15 mutations. In CARD15 wild type patients, high TNFa producer genotypes protect against IgA-ASCA development, whereas an inverse association was observed in autoantibody synthesis (PAB). These associations were not observed in patients with CARD15 mutations, probably due to the lack of TNFa release as a consequence of the failure of CARD15 protein to recognize the peptidoglycan. Thus, we proposed a CARD15— TNFa circuit that might play a role in mucosal immune surveillance. D 2006 Elsevier Inc. All rights reserved.
Introduction
Abbreviations: IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; TNFa, tumor necrosis factor a; IL-10, interleukin 10; ASCA, anti-Saccharomyces cerevisiae antibodies; PAB, anti-exocrine pancreas antibodies; SNP, single nucleotide polymorphism. * Corresponding author. Fax: +34 985 106 195. E-mail address: [email protected] (A. Sua ´rez).
Crohn’s disease (CD) is a chronic/relapsing intestinal inflammation causing important alterations in the digestive mucosa [1]. A typical feature in these patients is the development of antibodies against colonist or dietary and self-antigens, the most prevalent and characteristic being anti-Saccharomyces cerevisiae (ASCA) and anti-exocrine pancreas (PAB) antibodies, which are also present at a low frequency in ulcerative colitis patients (UC) [2], another variant of inflammatory bowel disease (IBD). Though the
1521-6616/$ — see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.07.006
306 origin of these antibodies is unknown, both environmental and genetic factors seem to be involved. The genetic influence is supported by the greater incidence of ASCA in patients’ relatives than in normal controls [3]. Among the environmental issues, exposure to neo-antigens as a consequence of intestinal tissue damage is assumed to play a predominant role in ASCA development. Mucosal injury has been associated with alterations in the cytokine pattern in the intestinal epithelium, particularly TNFa and IL-10, two reciprocally regulated cytokines that have opposing roles in triggering and maintaining the inflammatory process [4,5] and tissue wound [6] in CD patients. It has been also hypothesized, although some authors did not agree with this theory [7], that ASCA production is the consequence of increased permeability of the small intestine, a phenomenon observed in Crohn’s disease. This impaired function of the intestinal barrier could result in increased antigen leakage and stimulation of local immune cells with both dietary yeast and yeast species that are either natural or pathological inhabitants of the gastrointestinal tract. It is known that the intestinal mucosa is particularly conductive to induction of helper T cells producing type 2 and type 3 cytokines, an environment that favors IgA isotype antibodies production [8]. Furthermore, IgA-ASCA could also be originated as a consequence of crossed reactions with bacterial antigens from the intestine [9]. Phosphopeptidomannans in the wall of yeast cells have been described as the epitopes responsible for the antigenic reactivity in ASCA+ CD patients’ sera [10]. Mannans are believed to be the major antigenic component of yeast cell walls, but they are also an important antigenic constituent of mycobacteria and other microorganisms [11]. Thus, although the etiology of IgAASCA synthesis is unknown, the presence of intestinal microorganisms may contribute to its origin. Once more, the cytokine network, and particularly IL-10/TNFa balance, plays an essential role in the ability to eliminate pathogens [12]. Genetic polymorphisms at the promoter of the IL-10 and TNFa genes have been associated with different constitutive and induced cytokine productions. In the 308(G/A) TNFa polymorphism, the uncommon TNF2 allele ( 308A*) was associated with high transcriptional activity and carriers of this allele were considered genetically high TNFa producers [13,14] Similarly, IL-10 basal and induced production presented interindividual variations that were genetically regulated by a single nucleotide polymorphism (SNP) at position 1082 (G/A) of the IL-10 promoter and allele 1082G* has been associated with high IL-10 production [15,16]. Although several studies have analyzed the possible involvement of these polymorphic variants in the appearance and outcome of IBD, most authors did not find any significant association with CD susceptibility [17,18]. In a previous study, we also did not find any association between these polymorphisms and CD in our population [19]. The IBD1 susceptibility locus, located on the pericentromeric region of chromosome 16 [20], has received the greatest support in replicate studies on susceptibility to Crohn’s disease. This region overlaps with the CARD15 gene, which encodes a protein mainly expressed in monocytes that has been associated with defense against bacterial infection through peptidoglycan recognition [21,22]. The protein CARD15 presents 10 leucine-rich
P. Castro-Santos et al. repeats (LRRs) involved in the interaction with peptidoglycan. Three mutations in this gene associated with CD susceptibility have been reported: Arg702Trp, Gly908Arg and Leu1007fs [23,24]. Carriers of these variations showed a lower capacity of response to peptidoglycan [25] and a lower ability to eliminate bacterial infection [26], leading to persistent mucosal injury. Several studies have associated carriage of CARD15 mutant alleles with specific clinical characteristics of CD, such as aggressive clinical course [27], early age at onset [28], stricturing phenotype, no colonic involvement [29,30], fibrostenosing disease [31] and delayed progression [32]. High prevalence of ASCA antibodies has been also reported in CD patients with the mutated CARD15 protein [33], although other authors did not find significant differences [32]. It has therefore been suggested that carriers of mutations in the CARD15 gene could represent a clinical subtype within the group of CD patients. In this study, we evaluated the influence of functional polymorphisms at the IL-10 and TNFa genes on the production of antibodies specific to CD patients and their relationship with the presence of mutant alleles at the CARD15 gene.
Subjects and methods Subjects Approval for this study was obtained from the Regional Ethics Committee for Clinical Investigation. The Crohn’s disease (CD) patient population included 146 individuals who were consecutively referred to a central specialized Immunology Unit (Central University Hospital of Asturias) from the Gastroenterology Clinics (San Agustin Hospital of Avile ´s) for immunology evaluation between March 2002 and September 2003. Clinical data were obtained by detailed review of case records. Patient diagnosis and disease extent were established on the basis of clinical, endoscopic, radiological and histological data according to standard criteria [34]. Disease phenotypes were defined following the Vienna Classification [35] for disease location (upper gastrointestinal, ileal, colonic or ileocolonic) and type of disease (penetrating, stricturing or inflammatory). Other parameters studied were: sex, age at diagnosis, familial disease, presence of extraintestinal manifestations (arthritis and/or dermatological involvement), presence of perianal disease (defined by the presence of perianal abscesses, fistulae and/or ulcers) and response to steroid therapy. Steroid dependence was defined as either two successive relapses during steroid tapering or two successive relapses within 2 months after steroid discontinuation. A few patients were steroid-resistant, as manifested by lack of response to steroid treatment. The clinical, serological and demographic characteristics of the CD patients are shown in Table 1. The healthy control population consisted of 343 matched for age and sex unrelated blood donors, all Caucasian in origin, collected between March 2002 and October 2002. Additionally, a group of 99 ulcerative colitis (UC) patients, of the same origin, was studied as the disease control group for antibody determinations. All determinations and genetic analyses were performed with fully
TNFa genotype influences development of Iga-ASCA antibodies Table 1 Crohn’s disease patient characteristics and disease parameters Total CD patients
146
Male/Female Age (years) (median, range) Disease duration (years) (median, range) Age at onset (years) (median, range) Familial history Clinical characteristics Location Ileum Colon Ileum + colon Upper gastrointestinal Disease type Inflammatory Stricturing Penetrating Extraintestinal manifestations: Arthritis Dermatological affection Perianal disease Steroid-dependent Steroid-resistant Serological profile ASCA+ (IgG or IgA) IgG-ASCA+ IgA-ASCA+ PAB+ ANCA+
87/59 42.0 (14—84) 8.5 (1—47) 28.5 (10—80) 28 (19.2)
57 15 70 4
(39.0) (10.3) (47.9) (2.7)
43 49 54 102 90 46 44 80 17
(29.5) (33.6) (37.0) (69.9) (61.6) (31.5) (30.1) (54.8) (11.6)
87 85 55 48 12
(59.6) (58.2) (37.7) (32.9) (8.2)
Values are n or n (%) unless stated otherwise.
informed written consent, while guaranteeing the anonymity of the data.
