Lammers K, Ouburg S, Morré S, Crusius J, Gionchetti P, Rizzello F, Morselli C, Caramelli E, Conte R, Poggioli G, Campieri M, Peña A. Combined carriership of TLR9 -1237C and CD14 -260T alleles enhances the risk of developing chronic relapsing pouchitis. World J Gastroenterol 2005; 11(46): 7323-7329 [PMID: 16437636 DOI: 10.3748/wjg.v11.i46.7323]
Corresponding Author of This Article
KM Lammers, Department Internal Medicine and Gastroenterology, Policlinic S. Orsola, University of Bologna, Nuove patologie-Pad. 5, Via Massarenti 9, 40138 Bologna, Italy. kmlammers@hotmail.com
Article-Type of This Article
Rapid Communication
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KM Lammers, P Gionchetti, F Rizzello, C Morselli, M Campieri, Department of Internal Medicine and Gastroenterology, Policlinico S. Orsola, University of Bologna, Bologna, Italy
S Ouburg, SA Morré, JBA Crusius, AS Peña, Laboratory of Immunogenetics, VU University Medical Center, Amsterdam, The Netherlands
E Caramelli, Institute of Histology and General Embryology, University of Bologna, Bologna, Italy
R Conte, Department of Immunohaematology and Blood Transfusion, Policlinico S. Orsola, University of Bologna, Bologna, Italy
AS Peña, Laboratory of Immunogenetics and Department of Gastroenterology, VU University Medical Center, Amsterdam, The Netherlands
G Poggioli, Department of Surgery and Organ Transplantation, Policlinic S. Orsola, University of Bologna, Bologna, Italy
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Correspondence to: KM Lammers, Department Internal Medicine and Gastroenterology, Policlinic S. Orsola, University of Bologna, Nuove patologie-Pad. 5, Via Massarenti 9, 40138 Bologna, Italy. kmlammers@hotmail.com
Telephone: +39-51-6364122 Fax: +39-51-392538
Received: May 2, 2005 Revised: June 2, 2005 Accepted: June 9, 2005 Published online: December 14, 2005
Abstract
AIM: To investigate the single nucleotide polymorphisms (SNPs) in genes involved in bacterial recognition and the susceptibility to pouchitis or pouchitis severity.
METHODS: Analyses of CD14 -260C>T, CARD15/NOD2 3020insC, Toll-like receptor (TLR)4 +896A>G, TLR9 -1237T>C, TLR9+2848G>A, and IRAKM + 22148G>A SNPs were performed in 157 ileal-pouch anal anastomosis (IPAA) patients (79 patients who did not develop pouchitis, 43 infrequent pouchitis patients, 35 chronic relapsing pouchitis patients) and 224 Italian Caucasian healthy controls.
RESULTS: No significant differences were found in SNP frequencies between controls and IPAA patients. However, a significant difference in carriership frequency of the TLR9-1237C allele was found between the infrequent pouchitis and chronic relapsing pouchitis groups [P = 0.028, odd’s ratio (OR) = 3.2, 95%CI = 1.2-8.6]. This allele uniquely represented a 4-locus TLR9 haplotype comprising both studied TLR9 SNPs in Caucasians. Carrier trait analysis revealed an enhanced combined carriership of the alleles TLR9 -1237C and CD14 -260T in the chronic relapsing pouchitis and infrequent pouchitis group (P = 0.018, OR = 4.1, 95%CI = 1.4 -12.3).
CONCLUSION: There is no evidence that the SNPs predispose to the need for IPAA surgery. The significant increase of the combined carriership of the CD14 -260T and TLR9 -1237C alleles in the chronic relapsing pouchitis group suggests that these markers identify a subgroup of IPAA patients with a risk of developing chronic or refractory pouchitis.
Patients with ulcerative colitis may need surgery for their disease and proctocolectomy with ileal-pouch anal anastomosis (IPAA) is the surgical procedure of choice for the management of these patients[1,2]. Most patients undergoing IPAA for severe colitis or chronic continuous disease achieve good functional results, but some patients develop pouchitis, a non-specific idiopathic inflammation of the ileal reservoir. Frequency rates of pouchitis are highly variable, ranging 10-59% depending on the length of follow-up and the diagnostic criteria used[3]. Though the origin of pouchitis remains unknown, genetic and immunological factors are likely to be involved in addition to ileal mucosa that needs to adapt to its new role as a reservoir[4]. This is illustrated by the fact that pouchitis occurs almost exclusively in patients with IPAA for ulcerative colitis and not in patients with IPAA for familial adenomatous polyposis, a hereditary non-inflammatory disease of the colon with high risk of developing colon cancer. An important role of luminal bacteria in the development of pouchitis is underscored by various reports on bacterial overgrowth and dysbiosis in pouchitis[5] and is further confirmed by the efficacy of antibiotic and probiotic therapy[6,7].
Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), are essential components of the innate immune system as recognition of microbial products occurs via PRRs that are expressed by innate effector cells. Microbial recognition results in a rapid and efficient immune response against invading microorganisms[8].
Given the role of luminal bacteria in driving the inflammatory response in pouchitis, identification and functional characterization of polymorphisms in innate immunity genes may provide insight in a possible genetically determined susceptibility to pouchitis and/or chronic relapsing pouchitis[9].
CD14 is part of the endotoxin/lipopolysaccharide (LPS) receptor complex[8] and is important in conjunction with TLR4 and with TLR2[10] in the recognition of LPS, a membrane glycolipid on Gram-negative bacteria. CD14 may also recognize cell membrane components of Gram-positive mycobacteria and viruses[11-15]. CD14 exists in a membrane form on monocytes and neutrophils and in a soluble form in serum[16-18]. The SNP at position -260C>T (also known as CD14-159C>T) in the promoter region of the CD14 gene (located on chromosome 5q31) is associated with enhanced transcriptional activity[19] and significantly higher CD14 serum levels[20]. Increased expression of CD14 in macrophages has been found in inflammatory bowel disease (IBD)[21]. An association of the CD14-260C>T gene polymorphism with IBD[22,23] and atherosclerosis[24] has been described. Genetically determined variation in CD14 serum levels may have functional consequences given the ability of soluble CD14 to confer pathogen responsiveness to cells such as intestinal, epithelial and endothelial cells that do not express CD14 on their membranes[25].
The TLR4 gene is located on chromosome 9q32-q33. The TLR4+896A>G SNP affects the leucine-rich repeat domain of TLR4 and is associated with hyporesponsiveness to LPS[26] with increased susceptibility to severe bacterial infections and IBD[27] and may predispose to septic shock with Gram-negative microorganisms[28,29].
TLR9 is required for the recognition of CpG motifs, short sequences of unmethylated DNA predominantly present in bacterial DNA. CpG motifs have immunostimulatory activity by inducing dendritic cell maturation, B-cell proliferation and production of cytokines, including interleukin-6 (IL-6) and interleukin-12 (IL-12)[30,31]. TLR9 signaling has been shown to mediate the resolution of intestinal inflammation in experimental colitis[32], suggesting that the release of bacterial DNA from the microflora might favor immune homeostasis. The promoter TLR9 -1237T>C SNP located on chromosome 3p21.3 is associated with susceptibility to asthma in European Americans, but not in Hispanic or African Americans[33] and the marker D3S1076 in this region shows association with IBD in a classical TDT test[42]. Török and colleagues[34] studied the TLR9 -1237T>C and TLR9+2848 G>A SNPs in German patients with Crohn’s disease, ulcerative colitis and healthy blood controls and found that the allele TLR9 -1237C carrier status is associated with Crohn’s disease compared to controls. TLR9 +2848 G>A allele frequencies are not different between the study groups.
CARD15/NOD2 is a cytoplasmatic bacterial PRR expressed in monocytes and intestinal epithelial cells and mediates response against muramyl dipeptide derived from peptidoglycan[35,36]. The CARD15/NOD2 gene is located on chromosome 16q12. Three major polymorphisms in this gene (R702W, G908R, and L1007 frameshift mutation) are associated with susceptibility to Crohn’s disease, possibly as a result of a defective response against muramyl dipeptide derived from peptidoglycan[37]. Recently, an increased frequency of the L1007 frameshift mutation has been observed in Italian patients with ulcerative colitis when compared to controls[38].
