TY - JOUR
AU - PhD, Rubén Francés,
AB - Abstract Background Blood translocation of bacterial-DNA has been described in patients with Crohn's disease (CD). The host's immune cell types cooperate to respond against bacterial insults. Some antimicrobial peptides are inducible after culture with bacterial products and a linkage has been established between them and NOD2/CARD15. The aim was to test whether defensins and cathelicidin (LL-37) expression and NOD2/CARD15 mutations in blood neutrophils are related to molecular bacterial translocation events in CD patients. Methods Fifty consecutively admitted CD patients and 15 healthy controls were included. Clinical and analytical characteristics of patients were considered. NOD2/CARD15 genotyping, presence of bacterial-DNA, defensin and cathelicidin gene, and protein levels in neutrophils and serum cytokine levels were studied. Results Twenty patients (40%) presented bacterial-DNA in blood. Eleven were active and 9 were in remission. Bacterial-DNA was not present in controls. NOD2/CARD15 mutations were identified in 25 patients (50%), 15 of which were in remission. Sixty percent of bacterial-DNA(+) and 43% of bacterial-DNA(−) patients showed a NOD2/CARD15 mutation. β-Defensin 2 and LL-37 mRNA and protein levels were upregulated in bacterial-DNA(+) patients. β-Defensin 2 and LL-37 expression correlated with bacterial-DNA concentration only in patients with a wildtype NOD2/CARD15 genotype. Cultured neutrophils of bacterial-DNA(-) patients confirmed the muramyl dipeptide-independent association between DEFB2 and LL-37 with bacterial-DNA concentration in wildtype NOD2/CARD15 patients. Cytokine levels were increased in bacterial-DNA(+) patients and correlated with bacterial-DNA concentration. NOD2/CARD15 genotype did not influence this correlation. Conclusions β-Defensin 2, LL-37, and proinflammatory cytokines are increased in CD patients with bacterial-DNA in a concentration-dependent manner. NOD2/CARD15 plays a key role in the regulation of this response. (Inflamm Bowel Dis 2010;) Crohn's disease, bacterial DNA translocation, NOD2/CARD15, defensin, cathelicidin, cytokines Crohn's disease (CD) is one of two major forms of inflammatory bowel disease (IBD), with an increasing incidence worldwide.1,2 It is primarily localized at ileum and/or colon and genetically conditioned, at least in part, through nucleotide-binding oligomerization domain containing 2 (NOD2/CARD15) single nucleotide polymorphism (SNP) mutations, which apparently confer a predisposition to develop CD.3,4 The host's innate immune response in CD is a complex system in which different immune cell types, such as neutrophils or macrophages, together with epithelial cells cooperate to mount an effective defense. Antimicrobial peptides like defensins and cathelicidins are increasingly recognized as an important part of this response. Defensins are small cysteine-rich cationic proteins with antimicrobial activity against bacteria, fungi, and many viruses. In mammals they are distributed into two main forms: α-defensins, primary expressed by neutrophils and by Paneth cells, and β-defensins, most widely distributed in epithelial cells. Several recent studies are revealing the modulatory activity of defensins, mainly at the local inflammatory level. For example, an increased expression of human β-defensin (DEFB)2 has been reported in colonic CD.5,–7 In the attempt to determine the role of defensins as a cause or a consequence of CD, studies were conducted reporting whether an association between NOD2/CARD15 status and mucosal α-defensin expression could be established,8,9 and the literature supporting that association is now abundant.10,–13 Others have demonstrated that NOD2/CARD15 regulates DEFB2 expression in primary epithelial cells in skin.14 From a systemic point of view, elevation of circulating DEFA1-3 versus DEFB2 levels has been reported in plasma of patients with IBD15 and some DEFB have shown an inducible expression in healthy controls after in vitro culture with bacterial products.16 On the other hand, cathelicidins also are a family of antimicrobial peptides structurally characterized