Detection of antibodies ASCA in both isotypes, IgA and IgG, were examined by ELISA tests (Orgentec Diagnostika GmbH, Mainz, Germany). None of the patients presented a selective total IgA deficit. PAB and ANCA were detected by standard indirect immunofluorescence (IIF). For PAB determination, unfixed frozen sections of human pancreas from a group 0 blood donor were used as substrate. Patients’ sera were initially diluted 1:10 in phosphate-buffered saline (PBS). As reported previously [36], two different patterns against exocrine pancreas were found: reticulogranular and droplet-like patterns. ANCA presence was tested using human ethanol-fixed granulocytes as substrate (Euroimmun, Lqbeck, Germany). For these determinations, patients’ sera were initially diluted 1:20 in PBS. The results were evaluated by two specialists, independently of one another, at a magnification of 400 under a Zeiss epifluorescence microscope.
TNFa and IL-10 promoter polymorphism genotyping DNA was obtained from the peripheral blood cells of 146 CD patients and 343 unrelated local Caucasian healthy blood donors by standard procedures. Single nucleotide poly-
307 morphisms at positions 1082 on the IL-10 gene and 308 on the TNFa gene were determined by analyzing the Tm of the probe/target duplex after PCR amplification and hybridization with fluorescent-labeled probes matched with one sequence variant (LightCycler, Roche Diagnostics, Mannheim, Germany), as previously reported [15]. The primers used were: 5V-ATC CAA GAC AAC ACT ACT AAG GC and 5V-ATG GGG TGG AAG AAG TTG AA for 1082 IL-10 and 5V-CCT GCA TCC TGT CTG GAA GTT A and 5V-CTG CAC CTT CTG TCT CGG TTT for 308 TNFa. The hybridization probes (designed by TIB MOLBIOL, Berlin, Germany) were: GGA TAG GAG GTC CCT TAC TTT CCT CTT ACC-F and LC Red 640-CCC TAC TTC CCC CTC CCA AA for 1082 IL-10 and AAC CCC GTC CCC ATG CCC C-F and LC Red 640-CCA AAC CTA TTG CCT CCA TTT CTT TTG GGG AC for 308 TNFa.
Determination of the Leu1007fs, Arg702Trp and Gly908Arg CARD15 mutations The presence of the three major variants of the CARD15 gene was determined in 178 healthy controls and 132 CD patients. Determination of the Leu1007fs variant was assessed by PCR amplification and hybridization with fluorescent-labeled allele-specific probes (LightCycler system) using the amplification primers 5V-TCT TCT TTT CCA GGT TGT CCA A-3V and 5V-TGA GGT TCG GAG AGC TAA AAC AG-3V and the hybridization probes AGG CCC CTT GAA AGG AAT GAC-F and LC Red640-CCA TCC TGG AAG TCT GGT AAG GCC [37]. The Arg702Trp and Gly908Arg CARD15 variants were genotyped by PCR-restriction digestion using the primers 5V-CGC ACA ACC TTC AGA TCA CA-3V and 5V-GGA TGG AGT GGA AGT GCT TG-3V for Arg702Trp and 5V-AGG CCA CTC TGG GAT TGA G-3Vand 5V-GTG ATC ACC CAA GGC TTC AG-3Vfor Gly908Arg and the restriction enzymes MspI for Arg702Trp and CfoI for Gly908Arg [38,39]. Following overnight digestion, PCR products were separated and visualized by ethidium bromide-stained agarose gel electrophoresis.
Statistical analysis The SPSS 11.5 statistical software package (SPSS Inc., Chicago; IL) was used for all calculations. Allelic and genotype frequencies were obtained by direct counting. A binary logistic regression was used to determine the influence of the functional genotypes (associated with low or high protein production) in the development of ASCA (IgG or IgA) and PAB in CD patients. A 2 2 contingence table, the m 2 test or the two-tailed Fisher’s Exact Test, when the number of expected cases was too low, were used to compare CARD15 mutation carriership frequency between the patient and control groups. Univariate and multivariate analysis were performed by unconditional logistic regression modeling in order to define the impact of a specific functional cytokine genotype on ASCA or PAB production (dependent variable). Covariates for the multivariate analyses included: sex, age and the clinical parameters duration of the disease, age at onset, location and type of disease, presence of extraintestinal manifestations, perianal disease and steroid dependence. Odds ratios (OR) and 95% confidence intervals
308 Table 2
P. Castro-Santos et al. TNFa and IL-10 genotypes of healthy controls and CD patients stratified by serological profile 308 TNFa
Healthy controls CD patients OR (95% CI) Serological profile IgA-ASCA IgA-ASCA+ OR (95% CI) IgG-ASCA IgG-ASCA+ OR (95% CI) PAB PAB+ OR (95% CI)
1082 IL-10
GG (low)
AG/AA (high)
265 (77.3) 108 (74.0) 1.20 (0.76 to 1.87)
78 (22.7) 38 (26.0)
62 46 0.42 42 66 0.64 77 31 2.01
29 (31.8) 9 (16.4)
(68.1) (83.6) (0.18 to 0.97) (68.8) (77.6) (0.30 to 1.34) (78.6) (64.5) (0.94 to 4.31)
p
AA (low)
GG/AG (high) 209 (60.9) 82 (56.2)
0.435
134 (39.1) 64 (43.8) 1.21 (0.82 to 1.80)
0.042 19 (31.1) 19 (22.4) 0.234 21 (21.4) 17 (35.5) 0.073
37 27 1.41 23 41 1.54 40 24 1.45
p
0.326
(40.7) (49.1) (0.72 to 2.76) (37.7) (48.2) (0.79 to 3.01) (40.8) (50.0) (0.72 to 2.90)
54 (59.3) 28 (50.9) 0.320 38 (62.3) 44 (51.8) 0.207 58 (59.2) 24 (50.0) 0.294
Values are n (%). Differences were evaluated by binary logistic regression.
(95% CI) were used as an estimate of risk in all cases. The level of significance was set at p b 0.05.
Results The high TNFA producer genotype associated with the low prevalence of IgA-ASCA antibodies in CD patients It has been previously reported that ASCA and PAB antibodies might be used as serological markers for CD, although they are also present in some UC patients. In our population, 59.6% of CD patients were positive for ASCA (IgG or IgA), whereas separate isotype analysis showed the presence of IgG-ASCA in 58.2% and of IgA-ASCA in 37.7% of CD patients. By means of IFI assays in human pancreas, we detected the presence of PAB in 32.9% of CD patients and in human ethanol-fixed granulocytes 8.2% of patients were positive for ANCA (Table 1). The prevalence of these antibodies in the ulcerative colitis control population (n = 99) was: 13.1% IgG-ASCA+, 1.0% IgA-ASCA+, 5.1% PAB+ and 45.5% ANCA+, indicating that IgA-ASCA and PAB are the most specific serological markers for CD. We consequently wished to evaluate the possible involvement of functional IL-10 and TNFa genetic polymorphisms in their production. Accordingly, after determination of the alleles present at the 1082 IL-10 and 308 TNFa gene promoters, we classified patients and controls in genetically high (AA/AG) or low (GG) TNFa producers and high (GG/AG) or low (AA) IL-10 producers (Table 2). As we have previously reported [19], no significant differences in allele or genotype frequencies
Table 3
were detected between CD patients and healthy controls in our population. However, when patients were stratified in accordance with their serological profile, slight differences in genotype distribution were detected. The high TNFa producer genotype was significantly underrepresented in the IgA-ASCA+ patient group when compared to patients lacking this serological marker (16.4% vs 31.8%; OR = 0.42; 95% CI: 0.18 to 0.97; p = 0.042), while IgG-ASCA did not show any significant association. In contrast, a clear trend towards an increased prevalence of high TNFa producers was observed among PAB+ patients (35.5% vs 21.4%; OR = 2.01, 95% CI = 0.94 to 4.31; p = 0.073). No association was found between 1082 IL-10 promoter polymorphisms and the development of either ASCA or PAB. These results suggest a possible involvement of functional TNFa genotypes in the specific antibodies production that was different for exogenous (S. cerevisiae) and self- (exocrine pancreas) antigens.