The intracellular domains of TLRs are homologous to the interleukin-1 receptor (IL-1R) type I intracellular domain and use a common pathway of intracellular signaling with shared components including the protein kinase IL-1R-associated kinase1 (IRAK1) and IRAK-M, as a negative regulator of TLR signaling. Cytokine production increases in IRAK-M-/- macrophages after TLR/IL-1 stimulation and bacterial challenge, while endotoxin tolerance reduces in these cells. Furthermore, IRAK-M-/- mice have increased inflammatory responses to bacterial infection and develop intestinal inflammation. These data suggest that IRAK-M has a regulatory function in TLR/IL-1R signaling and innate immune homeostasis[39]. The IRAKM (or IRAK3) gene is located at chromosome 12q14.2 within the IBD2 region associated with ulcerative colitis[40,41]. Genetic variation in the IRAKM gene may be involved in the development of chronic intestinal inflammation. For this reason, we chose to analyze a non-synonymous SNP in exon 5 resulting in an Ile/Val substitution.
A candidate gene approach, based on the determination of frequencies of functional SNPs, can be used to investigate the relevance of genes to the disease susceptibility and severity. Carrier trait analysis investigates combinations of SNPs and allows studying the implication of different SNPs in disease susceptibility and severity as a result of their synergistic action.
The aim of this study was whether SNPs in innate immunity genes could contribute to the susceptibility to pouchitis and/or severity of pouchitis. We chose candidate genes of CD14, TLR4, TLR9, NOD2/CARD15, and IRAKM for their involvement in bacterial recognition and intracellular signaling pathways.
MATERIALS AND METHODS
Patients
One hundred and fifty-seven unrelated patients with IPAA for ulcerative colitis and 224 healthy blood donors were studied. All individuals were Italian Caucasians. Consent was obtained and the local ethics committee approved the protocol. Their demographic and clinical information is described in Table 1. IPAA patients were subdivided into three test groups according to the pouchitis pattern. One group consisted of IPAA patients who had never developed pouchitis, another group consisted of IPAA patients who had up to two episodes of pouchitis during IPAA (infrequent pouchitis) and third group consisted of IPAA patients who developed three or more episodes of pouchitis (chronic relapsing pouchitis).
Table 1 Demographic features of IPAA patients and healthy controls.
IPAA patients
Healthy controls
Total number (n)
157
224
Gender M/F
88/69
118/106
Mean age in yr (SD)
42.9 (11.8)
45.8 (12.8)
Range
17-73
21-77
Median
41
45.5
DNA isolation
Venous blood (5-10 mL) was drawn and genomic DNA was isolated using standard protocols. Polymorphisms of the CD14, TLR4, TLR9, CARD15/NOD2, and IRAKM genes in these three groups were analyzed. Genomic DNA (5-100 ng) was used for each genotyping.
Analysis of gene polymorphisms
Polymerase chain reaction (PCR) for RFLP analyses was performed on a thermal cycler GeneAmp 9700 (Perkin-Elmer Cetus, Norwalk, CT, USA). Digested fragments were analyzed on a 4% agarose gel except for the IRAKM SNP analyzed on a 2% agarose gel and visualized with an UV-illuminator after ethidium bromide staining. SNPs were analyzed with the TaqMan assay (Applied Biosystems, Foster City, CA, USA). MGB TaqMan probes and primer pairs were designed with Primer Express software (version 2.0). TaqMan thermocycling consisted of an initial step at 50 °C for 2 min and denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. We used the ABI Prism 7000 sequence detector (Applied Biosystems) for data acquisition.
Genotyping
CD14-260C>T genotyping (NCBI SNP CLUSTER ID: rs2569190) was performed by PCR. Primers used were: forward primer 5’-TCA CCT CCC CAC CTC TCT T-3’ and reverse primer 5’-CCT GCA GAA TCC TTC CTG TT-3’. The PCR conditions were initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min followed by cooling to 4 °C. The 107-bp amplicons were digested overnight with HaeIII (New England Biolabs, UK). Digestion resulted in two fragments of 83 and 24 bp (C allele) or 107 bp (T allele), respectively.
Genotyping of the TLR4+896 A>G SNP (NCBI SNP CLUSTER ID: rs4986790) was performed with forward primer 5’-TTT ACC CTT TCA ATA GTC ACA CTC A-3’ and reverse primer 5’-AGC ATA CTT AGA CTA CTA CCT CCA TG-3’. PCR conditions were: initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s and a final extension at 72 °C for 5 min followed by cooling to 4 °C. The 102-bp amplicons were digested overnight with NcoI (New England Biolabs, UK). Digestion resulted in two fragments of 80 and 22 bp (G allele) or 102 bp (A allele), respectively.