by the presence of a highly conserved cathelin domain. Neutrophils are potent producers of the cathelicidin LL-37,17,18 a 37-residue peptide with broad-spectrum bactericidal activity.19 Studies have shown that a vitamin D pathway activates antimicrobial peptides in functional NOD2/CARD15 cells from CD patients in response to muramyl dipeptide (MDP), whereas it fails to induce these agents in cells homozygous for an inactivating NOD2/CARD15 mutation.3,20,–22 We recently described the frequent translocation of bacterial genomic fragments (bactDNA) into blood of patients with IBD, irrespective of disease activity.23 Systemic inflammatory consequences include an enhanced Th1-driven immune response by activation of nuclear factor (NF)-κB through Toll-like receptor (TLR)-9,24,–27 similar to what muramyl dipeptide does through NOD2/CARD15.26,–28 Since a linkage has been suggested between defensins, LL-37, and NOD2/CARD15, and some of these agents have shown an inducible expression in vitro after culture with bacterial products,16 we aimed to test the hypothesis of whether an association between defensins and LL-37 expression, bactDNA presence, and NOD2/CARD15 mutations can be established in peripheral blood neutrophils from CD patients. Knowing if NOD2/CARD15 mutations facilitate bacterial DNA translocation, and therefore an exacerbated immune reaction, may be of great interest not only to treat patients but also to prevent complications. Patients and Methods Patients Fifty consecutive patients previously diagnosed with CD were included in this study. The diagnosis of CD was established on standard clinical, endoscopic, histological, and radiographic criteria.29 Patients who had received antibiotics in the previous 4 weeks were not considered for inclusion. All patients were classified according to the Montreal Classification.30 Fifteen healthy controls were included in the study. Blood samples from each patient and from each healthy donor were obtained for routine hematological and biochemical studies and inoculated in aerobic and anaerobic blood culture bottles, 10 mL each. Simultaneously, two separate blood samples were inoculated, under aseptic conditions, in rubber-sealed sterile Vacutainer SST II and K3E tubes (BD Diagnostics, Belgium) that were never exposed to free air. The Ethics Committee of the Hospital General Universitario de Alicante approved the study protocol and all controls and patients gave informed consent for inclusion in the study. Isolation and Culture of Human PMN Cells Polymorphoniclear's (PMNs), from peripheral blood samples were isolated with PolymorphPrep (Axis-Shield PoC, Oslo, Norway) according to the manufacturer's instructions. After PMN isolation, cells were washed twice with freshly made phosphate-buffered saline (PBS) at 4°C. Cell viability was evaluated by Trypan blue (Sigma, Madrid, Spain). Cells were aliquoted and 10 × 106 used for genomic DNA and total RNA isolation, as described below. In all patients without bactDNA, the rest was resuspended in phenol red free RPMI 1640 medium (Gibco BRL, Life Technologies, Paisley, UK) supplemented with 10% human serum AB (BioWhittaker, Walkersville, MD), 100 IU/mL penicillin/streptomycin, and 2.5 mg/mL amphotericin B (Gibco BRL). Two-million cells per well were cultured for 24 hours without stimuli, with 10, 20, and 40 ng/1×106 cells of bactDNA obtained from a pool of bacterial species and with 1 μg/1×106 cells of MDP (N-acetylmuramyl-L-Alanyl-D-Isoglutamine Hydrate, Sigma-Aldrich, Madrid, Spain) as control for positive response. After that, cells and supernatants were collected for gene and protein assays. NOD2/CARD15 Genotyping, Defensin, and LL-37 mRNA Expression Genomic DNA was isolated from 5 × 106 cells with the QIAmp DNA Blood Minikit (Qiagen, Hilden, Germany). NOD2/CARD15 mutations were evaluated as previously described.31 Total cellular RNA was isolated from 5 × 106 cells with the QIAmp RNA Blood Minikit (Qiagen). Quantitec SYBR Green (Qiagen) was used to perform gene expression with an IQ5 Real-Time polymerase chain reaction (PCR) (BioRad, Hercules, CA). DEFA1-3 gene expression was evaluated using 5′-ATCCTTGCTGCCATTCTC