The association between antibody synthesis and TNFA genotype is not present in carriers of CARD15 risk alleles The Arg702Trp, Gly908Arg and Leu1007fs CARD15 variations have been strongly associated with CD development. We wanted to know whether or not the slight influence of cytokine polymorphisms previously detected on the development of specific antibodies occurred both in patients with and without CARD15 risk alleles. To do so, we first determined the prevalence of the Arg702Trp, Gly908Arg and Leu1007fs CARD15 mutations in our population of CD
Frequencies of the Arg702Trp, Gly908Arg and Leu1007fs CARD15 genetic variants in healthy controls and CD patients
CARD15 carriership
Controls (n = 178)
CD patients (n = 132)
OR (95% CI)
Arg702Trp Gly908Arg Leu1007fs At least one variant
7 3 6 14
22 6 16 39
4.98 2.78 3.94 4.8
(3.9) (1.7) (3.2) (8.0)
(16.6) (4.5) (12.1) (29.5)
Values are n (%). Differences were evaluated by the m 2 test and estimation of risk.
(2.06 (0.68 (1.50 (2.49
to to to to
p 12.05) 11.32) 10.40) 9.35)
b 0.0001 0.177 0.003 b 0.00001
TNFa genotype influences development of Iga-ASCA antibodies Table 4
309
TNFa and IL-10 genotype distribution of CD patients without CARD15 mutations and with different serological markers ASCA IgA
ASCA IgA+
308 TNFa GG (low) AA/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
40 19 0.20 0.12
(67.8) (32.2) (0.05 to 0.73) (0.02 to 0.79)
31 (91.2) 3 (8.8)
1082 IL-10 AA (low) GG/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
22 37 1.68 1.40
(37.3) (62.7) (0.72 to 3.95) (0.51 to 3.88)
17 (50.0) 17 (50.0)
p
PAB
PAB+ (82.0) (18.0) (0.09 to 6.34) (1.31 to 17.83)
21 (65.6) 11 (34.4)
0.009 0.011
50 11 2.38 4.82
(36.1) (63.9) (0.84 to 4.80) (0.98 to 8.78)
17 (53.1) 15 (46.9)
0.232 0.511
22 39 2.01 2.94
p
0.078 0.018
0.113 0.054
Values are n (%).Unconditional logistic regression model using the presence of antibody as dependent variable and the presence of the low producer genotype as reference. a Adjusted for sex, age and the clinical parameters: disease duration, age at onset, location, disease type, extraintestinal manifestations, steroid dependence, steroid resistance and perianal disease.
patients and healthy controls. As expected, we found a significantly higher frequency in CD patients with respect to controls of the Arg702Trp allele (0.096 vs 0.020; OR = 5.30; 95% CI: 2.26 to 12.46; p b 0.0001) and the Leu1007fs allele (0.055 vs 0.016; OR = 3.56; 95% CI: 1.38 to 9.22; p = 0.005). Thus, the percentage of carriers of these alleles in controls and patients also showed significant differences (Arg702Trp 3.9 vs 16.6, p b 0.0001 and Leu1007fs 3.2 vs 12.1, p = 0.003) (Table 3). Gly908Arg allele frequency was also increased in the CD population compared to controls (0.023 vs 0.008), although the differences were not significant, probably due to the low frequency of this mutation in our population. Thus, 29.5% of CD patients (39 of 132) and 8.0% of controls (14 of 175) presented at least one CARD15 mutant allele, the strength of the association being very highly significant (OR = 4.8; 95% CI: 2.49 to 9.35, p b 0.00001). CD patients were subsequently classified into two groups according to the presence or absence of at least one CARD15 risk allele, and genotype frequencies of the TNFa and IL-10 polymorphisms were compared between
patients found to be positive and negative for IgA-ASCA, IgG-ASCA and PAB serological markers. To exclude skewed stratification resulting from the division of the patient’s cohort into two smaller cohorts, we tested the independence between TNFa and IL-10 genotypes and the CARD15 variations (TNFa/CARD15 p = 0.582; IL-10/CARD15 p = 0.923, m 2 test). We found that the proposed protective role of the high TNFa producer genotype on IgA-ASCA production was highly noteworthy in the group of patients with the CARD15 wild genotype (OR = 0.20, 95% CI = 0.05 to 0.73, p = 0.009) (Table 4). This association was maintained in the multivariate analysis after adjusting for clinical and serological parameters (OR = 0.12, 95%IC: 0.02 to 0.79; p = 0.011). However, this association did not appear in the patients’ group with at least one CARD15 mutant allele (Table 5), indicating the absence of the influence of TNFa on IgAASCA development in patient carriers of CARD15 mutations. Similarly, the association trend found previously in the total CD population between high TNFa producer genotypes and the presence of PAB was also observed in the
Table 5 TNFa and IL-10 genotype distribution of CD patients with at least 1 CARD15 mutant allele and with different serological markers ASCA IgA
ASCA IgA+
308 TNFa GG (low) AA/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
17 6 1.28 0.81
(73.9) (26.1) (0.31 to 5.27) (0.08 to 8.06)
11 (68.7) 5 (31.3)
1082 IL-10 AA (low) GG/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
9 14 1.21 1.46
(39.1) (60.9) (0.33 to 4.42) (0.17 to 12.07)
7 (43.8) 9 (56.2)
p
PAB
PAB+ (78.3) (21.7) (0.52 to 8.90) (0.99 to 7.92)
10 (62.5) 6 (37.5)
0.734 0.856
18 5 2.16 0.91
(39.1) (60.9) (0.33 to 4.42) (0.10 to 2.66)
7 (43.8) 9 (56.2)
0.773 0.726
9 14 1.21 0.17
p
0.307 0.884
0.773 0.204
Values are n (%).Unconditional logistic regression model using the presence of antibody as dependent variable and the presence of the low producer genotype as reference. a Adjusted for sex, age and the clinical parameters: disease duration, age at onset, location, disease type, extraintestinal manifestations, steroid dependence, steroid resistance and perianal disease.
310 group of CARD15 wild genotype patient group, arising statistical significance at multivariate analysis (OR = 4.82; 95% CI = 1.31 to 17.83; p = 0.018) (Table 4); however, again this association did not appear in carriers of one or more of the CARD15 variations (Table 5). Polymorphisms at the IL10 gene did not influence antibody production in either mutated or CARD15 wild type patients. Taken as a whole, these results suggest an involvement of the interaction CARD15—TNFa genotype in the specific antibodies production in CD patients. Finally, analysis of the clinical features of patients without CARD15 mutated alleles suggested that the joint presence of IgA-ASCA and the low TNFa producer genotype could be indicative of a more severe disease as this patient group (n = 31) was characterized by a higher frequency of surgery (51.6% vs 27.4%, p = 0.022), stricturing/penetrating disease (83.9% vs 62.9%, p = 0.038) and ileal involvement (100% vs 85.5% p = 0.026) when compared with the remainder of CARD15 wild type patients (n = 62). A very low prevalence of PAB+ was also characteristic of these patients (13.1% vs 43.5%, p = 0.009). Interestingly, among carriers of the CARD15 mutations, any clinical or serological feature was overrepresented in patients with these serological and genetic markers (IgA-ASCA+ and low TNFa genotype), supporting the role of TNFa in disease outcome of CD patients without CARD15 mutations.