CARD15/NOD2 3020InsC (CARD15 L1007fs) (NCBI SNP CLUSTER ID: rs2066847) genotyping was performed with forward primer 5’-GGC AGA AGC CCT CCT GCA GGG CC-3’ and reverse primer 5’-CCT CAA AAT TCT GCC ATT CC-3’. PCR conditions were: initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 45 s and a final extension at 72 °C for 5 min followed by cooling to 4 °C. The 150-bp amplicons were digested overnight with ApaI. Digestion resulted in a fragment of 150 bp (no insertion) or 128 bp and 22 bp (insertion C), respectively.
TLR9-1237T>C (NCBI SNP CLUSTER ID: rs5743836) genotyping was performed with TaqMan method. Primers used were: forward primer 5’-GGC CTT GGG ATG TGC TGT T-3’ and reverse primer 5’-GGT GAC ATG GGA GCA GAG ACA-3’. Dual-labeled fluorogenic hybridization MGB-probes used were: CTGCCTGAAAACT 5’ Fluor Label (FAM, 6-carboxyfluorescein) and CTGGAAACTCCCC 5’ Fluor Label (VIC).
TLR9+2848G>A genotyping (NCBI SNP CLUSTER ID: rs352140) was performed with TaqMan method. Primers used were: forward primer 5’-CCG GTC TGC AGG TGC TAG AC-3’ and reverse primer 5’-CCA AAG GGC TGG CTG TTG TA-3’. Dual-labeled fluorogenic hybridization MGB probes used were: AGCTACCGCGACTGG 5’ Fluor Label (FAM) and AGCTACCACGACTGGA 5’ Fluor Label (VIC).
Genotyping of the IRAKM+22148G>A exon 5 SNP (NCBI SNP CLUSTER ID: rs1152888) was performed by PCR with forward primer 5´-AGT GGA AC T GAT GTC CTG TGA CAG-3´ and reverse primer 5´-GCA ACA CAT TGA CCT AAT GAC CAG-3´.
The PCR conditions were: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 50 s, at 60 °C for 50 s, at 72 °C for 150 s and a final extension at 72 °C for 5 min followed by cooling to 4 °C. Digestion overnight with RsaI (Invitrogen Life Technologies) of the 505-bp amplicons resulted in two fragments of 188+317 bp (allele G) or 505 bp (allele A).
Statistical analysis
Hardy-Weinberg equilibrium was determined in healthy controls to assess the Mendelian inheritance. Comparisons of the genotypes between control and different groups of IPAA patients were performed by Fisher’s exact or χ2 two-tailed tests where appropriate. Carrier trait analysis was performed to determine whether combinations of SNPs were acting synergistically on the risk of developing pouchitis or of predisposing to chronic relapsing pouchitis. Adjusted odd’s ratio (OR) and 95% confidence intervals (95%CI) were calculated. P<0.05 was considered statistically significant.
RESULTS
Characteristics of patients and control groups
The demographic features of IPAA patients and healthy controls are shown in Table 1. No statistical differences were found in the variables including gender and age between the IPAA group and healthy controls.
The clinical characteristics of patients with IPAA for ulcerative colitis were summarized. Information on the pattern of pouchitis within the group of IPAA patients for ulcerative colitis (patients who did not develop a pouchitis, patients who had infrequent pouchitis and patients who suffered from chronic relapsing pouchitis, respectively) is shown in Table 2. No statistically significant differences were found in gender and age between controls and IPAA patients as well as in the duration of pouch and the first episode of pouchitis after IPAA, respectively and among IPAA groups and between infrequent pouchitis and chronic relapsing pouchitis groups.
Table 2 Clinical data obtained from IPAA patients.
IPAA patients
Mean (SD)
Median
Range
Age (yr) at diagnosis of ulcerative colitis
29.7 (11.7)
27
8-61
Time (yr) from diagnosis of ulcerative colitis to IPAA surgery
6.3 (5.5)
5
0-34
Time from IPAA surgery to first episode of pouchitis
Total pouchitis group (n = 78)
2.8 (3.1)
2
0-12
Infrequent pouchitis ( ≤2 episodes, n = 43)
3.7 (3.3)
3
0-12
Chronic relapsing pouchitis (≥3 episodes, n = 35)
2 (2.6)
1
0-12
Pattern of pouchitis
Duration of pouch (yr)
No episodes of pouchitis (n = 79)
6.3 (4.1)
5
0-14
Infrequent pouchitis ( ≤2 episodes, n = 43)
7.6 (3.8)
7
1-16
Chronic relapsing pouchitis (≥3 episodes, n = 35)
7.9 (3.6)
7
2–14
Genotyping
The genotype frequencies in the control group were in Hardy-Weinberg equilibrium for the CD14, TLR4, TLR9, CARD15/NOD2, and IRAKM gene polymorphisms. The genotype frequencies of these polymorphisms are described in Table 3. No significant differences in allele-, genotype- or carrier frequencies of the gene polymorphisms were found between the healthy controls and IPAA patients.