CT-3′ as forward primer and 5′-TCCCTGTAGTCTCAAA GCAA-3′ as reverse primer. Other primers used were: for DEFB1, 5′-TGCTGTTTACTCTCTGCTTACT-3′ as forward and 5′-TCATTTCTTCTGGTCACTCTCA-3′ as reverse; for DEFB2, 5′-CCAGCCATCAGCCATGAGGGT-3′ as forward and 5′-GGAGCCCTTTCTGAATCCGCA-3′ as reverse; for DEFB3, 5′-AGCCTAGCAGCTATGAGGATC-3′ as forward and 5′-CTTCGGCAGCATTTTCGGCCA-3′ as reverse; for LL37 5′-GTGCCCCAGGACGACACAGC-3′ as forward and 5′-CCCCTGGCCTGGTTGAGGGT-3′ as reverse; and for β2-microglobulin, 5′-GACTTTGTCACAGCCCAAGA-3′ as forward and 5′-CAAGCAAGCAGAATTTGGAA-3′ as reverse. Ratios of defensins and LL37 to β2-microglobulin expression represented their level of mRNA relative expression. Quantification of Serum Cytokine, Defensin, and LL-37 Protein Levels Enzyme-linked immunosorbent assays (ELISAs) were carried out for IL-21, TNF-α, and IFN-γ (Human Quantikine kits, R&D Systems, Minneapolis, MN), DEFB2 and DEFB3 (Phoenix Pharmaceuticals, Karlsruhe, Germany), DEFB1 (Promokine, Heidelberg, Germany), DEFA1-3, and LL-37 (Hycult Biotechnology, Uden, Netherlands) according to the manufacturers' instructions. All samples were tested in triplicate and read with a Sunrise Microplate Reader (Tecan, Männedorf, Switzerland). The lower limit of detection for each assay was: 2–5 pg/mL for all cytokines; 40 pg/mL for DEFA1-3; 10 pg/mL for DEFB1 and DEFB2; and 0.1 ng/mL for DEFB3 and LL-37. Standard curves were generated for every plate and the average zero standard optical densities were subtracted from the rest of the standards, controls, and samples to obtain a corrected concentration for all parameters. Identification of BactDNA Fragments and Measurement of Serum LPS Levels BactDNA was identified in blood samples by running a broad-range PCR followed by nucleotide sequencing of a conserved region of 16SrRNA gene, as previously described.32 PCR amplicons were loaded onto DNA Laboratory-on-chips (Agilent Technologies, Palo Alto, CA) and analyzed with an Agilent 2100 BioAnalyzer. Statistical Analysis Continuous variables are reported as mean ± standard deviation and categorical variables as frequency or percentages. The Kolmogorov–Smirnov test was used to test the normality of the distribution of continuous variables. Statistical differences of basal characteristics between groups were analyzed using the chi test for categorical data and an analysis of variance (ANOVA) test with Bonferroni's correction for multiple comparisons for quantitative data showing normal distribution or the Mann–Whitney U-test for quantitative data showing nonnormal distribution. Bivariate correlations were analyzed using the Spearman test. Analysis of covariance (ANCOVA) was used to further examine the most contributing factors (NOD2/CARD15 genotype and CDAI) and covariates (bacterial DNA concentration and serum cytokines levels) explaining defensin and LL37 expression levels in human peripheral blood neutrophils. All reported P values are two-sided, and P values lower than 0.05 were considered to indicate significance. All calculations were performed using the SPSS 16.0 software (Chicago, IL). Results Characteristics of Patients Clinical and analytical characteristics of patients included in the study are detailed in Table 1, distributed according to the Crohn's Disease Activity Index (CDAI). No statistically significant clinical or epidemiological differences were observed between patients according to their clinical status. Disease was located in the ileum (62%) versus colon (20%) (P < 0.05), according to the Montreal location score. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were significantly increased in active patients. No statistically significant associations between immunosuppressive therapies or use of biologics and evidences of bacterial translocation, NOD2/CARD15 status, defensin expression, or cytokine levels could be established in active CD patients. Among healthy controls the mean age was 32 ± 11 years, male/female ratio was 9/6, and 35% of them were smokers. No analytical differences between controls and patients in remission were observed. All patients and controls included in the study showed a negative blood microbiological culture at inclusion in the study. Table 1 Clinical and Analytical Characteristics of Patients Included in the study     View Large Table 1 Clinical and Analytical Characteristics of Patients Included in the study     View Large All patients were followed up for a 6 months. Among patients in remission, 7 out of 30 (23%) relapsed in this period. Six (85%) presented bactDNA in blood and a mutated NOD2/CARD15 genotype. Serum BactDNA and NOD2/CARD15 Status in CD Patients From the overall series of patients, 20 out of 50 (40%) showed the presence of bactDNA in blood samples. By groups, 11 out of 20 patients clinically active (55%) and 9 out of 30 patients in remission (30%) showed bactDNA. Identified bacterial species included: Escherichia coli (n = 8), Staphylococcus aureus (n = 6), Klebsiella pneumoniae (n = 3), Streptococcus pneumoniae (n = 2), and Shigella spp. (n = 1). The mean amplified bactDNA concentration was 27.63 ± 8.06 ng/μL, with no statistically significant differences between groups in Gram-negative and Gram-positive species. No clinical or analytical differences were appreciated when subdividing the overall series of CD patients in remission according to the presence bactDNA in serum. BactDNA was not present in blood of any of the healthy controls. NOD2/CARD15 mutations were identified in a total of 25 patients (50%). By groups, 10 out 20 active patients (50%) and 15 out of 30 patients in remission (50%) showed a mutated NOD2/CARD15 genotype. SNP mutations distribution in the overall series of patients was: SNP-8 (n = 17), SNP-12 (n = 4), and SNP-13 (n = 4), with no significant differences according to the CDAI. Two out of 15 healthy controls (13%) presented any NOD2/CARD15 mutations. Sixty percent of patients with bactDNA and 43% of patients without bactDNA showed NOD2/CARD15 mutations (P = n.s.). A mutated NOD2/CARD15 genotype was found in 58% of patients with pure ileal (L1) disease and in 44% of patients with a penetrating (B3) behavior. No significant differences were observed between specific mutated NOD2/CARD15 genotypes and any clinical characteristic of patients. BactDNA Affects DEFB2 and LL-37 Expression in Neutrophils of CD Patients DEFA1-3 mRNA was constitutively expressed through the overall series of controls and patients, irrespective of the presence of bactDNA or its concentration and no statistically significant differences were found in DEFB1 relative expression between controls and patients, even in the presence of bactDNA (Supporting Fig. 1). Relative gene expression of DEFB2 was lower in controls and in patients without bactDNA than in patients with bactDNA, irrespective of their disease activity. However, patients with bactDNA showed a variable expression of DEFB2 that was affected by bactDNA concentration, describing a two-group pattern (Fig. 1A). LL37 mRNA expression was significantly higher in patients with bactDNA than in the rest of the groups and, as shown for DEFB2, the expression of LL-37 seemed to describe two different subpopulations when represented against bactDNA concentration (Fig. 1B). DEFB3 mRNA was not detectable either in blood neutrophils of CD patients or healthy controls. Figure 1 View largeDownload slide (A) DEFB2 and LL-37 mRNA relative expression in controls and CD patients, distributed according to the presence of bactDNA. (B) DEFB2 and LL-37 mRNA relative expression in patients with bactDNA, according to bactDNA concentration. mRNA levels are normalized to constitutive β2-microglobulin expression. DEFB2: β-Defensin 2; LL-37: 37-residue antimicrobial peptide; bactDNA: bacterial DNA. Figure 1 View largeDownload slide (A) DEFB2 and LL-37 mRNA relative expression in controls and CD patients, distributed according to the presence of bactDNA. (B) DEFB2 and LL-37 mRNA relative expression in patients with bactDNA, according to bactDNA concentration. mRNA levels are normalized to constitutive β2-microglobulin expression. DEFB2: β-Defensin 2; LL-37: 37-residue antimicrobial peptide; bactDNA: bacterial DNA. The results were confirmed at a protein level in all cases and can be found in the Supporting Material that describe