Discussion In this study, we have found a relationship between functional polymorphisms at the TNFA gene and the presence of anti-S. cerevisiae antibodies of the IgA isotype (IgA-ASCA) and anti-exocrine pancreas antigen antibodies (PAB) in Crohn’s disease patients. Specifically, carriage of the rare high TNFa producer allele at the 308 position (TNF2) protects against the generation of IgA-ASCA, whereas it increases the risk of PAB production, suggesting a different effect of TNFa on the production of antibodies against self- and exogenous antigens. High TNFa producer genotypes not only are a risk factor for the appearance of several autoimmune diseases [40,41], but also they have been previously associated with increased production of antibodies against self-antigens such as ANCA in ulcerative colitis [42,43], SSa and SSb in systemic lupus erythematosus [40] or SSb in Sjfgren syndrome [44]. However, to the best of our knowledge, this is the first study reporting an association between the 308GG TNFa low producer genotype and the generation of antibodies against dietary or colonist microorganisms. The elevated specificity of IgA-ASCA found in our work supports the relevance of GALT (gut-associated lymphoid tissues) immune responses to exogenous antigens in CD patients. A higher discriminating value between CD and other bowel diseases has been previously described for IgA when compared to IgG responses [45,46]. It is know that most of the plasma cells in the intestinal tract produce IgA, indicating an individual cytokine profile that promoted this type of response. Additionally, it has been reported that IgA production is dependent on microorganism presence since germfree animals lack intestinal IgA [47]. Thus, mucosal specific response against intestinal colonists is probably mediated through IgA production. IgG isotype ASCA, how-
P. Castro-Santos et al. ever, are most likely produced by plasma cells generated in lymphoid follicles away from the Peyer’s patches, where environmental conditions and cytokine network are different from the intestinal mucosa. Given the genetic origin proposed for ASCA [48], association with TNFa genotype might be explained by linkage disequilibrium with a gene related to ASCA expression located on chromosome 6, in the vicinity of the major histocompatibility complex. However, this is not the case since the association between TNFa genotype and antibody production disappeared completely in patients expressing a CARD15 mutated protein. The CARD15 gene encodes a molecule involved in bacterial recognition and the subsequent proinflammatory response through NF-nB activation. Presence of Arg702Trp, Gly908Arg or Leu1007fs CARD15 mutations modifies the structure of the leucine-rich repeat domain of the protein, determining a different degree of loss of function. These three major variants have been associated with a poor response to microorganism infection [21] and with an inability to eliminate pathogens [26] especially in the intestine [49]. Therefore, the lack of recognition of bacterial peptidoglycan caused by these CARD15 variations decreased NF-nB activation [25] and the subsequent TNFa release [50], which is known to be dependent on promoter polymorphisms. Thus, among carriers of CARD15 mutations, differences between genetically high and low TNFa producers in response to bacterial stimulation could be unapparent, explaining the lack of association between TNFa genotypes and clinical or serological parameters. However, in the absence of an adequate TNFa response, other mechanisms might be activated to eliminate bacterial infection. Hence, the higher incidence of ASCA among CARD15 mutated CD patients previously reported by several authors [51] might be explained by the low TNFa production in response to bacterial stimulation. In our CARD15 mutated CD group, an increased prevalence of IgA-ASCA was also found, although this was not statistically significant, probably due to the low percentage of carriers of CARD15 variations (29.5%) and the relatively low prevalence of IgA-ASCA+ (37.7%). TNFa is a proinflammatory and a proapoptotic cytokine mainly secreted by monocytes/macrophages during innate immune responses and by effector Th1 cells. Interestingly, low levels of TNFa and carriage of the 308GG genotype have been widely related to less efficient elimination of pathogens and to tuberculosis [52] and leprosy [53]. The immunoregulatory function of TNFa in the intestinal mucosa of CD patients could explain the association found between CARD15—TNFa genotype and IgA-ASCA production. Fig. 1 summarizes the putative interactions we propose at the cellular and molecular level. CARD15 receptor, usually highly expressed in monocytes, macrophages and epithelial and Paneth cells from CD patients, recognizes bacteria that penetrated the mucosa as a result of breaks in the integrity of the epithelium, leading to NF-nB activation and TNFa production. Thus, in genetically high TNFa producers, the elevated TNFa levels may be able to reduce the generation of specific immune responses against bacteria and yeast, dietary or intestinal colonist, either by the activation of neutrophil phagocytosis and the subsequent clearance of microorganisms and antigenic products or by compromising antigen uptake and signaling by leukocytes that interfaced
TNFa genotype influences development of Iga-ASCA antibodies
311
Figure 1 Proposed involvement of CARD15—TNFa circuit in IgA-ASCA production. Breaks in the integrity of the mucosal epithelium allow the internalization of exogenous antigens (from dietary or colonist bacteria and yeast). Interaction of bacterial peptidoglycan with CARD15 receptor leads to NF-nB activation and TNFa transcription at high or low levels depending on promoter genotype. High TNFa levels reduce the generation of adaptive immune responses against exogenous antigens by the activation of neutrophil phagocytosis and the subsequent clearance of microorganisms and antigenic products and/or by compromising antigen uptake and signaling by leukocytes that interfaced with intestinal antigens. Lamina propia dendritic cells (DC) uptake exogenous antigens and migrate into lymph follicles where T and B cells were activated and initiate a specific immune response. Humoral immune responses at the intestinal mucosa (Peyer’s patches) are particularly conductive to the induction of IgA, probably, under the influence of Th2 and Th3 cytokines. Part of the lamina propia DC migrate to mesenteric lymph nodes where, in the absence of mucosal environment, humoral immune response leads to IgG production.
with intestinal antigens. On the contrary, low TNFa producers might present an impaired capacity to efficiently eliminate microorganisms that enter the mucosal epithelium. It allows dendritic cells of the lamina propia to uptake exogenous antigens and migrate into local lymph follicles, where T and B lymphocytes are activated and IgA-ASCA generation takes place, under the influence of mucosal cytokines. Moreover, since Ig class switch depends on environmental cytokines, diminished Th1 cell-mediated immune responses, as a consequence of low TNFa production, may predispose to the generation of a predominant Th2/Th3 profile in the intestinal mucosa, favoring the generation of IgA antibodies to yeast antigens. On the other hand, part of the lamina propia dendritic cells, loaded with exogenous Ag, migrate into lymph nodes away to the GALT, where they initiate an immune response that might lead, in the absence of the cytokine mucosal environment, to the generation of plasma cells producers of IgG [54].
On the contrary to IgA-ASCA development, the generation of PAB seems to be promoted by high TNFa production. This is in accordance to previously reported associations between ANCA and other autoantibodies synthesis and the TNF2 allele [42—44]. It suggests that autoantibodies production might be the result of autoantigen exposure due to chronic inflammation and increased apoptosis. Furthermore, it has been reported that patients with a combination of positive pANCA and negative ASCA, serological markers that were suggestive of high TNFa levels, showed the least favorable response to anti-TNFa therapy (infliximab). Many studies have examined TNFa gene polymorphisms as possible predictors for the response to infliximab treatment. Several of these studies showed a slightly higher frequency of the rare TNF2 allele among non-responders. However, despite the large number of works, results were inconclusive [55,56]. According to our results, the reason of this controversy may be the lower
312 influence of the TNFa genotype on the intestinal mucosa TNFa levels among patient carriers of CARD15 mutated alleles. Thus, relationship between the lack of response to anti-TNFa therapy and absence of ASCA, associated with high TNFa production, could implicate that TNFa may not play a pathological role in ASCA negative patients. Consequently, studies on the predictor value of genetic and serological markers on the response to TNFa blocking agents should be performed separately for CD patients carrying either the mutant or the wild type CARD15 gene. In summary, our results indicate that the TNFa genotype influences the development of antibodies in CD patients without CARD15 mutations, supporting the role of TNFa in the surveillance of immune interface with intestinal colonist. Moreover, the CARD15—TNFa circuit proposed by this study is likely to play a significant role in mucosal immune regulation and when perturbed may affect disease outcome. Consequently, presence of CARD15 variations should be taken into account to classify CD patients in order to study pathological mechanisms.