Table 3 Genotypes of the CD14, CARD15, TLR4, TLR9, and IRAKM gene polymorphisms in subgroups of patients with IPAA and controls.
Polymorphisms
Genotype
Controls
No pouchitis
Infrequent pouchitis
Chronic relapsing pouchitis
n = 224 (%)
n = 79 (%)
n = 43 (%)
n = 35 (%)
CD14–260 C>T
CC
55 (24.6)
24 (30.4)
11 (25.6)
7 (20)
CT
102 (45.5)
34 (43)
21 (48.8)
22 (62.9)
TT
67 (29.9)
21 (26.6)
11 (25.6)
6 (17.1)
CARD15 3020InsC
WT/WT
218 (97.3)
77 (97.5)
43 (100)
34 (97.1)
WT/InsC
6 (2.7)
2 (2.5)
0 (0)
1 (2.9)
InsC/InsC
0 (0)
0 (0)
0 (0)
0 (0)
TLR4 +896 A>G
AA
208 (92.9)
73 (92.4)
38 (88.4)
33 (94.3)
AG
16 (7.1)
5 (6.3)
5 (11.6)
2 (5.7)
GG
0 (0)
1 (1.3)
0 (0)
0 (0)
TLR9–1237 T>C
TT
158 (70.5)
52 (65.8)
34 (79.1)
19 (54.3)
TC
60 (26.8)
24 (30.4)
9 (20.9)
14 (40)
CC
6 (2.7)
3 (3.8)
0 (0)
2 (5.7)
TLR9 +2848 G>A
GG
40 (17.9)
20 (25.3)
9 (20.9)
6 (17.1)
GA
104 (46.4)
38 (48.1)
22 (51.2)
16 (45.7)
AA
80 (35.7)
21 (26.6)
12 (27.9)
13 (37.2)
IRAKM +22148G>A
GG
181 (80.8)
64 (81)
33 (76.7)
28 (80)
GA
42 (18.8)
14 (17.7)
7 (16.3)
7 (20)
AA
1 (0.4)
1 (1.3)
3 (7)
0 (0)
No significant differences in allele-, genotype- or carrier frequencies of the gene polymorphisms were found between the three subgroups of IPAA patients except that the carriership of allele TLR9 -1237 C was more frequent in patients with chronic relapsing pouchitis (45.7%) as compared to those with infrequent pouchitis (20.9%) (P =0.028, OR = 3.2, 95%CI = 1.2-8.6). When the combined groups of infrequent pouchitis and chronic relapsing pouchitis (i.e. total pouchitis group) were compared to the patients without pouchitis, carriership of this allele was not significantly different (P = 0.87, OR = 1.1, 95%CI = 0.6-2.1).
TLR9 haplotype
The two analyzed TLR9 SNPs were chosen based on the study of Lazarus et al[33] in which a set of four frequent TLR9 SNPs designated as TLR9 -1486, TLR9 -1237, TLR9 +1174, and TLR9 +2848, were described. Genotyping of both TLR9 -1237 and TLR9 +2848 could distinguish all four-locus haplotypes commonly present in the European American population. We therefore calculated the haplotypic genotypes in Italian Caucasians (Table 4). The haplotype frequencies in the healthy controls were identical to the European-Americans as reported by Lazarus et al[33]. Haplotype III was more frequent in chronic relapsing pouchitis as compared to infrequent pouchitis (P = 0.018; OR = 3.0, 95%CI = 1.2-7.1). This haplotype, however, did not show nucleotides uniquely present (tag SNPs) on position -1486 (allele T present in haplotypes I and III) or on position +1174 (allele G present in haplotypes II and III), indicating that allele TLR9-1237C could provide the strongest association.
Table 4 Frequencies of TLR9 haplotypes formed by -1237 T>C and +2848 G>A SNPs.