defensin and LL-37 protein levels in all patients and controls included in the study according to CDAI distribution and NOD2/CARD15 genotype (Supporting Table 1A) or presence of bactDNA (Supporting Table 2A). DEFB2 levels in active CD patients with wildtype NOD2/CARD15 were significantly higher than in patients with mutated NOD2/CARD15. To further study differences in DEFB2 and LL-37 expression, bactDNA(+) patients were distributed according to the presence of NOD2/CARD15 mutations. A clear-cut correlation was observed between these two molecules and bactDNA concentration in patients with a wildtype NOD2/CARD15 genotype, whereas this correlation was not found in the presence of NOD2/CARD15 mutations (Figs. 2, 3, respectively). These results were unaltered when statistically controlled by CDAI. A positive correlation was observed between DEFB2 and LL-37 in bactDNA(+) patients with wildtype NOD2/CARD15 genotype both at the gene expression (r = 0.84, P = 0,01) and protein level (r = 0,81, P = 0,01). Figure 2 View largeDownload slide DEFB2 relative mRNA and protein levels in the overall series of bactDNA-positive patients distributed according to the NOD2/CARD15 genotype. DEFB2 is also represented against bactDNA concentration according to NOD2/CARD15 genotype in bactDNA-positive patients. mRNA levels are normalized to constitutive β2-microglobulin expression. DEFB2: β-defensin 2; bactDNA: bacterial DNA; wt: wildtype; mut: mutated. Figure 2 View largeDownload slide DEFB2 relative mRNA and protein levels in the overall series of bactDNA-positive patients distributed according to the NOD2/CARD15 genotype. DEFB2 is also represented against bactDNA concentration according to NOD2/CARD15 genotype in bactDNA-positive patients. mRNA levels are normalized to constitutive β2-microglobulin expression. DEFB2: β-defensin 2; bactDNA: bacterial DNA; wt: wildtype; mut: mutated. Figure 3 View largeDownload slide LL-37 relative mRNA and protein levels in bactDNA-positive patients, according to bactDNA concentration and distributed by NOD2/CARD15 status. LL-37 is also represented against bactDNA concentration according to NOD2/CARD15 genotype in bactDNA-positive patients. mRNA levels are normalized to constitutive β2-microglobulin expression. BactDNA: bacterial DNA; wt: wildtype; mut: mutated; LL-37: 37-residue antimicrobial peptide. Figure 3 View largeDownload slide LL-37 relative mRNA and protein levels in bactDNA-positive patients, according to bactDNA concentration and distributed by NOD2/CARD15 status. LL-37 is also represented against bactDNA concentration according to NOD2/CARD15 genotype in bactDNA-positive patients. mRNA levels are normalized to constitutive β2-microglobulin expression. BactDNA: bacterial DNA; wt: wildtype; mut: mutated; LL-37: 37-residue antimicrobial peptide. A bactDNA independent effect on DEFB2 and LL-37 expression was evaluated in vitro on blood neutrophils of bactDNA(−) patients. Increasing concentrations of bactDNA showed increased levels of DEFB2 and LL37 mRNA (Fig. 4A) and protein (Fig. 4B) after a 24-hour culture. Neutrophils without stimuli remained unaltered. The addition of MDP, as a positive control for DEFB2 and LL-37 stimulation, induced similar levels as bactDNA. A positive correlation between mRNA expression and protein levels was obtained for DEFB2 and LL-37 in the overall series of samples with different concentrations of bactDNA (Fig. 4C). A separate analysis of each NOD2/CARD15 SNP revealed no differences between them in their effect on DEFB2 and LL-37 expression in cultured neutrophils of bactDNA(−) patients (Supporting Table 3). Figure 4 View largeDownload slide (A) DEFB2 and LL-37 mRNA relative expression in cultured neutrophils of bactDNA-negative patients. (B) DEFB2 and LL-37 protein levels in the supernatants of cultured neutrophils of bactDNA-negative patients. (C) Correlations between mRNA relative expression and protein levels for DEFB2 and LL-37 in the overall series of cultured neutrophils treated with different bactDNA concentrations. mRNA levels are normalized to constitutive β2-microglobulin expression per 106 cells. DEFB2: β-defensin 2; LL-37: 