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TNFA genotype influences development of IgA-ASCA antibodies in Crohn’s disease patients with CARD15 wild type Patricia Castro-Santos a, Lourdes Mozo a, Carmen Gutie ´rrez a,b, Ana Sua ´rez b,* a
Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain Department of Functional Biology, Immunology Area, University of Oviedo, C/Julia´n Claverı´a s/n, 33006 Oviedo, Spain
b
Received 11 April 2006; accepted with revision 18 July 2006 Available online 6 September 2006
KEYWORDS Inflammatory bowel disease; CD; Cytokine polymorphisms; ASCA; PAB; TNFa; IL-10; CARD15
Abstract A typical feature of Crohn’s disease (CD) patients is the development of antibodies against self- (PAB) or exogenous (ASCA) antigens, a process in which mucosal cytokine expression pattern might be involved. On the other hand, mutations in CARD15, a genetic risk factor for CD, alter cytokine production in response to bacterial infection. In the present study, we evaluated the role of functionally relevant IL-10 and TNFa gene polymorphisms in the synthesis of these antibodies and their relationship with CARD15 mutations. In CARD15 wild type patients, high TNFa producer genotypes protect against IgA-ASCA development, whereas an inverse association was observed in autoantibody synthesis (PAB). These associations were not observed in patients with CARD15 mutations, probably due to the lack of TNFa release as a consequence of the failure of CARD15 protein to recognize the peptidoglycan. Thus, we proposed a CARD15— TNFa circuit that might play a role in mucosal immune surveillance. D 2006 Elsevier Inc. All rights reserved.
Introduction
Abbreviations: IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; TNFa, tumor necrosis factor a; IL-10, interleukin 10; ASCA, anti-Saccharomyces cerevisiae antibodies; PAB, anti-exocrine pancreas antibodies; SNP, single nucleotide polymorphism. * Corresponding author. Fax: +34 985 106 195. E-mail address: [email protected] (A. Sua ´rez).
Crohn’s disease (CD) is a chronic/relapsing intestinal inflammation causing important alterations in the digestive mucosa [1]. A typical feature in these patients is the development of antibodies against colonist or dietary and self-antigens, the most prevalent and characteristic being anti-Saccharomyces cerevisiae (ASCA) and anti-exocrine pancreas (PAB) antibodies, which are also present at a low frequency in ulcerative colitis patients (UC) [2], another variant of inflammatory bowel disease (IBD). Though the
1521-6616/$ — see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.07.006
306 origin of these antibodies is unknown, both environmental and genetic factors seem to be involved. The genetic influence is supported by the greater incidence of ASCA in patients’ relatives than in normal controls [3]. Among the environmental issues, exposure to neo-antigens as a consequence of intestinal tissue damage is assumed to play a predominant role in ASCA development. Mucosal injury has been associated with alterations in the cytokine pattern in the intestinal epithelium, particularly TNFa and IL-10, two reciprocally regulated cytokines that have opposing roles in triggering and maintaining the inflammatory process [4,5] and tissue wound [6] in CD patients. It has been also hypothesized, although some authors did not agree with this theory [7], that ASCA production is the consequence of increased permeability of the small intestine, a phenomenon observed in Crohn’s disease. This impaired function of the intestinal barrier could result in increased antigen leakage and stimulation of local immune cells with both dietary yeast and yeast species that are either natural or pathological inhabitants of the gastrointestinal tract. It is known that the intestinal mucosa is particularly conductive to induction of helper T cells producing type 2 and type 3 cytokines, an environment that favors IgA isotype antibodies production [8]. Furthermore, IgA-ASCA could also be originated as a consequence of crossed reactions with bacterial antigens from the intestine [9]. Phosphopeptidomannans in the wall of yeast cells have been described as the epitopes responsible for the antigenic reactivity in ASCA+ CD patients’ sera [10]. Mannans are believed to be the major antigenic component of yeast cell walls, but they are also an important antigenic constituent of mycobacteria and other microorganisms [11]. Thus, although the etiology of IgAASCA synthesis is unknown, the presence of intestinal microorganisms may contribute to its origin. Once more, the cytokine network, and particularly IL-10/TNFa balance, plays an essential role in the ability to eliminate pathogens [12]. Genetic polymorphisms at the promoter of the IL-10 and TNFa genes have been associated with different constitutive and induced cytokine productions. In the 308(G/A) TNFa polymorphism, the uncommon TNF2 allele ( 308A*) was associated with high transcriptional activity and carriers of this allele were considered genetically high TNFa producers [13,14] Similarly, IL-10 basal and induced production presented interindividual variations that were genetically regulated by a single nucleotide polymorphism (SNP) at position 1082 (G/A) of the IL-10 promoter and allele 1082G* has been associated with high IL-10 production [15,16]. Although several studies have analyzed the possible involvement of these polymorphic variants in the appearance and outcome of IBD, most authors did not find any significant association with CD susceptibility [17,18]. In a previous study, we also did not find any association between these polymorphisms and CD in our population [19]. The IBD1 susceptibility locus, located on the pericentromeric region of chromosome 16 [20], has received the greatest support in replicate studies on susceptibility to Crohn’s disease. This region overlaps with the CARD15 gene, which encodes a protein mainly expressed in monocytes that has been associated with defense against bacterial infection through peptidoglycan recognition [21,22]. The protein CARD15 presents 10 leucine-rich
P. Castro-Santos et al. repeats (LRRs) involved in the interaction with peptidoglycan. Three mutations in this gene associated with CD susceptibility have been reported: Arg702Trp, Gly908Arg and Leu1007fs [23,24]. Carriers of these variations showed a lower capacity of response to peptidoglycan [25] and a lower ability to eliminate bacterial infection [26], leading to persistent mucosal injury. Several studies have associated carriage of CARD15 mutant alleles with specific clinical characteristics of CD, such as aggressive clinical course [27], early age at onset [28], stricturing phenotype, no colonic involvement [29,30], fibrostenosing disease [31] and delayed progression [32]. High prevalence of ASCA antibodies has been also reported in CD patients with the mutated CARD15 protein [33], although other authors did not find significant differences [32]. It has therefore been suggested that carriers of mutations in the CARD15 gene could represent a clinical subtype within the group of CD patients. In this study, we evaluated the influence of functional polymorphisms at the IL-10 and TNFa genes on the production of antibodies specific to CD patients and their relationship with the presence of mutant alleles at the CARD15 gene.
Subjects and methods Subjects Approval for this study was obtained from the Regional Ethics Committee for Clinical Investigation. The Crohn’s disease (CD) patient population included 146 individuals who were consecutively referred to a central specialized Immunology Unit (Central University Hospital of Asturias) from the Gastroenterology Clinics (San Agustin Hospital of Avile ´s) for immunology evaluation between March 2002 and September 2003. Clinical data were obtained by detailed review of case records. Patient diagnosis and disease extent were established on the basis of clinical, endoscopic, radiological and histological data according to standard criteria [34]. Disease phenotypes were defined following the Vienna Classification [35] for disease location (upper gastrointestinal, ileal, colonic or ileocolonic) and type of disease (penetrating, stricturing or inflammatory). Other parameters studied were: sex, age at diagnosis, familial disease, presence of extraintestinal manifestations (arthritis and/or dermatological involvement), presence of perianal disease (defined by the presence of perianal abscesses, fistulae and/or ulcers) and response to steroid therapy. Steroid dependence was defined as either two successive relapses during steroid tapering or two successive relapses within 2 months after steroid discontinuation. A few patients were steroid-resistant, as manifested by lack of response to steroid treatment. The clinical, serological and demographic characteristics of the CD patients are shown in Table 1. The healthy control population consisted of 343 matched for age and sex unrelated blood donors, all Caucasian in origin, collected between March 2002 and October 2002. Additionally, a group of 99 ulcerative colitis (UC) patients, of the same origin, was studied as the disease control group for antibody determinations. All determinations and genetic analyses were performed with fully
TNFa genotype influences development of Iga-ASCA antibodies Table 1 Crohn’s disease patient characteristics and disease parameters Total CD patients
146
Male/Female Age (years) (median, range) Disease duration (years) (median, range) Age at onset (years) (median, range) Familial history Clinical characteristics Location Ileum Colon Ileum + colon Upper gastrointestinal Disease type Inflammatory Stricturing Penetrating Extraintestinal manifestations: Arthritis Dermatological affection Perianal disease Steroid-dependent Steroid-resistant Serological profile ASCA+ (IgG or IgA) IgG-ASCA+ IgA-ASCA+ PAB+ ANCA+
87/59 42.0 (14—84) 8.5 (1—47) 28.5 (10—80) 28 (19.2)
57 15 70 4
(39.0) (10.3) (47.9) (2.7)
43 49 54 102 90 46 44 80 17
(29.5) (33.6) (37.0) (69.9) (61.6) (31.5) (30.1) (54.8) (11.6)
87 85 55 48 12
(59.6) (58.2) (37.7) (32.9) (8.2)
Values are n or n (%) unless stated otherwise.
informed written consent, while guaranteeing the anonymity of the data.