To investigate if SNPs in different genes could act synergistically on disease susceptibility and/or severity, carrier trait analysis with the associated TLR9 allele was performed. Simultaneous carriership of alleles TLR9 -1237C and CD14 -260T was more frequent in patients with chronic relapsing pouchitis as compared to those with infrequent pouchitis (P = 0.018, OR = 4.1, 95%CI = 1.4-12.3), which was more significant as compared to the analysis of allele TLR9 -1237C alone (P = 0.028, OR = 3.2, 95%CI = 1.2-8.6). No other significant carrier traits were observed.
DISCUSSION
There is convincing evidence that enteric bacteria play a role in driving the inflammatory response in IBD and that genetic factors contribute not only to the pathogenesis but also to the course and extent of these disorders. Given these processes, we investigated whether polymorphisms in the following genes encoding for proteins involved in innate immunity, TLR4 +896 A>G, TLR9 +2848 G>A, TLR9 -1237T>C, CD14 -260C>T, CARD15/NOD2 3020insC, and IRAKM +22148 G>A, were associated with the development of pouchitis, disease frequency or severity.
Analysis of the three subgroups of IPAA patients (i.e. patients who never developed pouchitis, patients with infrequent pouchitis and patients with a chronic refractory form of pouchitis) revealed a positive association of allele TLR9 -1237C with the risk of developing chronic refractory pouchitis, once these patients developed pouchitis. Haplotype analysis showed that out of the four SNPs defining TLR9 haplotypes, this allele was uniquely responsible for this finding. Subsequently, we investigated whether interactions of allele TLR9 -1237C with SNPs in the other candidate genes might strengthen this association. Carrier trait analysis revealed that an even stronger association was apparent with the combination of alleles TLR9 -1237C and CD14 -260T. These data suggest that this combination of alleles might be a valuable genetic marker to identify a clinical subgroup of IPAA patients with an enhanced risk of developing chronic pouchitis, compared to alleles of the SNPs TLR4 +896 A>G, TLR9 +2848 G>A, CARD15/NOD2 3020insC, and IRAKM +22148 G>A.
It cannot be excluded that the group of patients who did not develop pouchitis consisted of a mixture of patients who never developed pouchitis on the one hand and patients who proceeded to the infrequent or chronic relapsing pouchitis group on the other hand. This could explain why we did not detect an association of allele TLR9 -1237C between the no-pouchitis and the chronic relapsing pouchitis groups. It should be noted that no significant differences were found in the mean duration of IPAA between the three groups.
At present it is unknown as to what effect of the TLR9-1237 T>C SNP exerts on the expression of TLR9 given its location in the far promoter region where no DNA-binding site for known transcription factors is apparent. The association observed might therefore result from linkage disequilibrium with another polymorphism(s) in a nearby gene.
The mechanism underlying increased risk of developing chronic relapsing pouchitis by a combined carriership of alleles TLR9 -1237C and CD14 -260T might be a dysfunction in bacterial recognition or a lack of an adequate immune response to bacterial challenge. This could start at the level of the plasmacytoid dendritic cells, which play a central role in bacterial recognition, selectively express TLR9 and have soluble CD14 facilitating reactivity to a broad array of bacterial components[43] or at the level of the intestinal epithelium. Soluble CD14 might confer epithelial cell responsiveness[25].
The regulatory role of dendritic cells is of particular importance in the intestine where the mucosal immune system is closely associated with the external environment[44]. Dendritic cells sample bacterial products either indirectly via M cells or directly by reaching between epithelial cells into the gut lumen[45]. In this perspective, it is noteworthy to mention a recent article that reported a lack of immature blood dendritic cells, which possibly migrate to the gut in IBD patients with active disease[46].
Recently, carriership of the TLR9 -1237C allele has been associated with Crohn’s disease[34]. Ileal involvement is present in about 60% of patients with Crohn’s disease. Since the pouch is an ileal reservoir that is more vulnerable to the continuous contact with high bacterial titers, it could be hypothesized that carriership of the allele TLR9 -1237C (with or without CD14 -260T) is associated with an impaired immune response at the level of the ileal tissue. Paneth cells are located in the crypts and are central in host defense to luminal bacteria by releasing antimicrobial substances[47,48].
If this is true, carriership of allele TLR9 -1237C may become an important predictive marker to the enhanced risk of developing refractory chronic pouchitis and eventually pouch failure.