Detection of antibodies ASCA in both isotypes, IgA and IgG, were examined by ELISA tests (Orgentec Diagnostika GmbH, Mainz, Germany). None of the patients presented a selective total IgA deficit. PAB and ANCA were detected by standard indirect immunofluorescence (IIF). For PAB determination, unfixed frozen sections of human pancreas from a group 0 blood donor were used as substrate. Patients’ sera were initially diluted 1:10 in phosphate-buffered saline (PBS). As reported previously [36], two different patterns against exocrine pancreas were found: reticulogranular and droplet-like patterns. ANCA presence was tested using human ethanol-fixed granulocytes as substrate (Euroimmun, Lqbeck, Germany). For these determinations, patients’ sera were initially diluted 1:20 in PBS. The results were evaluated by two specialists, independently of one another, at a magnification of 400 under a Zeiss epifluorescence microscope.
TNFa and IL-10 promoter polymorphism genotyping DNA was obtained from the peripheral blood cells of 146 CD patients and 343 unrelated local Caucasian healthy blood donors by standard procedures. Single nucleotide poly-
307 morphisms at positions 1082 on the IL-10 gene and 308 on the TNFa gene were determined by analyzing the Tm of the probe/target duplex after PCR amplification and hybridization with fluorescent-labeled probes matched with one sequence variant (LightCycler, Roche Diagnostics, Mannheim, Germany), as previously reported [15]. The primers used were: 5V-ATC CAA GAC AAC ACT ACT AAG GC and 5V-ATG GGG TGG AAG AAG TTG AA for 1082 IL-10 and 5V-CCT GCA TCC TGT CTG GAA GTT A and 5V-CTG CAC CTT CTG TCT CGG TTT for 308 TNFa. The hybridization probes (designed by TIB MOLBIOL, Berlin, Germany) were: GGA TAG GAG GTC CCT TAC TTT CCT CTT ACC-F and LC Red 640-CCC TAC TTC CCC CTC CCA AA for 1082 IL-10 and AAC CCC GTC CCC ATG CCC C-F and LC Red 640-CCA AAC CTA TTG CCT CCA TTT CTT TTG GGG AC for 308 TNFa.
Determination of the Leu1007fs, Arg702Trp and Gly908Arg CARD15 mutations The presence of the three major variants of the CARD15 gene was determined in 178 healthy controls and 132 CD patients. Determination of the Leu1007fs variant was assessed by PCR amplification and hybridization with fluorescent-labeled allele-specific probes (LightCycler system) using the amplification primers 5V-TCT TCT TTT CCA GGT TGT CCA A-3V and 5V-TGA GGT TCG GAG AGC TAA AAC AG-3V and the hybridization probes AGG CCC CTT GAA AGG AAT GAC-F and LC Red640-CCA TCC TGG AAG TCT GGT AAG GCC [37]. The Arg702Trp and Gly908Arg CARD15 variants were genotyped by PCR-restriction digestion using the primers 5V-CGC ACA ACC TTC AGA TCA CA-3V and 5V-GGA TGG AGT GGA AGT GCT TG-3V for Arg702Trp and 5V-AGG CCA CTC TGG GAT TGA G-3Vand 5V-GTG ATC ACC CAA GGC TTC AG-3Vfor Gly908Arg and the restriction enzymes MspI for Arg702Trp and CfoI for Gly908Arg [38,39]. Following overnight digestion, PCR products were separated and visualized by ethidium bromide-stained agarose gel electrophoresis.
Statistical analysis The SPSS 11.5 statistical software package (SPSS Inc., Chicago; IL) was used for all calculations. Allelic and genotype frequencies were obtained by direct counting. A binary logistic regression was used to determine the influence of the functional genotypes (associated with low or high protein production) in the development of ASCA (IgG or IgA) and PAB in CD patients. A 2 2 contingence table, the m 2 test or the two-tailed Fisher’s Exact Test, when the number of expected cases was too low, were used to compare CARD15 mutation carriership frequency between the patient and control groups. Univariate and multivariate analysis were performed by unconditional logistic regression modeling in order to define the impact of a specific functional cytokine genotype on ASCA or PAB production (dependent variable). Covariates for the multivariate analyses included: sex, age and the clinical parameters duration of the disease, age at onset, location and type of disease, presence of extraintestinal manifestations, perianal disease and steroid dependence. Odds ratios (OR) and 95% confidence intervals
308 Table 2
P. Castro-Santos et al. TNFa and IL-10 genotypes of healthy controls and CD patients stratified by serological profile 308 TNFa
Healthy controls CD patients OR (95% CI) Serological profile IgA-ASCA IgA-ASCA+ OR (95% CI) IgG-ASCA IgG-ASCA+ OR (95% CI) PAB PAB+ OR (95% CI)
1082 IL-10
GG (low)
AG/AA (high)
265 (77.3) 108 (74.0) 1.20 (0.76 to 1.87)
78 (22.7) 38 (26.0)
62 46 0.42 42 66 0.64 77 31 2.01
29 (31.8) 9 (16.4)
(68.1) (83.6) (0.18 to 0.97) (68.8) (77.6) (0.30 to 1.34) (78.6) (64.5) (0.94 to 4.31)
p
AA (low)
GG/AG (high) 209 (60.9) 82 (56.2)
0.435
134 (39.1) 64 (43.8) 1.21 (0.82 to 1.80)
0.042 19 (31.1) 19 (22.4) 0.234 21 (21.4) 17 (35.5) 0.073
37 27 1.41 23 41 1.54 40 24 1.45
p
0.326
(40.7) (49.1) (0.72 to 2.76) (37.7) (48.2) (0.79 to 3.01) (40.8) (50.0) (0.72 to 2.90)
54 (59.3) 28 (50.9) 0.320 38 (62.3) 44 (51.8) 0.207 58 (59.2) 24 (50.0) 0.294
Values are n (%). Differences were evaluated by binary logistic regression.
(95% CI) were used as an estimate of risk in all cases. The level of significance was set at p b 0.05.
Results The high TNFA producer genotype associated with the low prevalence of IgA-ASCA antibodies in CD patients It has been previously reported that ASCA and PAB antibodies might be used as serological markers for CD, although they are also present in some UC patients. In our population, 59.6% of CD patients were positive for ASCA (IgG or IgA), whereas separate isotype analysis showed the presence of IgG-ASCA in 58.2% and of IgA-ASCA in 37.7% of CD patients. By means of IFI assays in human pancreas, we detected the presence of PAB in 32.9% of CD patients and in human ethanol-fixed granulocytes 8.2% of patients were positive for ANCA (Table 1). The prevalence of these antibodies in the ulcerative colitis control population (n = 99) was: 13.1% IgG-ASCA+, 1.0% IgA-ASCA+, 5.1% PAB+ and 45.5% ANCA+, indicating that IgA-ASCA and PAB are the most specific serological markers for CD. We consequently wished to evaluate the possible involvement of functional IL-10 and TNFa genetic polymorphisms in their production. Accordingly, after determination of the alleles present at the 1082 IL-10 and 308 TNFa gene promoters, we classified patients and controls in genetically high (AA/AG) or low (GG) TNFa producers and high (GG/AG) or low (AA) IL-10 producers (Table 2). As we have previously reported [19], no significant differences in allele or genotype frequencies
Table 3
were detected between CD patients and healthy controls in our population. However, when patients were stratified in accordance with their serological profile, slight differences in genotype distribution were detected. The high TNFa producer genotype was significantly underrepresented in the IgA-ASCA+ patient group when compared to patients lacking this serological marker (16.4% vs 31.8%; OR = 0.42; 95% CI: 0.18 to 0.97; p = 0.042), while IgG-ASCA did not show any significant association. In contrast, a clear trend towards an increased prevalence of high TNFa producers was observed among PAB+ patients (35.5% vs 21.4%; OR = 2.01, 95% CI = 0.94 to 4.31; p = 0.073). No association was found between 1082 IL-10 promoter polymorphisms and the development of either ASCA or PAB. These results suggest a possible involvement of functional TNFa genotypes in the specific antibodies production that was different for exogenous (S. cerevisiae) and self- (exocrine pancreas) antigens.
The association between antibody synthesis and TNFA genotype is not present in carriers of CARD15 risk alleles The Arg702Trp, Gly908Arg and Leu1007fs CARD15 variations have been strongly associated with CD development. We wanted to know whether or not the slight influence of cytokine polymorphisms previously detected on the development of specific antibodies occurred both in patients with and without CARD15 risk alleles. To do so, we first determined the prevalence of the Arg702Trp, Gly908Arg and Leu1007fs CARD15 mutations in our population of CD
Frequencies of the Arg702Trp, Gly908Arg and Leu1007fs CARD15 genetic variants in healthy controls and CD patients
CARD15 carriership
Controls (n = 178)
CD patients (n = 132)
OR (95% CI)
Arg702Trp Gly908Arg Leu1007fs At least one variant
7 3 6 14
22 6 16 39
4.98 2.78 3.94 4.8
(3.9) (1.7) (3.2) (8.0)
(16.6) (4.5) (12.1) (29.5)
Values are n (%). Differences were evaluated by the m 2 test and estimation of risk.
(2.06 (0.68 (1.50 (2.49
to to to to
p 12.05) 11.32) 10.40) 9.35)
b 0.0001 0.177 0.003 b 0.00001
TNFa genotype influences development of Iga-ASCA antibodies Table 4
309
TNFa and IL-10 genotype distribution of CD patients without CARD15 mutations and with different serological markers ASCA IgA
ASCA IgA+
308 TNFa GG (low) AA/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
40 19 0.20 0.12
(67.8) (32.2) (0.05 to 0.73) (0.02 to 0.79)
31 (91.2) 3 (8.8)
1082 IL-10 AA (low) GG/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
22 37 1.68 1.40
(37.3) (62.7) (0.72 to 3.95) (0.51 to 3.88)
17 (50.0) 17 (50.0)
p
PAB
PAB+ (82.0) (18.0) (0.09 to 6.34) (1.31 to 17.83)
21 (65.6) 11 (34.4)
0.009 0.011
50 11 2.38 4.82
(36.1) (63.9) (0.84 to 4.80) (0.98 to 8.78)
17 (53.1) 15 (46.9)
0.232 0.511
22 39 2.01 2.94
p
0.078 0.018
0.113 0.054
Values are n (%).Unconditional logistic regression model using the presence of antibody as dependent variable and the presence of the low producer genotype as reference. a Adjusted for sex, age and the clinical parameters: disease duration, age at onset, location, disease type, extraintestinal manifestations, steroid dependence, steroid resistance and perianal disease.
patients and healthy controls. As expected, we found a significantly higher frequency in CD patients with respect to controls of the Arg702Trp allele (0.096 vs 0.020; OR = 5.30; 95% CI: 2.26 to 12.46; p b 0.0001) and the Leu1007fs allele (0.055 vs 0.016; OR = 3.56; 95% CI: 1.38 to 9.22; p = 0.005). Thus, the percentage of carriers of these alleles in controls and patients also showed significant differences (Arg702Trp 3.9 vs 16.6, p b 0.0001 and Leu1007fs 3.2 vs 12.1, p = 0.003) (Table 3). Gly908Arg allele frequency was also increased in the CD population compared to controls (0.023 vs 0.008), although the differences were not significant, probably due to the low frequency of this mutation in our population. Thus, 29.5% of CD patients (39 of 132) and 8.0% of controls (14 of 175) presented at least one CARD15 mutant allele, the strength of the association being very highly significant (OR = 4.8; 95% CI: 2.49 to 9.35, p b 0.00001). CD patients were subsequently classified into two groups according to the presence or absence of at least one CARD15 risk allele, and genotype frequencies of the TNFa and IL-10 polymorphisms were compared between
patients found to be positive and negative for IgA-ASCA, IgG-ASCA and PAB serological markers. To exclude skewed stratification resulting from the division of the patient’s cohort into two smaller cohorts, we tested the independence between TNFa and IL-10 genotypes and the CARD15 variations (TNFa/CARD15 p = 0.582; IL-10/CARD15 p = 0.923, m 2 test). We found that the proposed protective role of the high TNFa producer genotype on IgA-ASCA production was highly noteworthy in the group of patients with the CARD15 wild genotype (OR = 0.20, 95% CI = 0.05 to 0.73, p = 0.009) (Table 4). This association was maintained in the multivariate analysis after adjusting for clinical and serological parameters (OR = 0.12, 95%IC: 0.02 to 0.79; p = 0.011). However, this association did not appear in the patients’ group with at least one CARD15 mutant allele (Table 5), indicating the absence of the influence of TNFa on IgAASCA development in patient carriers of CARD15 mutations. Similarly, the association trend found previously in the total CD population between high TNFa producer genotypes and the presence of PAB was also observed in the
Table 5 TNFa and IL-10 genotype distribution of CD patients with at least 1 CARD15 mutant allele and with different serological markers ASCA IgA
ASCA IgA+
308 TNFa GG (low) AA/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
17 6 1.28 0.81
(73.9) (26.1) (0.31 to 5.27) (0.08 to 8.06)
11 (68.7) 5 (31.3)
1082 IL-10 AA (low) GG/AG (high) Univariate analysis OR (95% CI) Multivariate analysisa OR (95% CI)
9 14 1.21 1.46
(39.1) (60.9) (0.33 to 4.42) (0.17 to 12.07)
7 (43.8) 9 (56.2)
p
PAB
PAB+ (78.3) (21.7) (0.52 to 8.90) (0.99 to 7.92)
10 (62.5) 6 (37.5)
0.734 0.856
18 5 2.16 0.91
(39.1) (60.9) (0.33 to 4.42) (0.10 to 2.66)
7 (43.8) 9 (56.2)
0.773 0.726
9 14 1.21 0.17
p
0.307 0.884
0.773 0.204
Values are n (%).Unconditional logistic regression model using the presence of antibody as dependent variable and the presence of the low producer genotype as reference. a Adjusted for sex, age and the clinical parameters: disease duration, age at onset, location, disease type, extraintestinal manifestations, steroid dependence, steroid resistance and perianal disease.
310 group of CARD15 wild genotype patient group, arising statistical significance at multivariate analysis (OR = 4.82; 95% CI = 1.31 to 17.83; p = 0.018) (Table 4); however, again this association did not appear in carriers of one or more of the CARD15 variations (Table 5). Polymorphisms at the IL10 gene did not influence antibody production in either mutated or CARD15 wild type patients. Taken as a whole, these results suggest an involvement of the interaction CARD15—TNFa genotype in the specific antibodies production in CD patients. Finally, analysis of the clinical features of patients without CARD15 mutated alleles suggested that the joint presence of IgA-ASCA and the low TNFa producer genotype could be indicative of a more severe disease as this patient group (n = 31) was characterized by a higher frequency of surgery (51.6% vs 27.4%, p = 0.022), stricturing/penetrating disease (83.9% vs 62.9%, p = 0.038) and ileal involvement (100% vs 85.5% p = 0.026) when compared with the remainder of CARD15 wild type patients (n = 62). A very low prevalence of PAB+ was also characteristic of these patients (13.1% vs 43.5%, p = 0.009). Interestingly, among carriers of the CARD15 mutations, any clinical or serological feature was overrepresented in patients with these serological and genetic markers (IgA-ASCA+ and low TNFa genotype), supporting the role of TNFa in disease outcome of CD patients without CARD15 mutations.