SNPs in different genes might work synergistically and constitute a small to moderate relative risk of developing diseases. Though the observations we described in this article were based on a relatively less number of patients, it should be realized that this study might represent one of the largest series available.
In conclusion, our data suggest that the alleles TLR9-1237C and CD14-260T synergistically enhance the risk of developing chronic relapsing pouchitis and eventually pouch failure in ulcerative colitis patients who need surgical intervention. Larger studies are required to determine whether this allelic combination becomes a valuable predictive marker and functional studies on the biological role of TLR9 and CD14 in pouchitis.
ACKNOWLEDGMENTS
We are indebted to Italian patients and healthy controls for their participation in this study. S.A. Morré was supported by Tramedico BV, the Netherlands, the Falk Foundation, Germany; the Foundation of Immunogenetics, The Netherlands; the Department of Internal Medicine of the VU University Medical Centre, the Netherlands. We thank Jolein Pleijster and Roel Heijmans for excellent technical assistance in the genotyping.
Footnotes
Science Editor Wang XL Language Editor Elsevier HK
Mimura T, Rizzello F, Helwig U, Poggioli G, Schreiber S, Talbot IC, Nicholls RJ, Gionchetti P, Campieri M, Kamm MA. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis.Gut. 2004;53:108-114.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 660][Cited by in F6Publishing: 578][Article Influence: 28.9][Reference Citation Analysis (0)]
Cleveland MG, Gorham JD, Murphy TL, Tuomanen E, Murphy KM. Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway.Infect Immun. 1996;64:1906-1912.
[PubMed] [DOI][Cited in This Article: ]
Vignal C, Guérardel Y, Kremer L, Masson M, Legrand D, Mazurier J, Elass E. Lipomannans, but not lipoarabinomannans, purified from Mycobacterium chelonae and Mycobacterium kansasii induce TNF-alpha and IL-8 secretion by a CD14-toll-like receptor 2-dependent mechanism.J Immunol. 2003;171:2014-2023.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 109][Cited by in F6Publishing: 111][Article Influence: 5.3][Reference Citation Analysis (0)]
Hailman E, Vasselon T, Kelley M, Busse LA, Hu MC, Lichenstein HS, Detmers PA, Wright SD. Stimulation of macrophages and neutrophils by complexes of lipopolysaccharide and soluble CD14.J Immunol. 1996;156:4384-4390.
[PubMed] [DOI][Cited in This Article: ]
Landmann R, Knopf HP, Link S, Sansano S, Schumann R, Zimmerli W. Human monocyte CD14 is upregulated by lipopolysaccharide.Infect Immun. 1996;64:1762-1769.
[PubMed] [DOI][Cited in This Article: ]
LeVan TD, Bloom JW, Bailey TJ, Karp CL, Halonen M, Martinez FD, Vercelli D. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity.J Immunol. 2001;167:5838-5844.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 245][Cited by in F6Publishing: 252][Article Influence: 11.0][Reference Citation Analysis (0)]
Franchimont D, Vermeire S, El Housni H, Pierik M, Van Steen K, Gustot T, Quertinmont E, Abramowicz M, Van Gossum A, Deviere J. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis.Gut. 2004;53:987-992 DOI : 10.1136/gut.2003.030205 PMCid: PMC1774122.
[PubMed] [DOI][Cited in This Article: ]
Lazarus R, Klimecki WT, Raby BA, Vercelli D, Palmer LJ, Kwiatkowski DJ, Silverman EK, Martinez F, Weiss ST. Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies.Genomics. 2003;81:85-91.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 164][Cited by in F6Publishing: 171][Article Influence: 8.1][Reference Citation Analysis (0)]
Hampe J, Lynch NJ, Daniels S, Bridger S, Macpherson AJ, Stokkers P, Forbes A, Lennard-Jones JE, Mathew CG, Curran ME. Fine mapping of the chromosome 3p susceptibility locus in inflammatory bowel disease.Gut. 2001;48:191-197.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 55][Cited by in F6Publishing: 58][Article Influence: 2.5][Reference Citation Analysis (0)]
Baumgart DC, Metzke D, Schmitz J, Scheffold A, Sturm A, Wiedenmann B, Dignass AU. Patients with active inflammatory bowel disease lack immature peripheral blood plasmacytoid and myeloid dendritic cells.Gut. 2005;54:228-236.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 94][Cited by in F6Publishing: 99][Article Influence: 5.2][Reference Citation Analysis (0)]