Discussion In this study, we have found a relationship between functional polymorphisms at the TNFA gene and the presence of anti-S. cerevisiae antibodies of the IgA isotype (IgA-ASCA) and anti-exocrine pancreas antigen antibodies (PAB) in Crohn’s disease patients. Specifically, carriage of the rare high TNFa producer allele at the 308 position (TNF2) protects against the generation of IgA-ASCA, whereas it increases the risk of PAB production, suggesting a different effect of TNFa on the production of antibodies against self- and exogenous antigens. High TNFa producer genotypes not only are a risk factor for the appearance of several autoimmune diseases [40,41], but also they have been previously associated with increased production of antibodies against self-antigens such as ANCA in ulcerative colitis [42,43], SSa and SSb in systemic lupus erythematosus [40] or SSb in Sjfgren syndrome [44]. However, to the best of our knowledge, this is the first study reporting an association between the 308GG TNFa low producer genotype and the generation of antibodies against dietary or colonist microorganisms. The elevated specificity of IgA-ASCA found in our work supports the relevance of GALT (gut-associated lymphoid tissues) immune responses to exogenous antigens in CD patients. A higher discriminating value between CD and other bowel diseases has been previously described for IgA when compared to IgG responses [45,46]. It is know that most of the plasma cells in the intestinal tract produce IgA, indicating an individual cytokine profile that promoted this type of response. Additionally, it has been reported that IgA production is dependent on microorganism presence since germfree animals lack intestinal IgA [47]. Thus, mucosal specific response against intestinal colonists is probably mediated through IgA production. IgG isotype ASCA, how-
P. Castro-Santos et al. ever, are most likely produced by plasma cells generated in lymphoid follicles away from the Peyer’s patches, where environmental conditions and cytokine network are different from the intestinal mucosa. Given the genetic origin proposed for ASCA [48], association with TNFa genotype might be explained by linkage disequilibrium with a gene related to ASCA expression located on chromosome 6, in the vicinity of the major histocompatibility complex. However, this is not the case since the association between TNFa genotype and antibody production disappeared completely in patients expressing a CARD15 mutated protein. The CARD15 gene encodes a molecule involved in bacterial recognition and the subsequent proinflammatory response through NF-nB activation. Presence of Arg702Trp, Gly908Arg or Leu1007fs CARD15 mutations modifies the structure of the leucine-rich repeat domain of the protein, determining a different degree of loss of function. These three major variants have been associated with a poor response to microorganism infection [21] and with an inability to eliminate pathogens [26] especially in the intestine [49]. Therefore, the lack of recognition of bacterial peptidoglycan caused by these CARD15 variations decreased NF-nB activation [25] and the subsequent TNFa release [50], which is known to be dependent on promoter polymorphisms. Thus, among carriers of CARD15 mutations, differences between genetically high and low TNFa producers in response to bacterial stimulation could be unapparent, explaining the lack of association between TNFa genotypes and clinical or serological parameters. However, in the absence of an adequate TNFa response, other mechanisms might be activated to eliminate bacterial infection. Hence, the higher incidence of ASCA among CARD15 mutated CD patients previously reported by several authors [51] might be explained by the low TNFa production in response to bacterial stimulation. In our CARD15 mutated CD group, an increased prevalence of IgA-ASCA was also found, although this was not statistically significant, probably due to the low percentage of carriers of CARD15 variations (29.5%) and the relatively low prevalence of IgA-ASCA+ (37.7%). TNFa is a proinflammatory and a proapoptotic cytokine mainly secreted by monocytes/macrophages during innate immune responses and by effector Th1 cells. Interestingly, low levels of TNFa and carriage of the 308GG genotype have been widely related to less efficient elimination of pathogens and to tuberculosis [52] and leprosy [53]. The immunoregulatory function of TNFa in the intestinal mucosa of CD patients could explain the association found between CARD15—TNFa genotype and IgA-ASCA production. Fig. 1 summarizes the putative interactions we propose at the cellular and molecular level. CARD15 receptor, usually highly expressed in monocytes, macrophages and epithelial and Paneth cells from CD patients, recognizes bacteria that penetrated the mucosa as a result of breaks in the integrity of the epithelium, leading to NF-nB activation and TNFa production. Thus, in genetically high TNFa producers, the elevated TNFa levels may be able to reduce the generation of specific immune responses against bacteria and yeast, dietary or intestinal colonist, either by the activation of neutrophil phagocytosis and the subsequent clearance of microorganisms and antigenic products or by compromising antigen uptake and signaling by leukocytes that interfaced
TNFa genotype influences development of Iga-ASCA antibodies
311
Figure 1 Proposed involvement of CARD15—TNFa circuit in IgA-ASCA production. Breaks in the integrity of the mucosal epithelium allow the internalization of exogenous antigens (from dietary or colonist bacteria and yeast). Interaction of bacterial peptidoglycan with CARD15 receptor leads to NF-nB activation and TNFa transcription at high or low levels depending on promoter genotype. High TNFa levels reduce the generation of adaptive immune responses against exogenous antigens by the activation of neutrophil phagocytosis and the subsequent clearance of microorganisms and antigenic products and/or by compromising antigen uptake and signaling by leukocytes that interfaced with intestinal antigens. Lamina propia dendritic cells (DC) uptake exogenous antigens and migrate into lymph follicles where T and B cells were activated and initiate a specific immune response. Humoral immune responses at the intestinal mucosa (Peyer’s patches) are particularly conductive to the induction of IgA, probably, under the influence of Th2 and Th3 cytokines. Part of the lamina propia DC migrate to mesenteric lymph nodes where, in the absence of mucosal environment, humoral immune response leads to IgG production.
with intestinal antigens. On the contrary, low TNFa producers might present an impaired capacity to efficiently eliminate microorganisms that enter the mucosal epithelium. It allows dendritic cells of the lamina propia to uptake exogenous antigens and migrate into local lymph follicles, where T and B lymphocytes are activated and IgA-ASCA generation takes place, under the influence of mucosal cytokines. Moreover, since Ig class switch depends on environmental cytokines, diminished Th1 cell-mediated immune responses, as a consequence of low TNFa production, may predispose to the generation of a predominant Th2/Th3 profile in the intestinal mucosa, favoring the generation of IgA antibodies to yeast antigens. On the other hand, part of the lamina propia dendritic cells, loaded with exogenous Ag, migrate into lymph nodes away to the GALT, where they initiate an immune response that might lead, in the absence of the cytokine mucosal environment, to the generation of plasma cells producers of IgG [54].
On the contrary to IgA-ASCA development, the generation of PAB seems to be promoted by high TNFa production. This is in accordance to previously reported associations between ANCA and other autoantibodies synthesis and the TNF2 allele [42—44]. It suggests that autoantibodies production might be the result of autoantigen exposure due to chronic inflammation and increased apoptosis. Furthermore, it has been reported that patients with a combination of positive pANCA and negative ASCA, serological markers that were suggestive of high TNFa levels, showed the least favorable response to anti-TNFa therapy (infliximab). Many studies have examined TNFa gene polymorphisms as possible predictors for the response to infliximab treatment. Several of these studies showed a slightly higher frequency of the rare TNF2 allele among non-responders. However, despite the large number of works, results were inconclusive [55,56]. According to our results, the reason of this controversy may be the lower
312 influence of the TNFa genotype on the intestinal mucosa TNFa levels among patient carriers of CARD15 mutated alleles. Thus, relationship between the lack of response to anti-TNFa therapy and absence of ASCA, associated with high TNFa production, could implicate that TNFa may not play a pathological role in ASCA negative patients. Consequently, studies on the predictor value of genetic and serological markers on the response to TNFa blocking agents should be performed separately for CD patients carrying either the mutant or the wild type CARD15 gene. In summary, our results indicate that the TNFa genotype influences the development of antibodies in CD patients without CARD15 mutations, supporting the role of TNFa in the surveillance of immune interface with intestinal colonist. Moreover, the CARD15—TNFa circuit proposed by this study is likely to play a significant role in mucosal immune regulation and when perturbed may affect disease outcome. Consequently, presence of CARD15 variations should be taken into account to classify CD patients in order to study pathological mechanisms.
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