37-residue antimicrobial peptide; bactDNA: bacterial DNA. Figure 4 View largeDownload slide (A) DEFB2 and LL-37 mRNA relative expression in cultured neutrophils of bactDNA-negative patients. (B) DEFB2 and LL-37 protein levels in the supernatants of cultured neutrophils of bactDNA-negative patients. (C) Correlations between mRNA relative expression and protein levels for DEFB2 and LL-37 in the overall series of cultured neutrophils treated with different bactDNA concentrations. mRNA levels are normalized to constitutive β2-microglobulin expression per 106 cells. DEFB2: β-defensin 2; LL-37: 37-residue antimicrobial peptide; bactDNA: bacterial DNA. Proinflammatory Cytokine Levels and BactDNA Concentration in Wildtype NOD2/CARD15 CD Patients Are Associated Serum levels of IL-21, TNF-α and IFN-γ were significantly increased in bactDNA(+) patients, irrespective or their disease activity, compared with controls and with bactDNA(−) patients. Cytokine levels were not statistically different between patients with the same activity grouped by NOD2/CARD15 status. However, when comparing patients with different disease activity, significantly increased TNF-α and IFN-γ levels were present in NOD2/CARD15 wildtype patients, whereas no differences were observed among mutated NOD2/CARD15 patients (Supporting Table 1B). However, the simultaneous presence of bactDNA and high activity index (CDAI >150) did not further increase the inflammatory response when compared with patients with bactDNA and low (CDAI <150) activity index (Supporting Table 2B). BactDNA(+) patients showed a positive correlation between all three studied cytokines and bactDNA concentration, irrespective of NOD2/CARD15 genotype (Fig. 5A). Since a correlation had been established between bactDNA concentration and the expression of DEFB2 and LL-37, further correlations with cytokine levels were evaluated in the subset of bactDNA(+) patients. Positive correlations were found between TNF-α and IFN-γ concentration and the expression of DEFB2 (Fig. 5B) and LL-37 (Fig. 5C) in wildtype NOD2/CARD15 patients. These correlations were not present in mutated NOD2/CARD15 patients. Statistically significant correlations were also observed between IL-21 levels and bactDNA concentration, irrespective of NOD2/CARD15 genotype and between IL-21 levels and the expression of DEFB2 and LL-37 in wildtype NOD2/CARD15 patients (Supporting Fig. 2). Correlations between cytokine levels and DEFA1-3 DEFB1 or DEFB3 could not be confirmed in any case (data not shown). An ANCOVA test revealed that bactDNA concentration and the interaction between bactDNA concentration and NOD2/CARD15 were the factors significantly influencing DEFB2 and LL-37 expression in peripheral blood neutrophils from IBD patients (Supporting Table 4). Figure 5 View largeDownload slide (A) Proinflammatory cytokine levels in bactDNA-positive patients, according to bactDNA concentration and distributed by NOD2/CARD15 status. (B) Correlations between DEFB2 protein levels and proinflammatory cytokines in bactDNA-positive patients, distributed by NOD2/CARD15 status. (C) Correlations between LL-37 protein levels and proinflammatory cytokines in bactDNA-positive patients, distributed by NOD2/CARD15 status. DEFB2: β-defensin 2; LL-37: 37-residue antimicrobial peptide; bactDNA: bacterial DNA; wt: wildtype; mut: mutated. Figure 5 View largeDownload slide (A) Proinflammatory cytokine levels in bactDNA-positive patients, according to bactDNA concentration and distributed by NOD2/CARD15 status. (B) Correlations between DEFB2 protein levels and proinflammatory cytokines in bactDNA-positive patients, distributed by NOD2/CARD15 status. (C) Correlations between LL-37 protein levels and proinflammatory cytokines in bactDNA-positive patients, distributed by NOD2/CARD15 status. DEFB2: β-defensin 2; LL-37: 37-residue antimicrobial peptide; bactDNA: bacterial DNA; wt: wildtype; mut: mutated. The bactDNA effect on cytokine levels was also evaluated in vitro on blood neutrophils of bactDNA(−) patients (Table 2). Treatment with bactDNA induced the upregulation of all proinflammatory cytokines irrespective of NOD2/CARD15 genotype status. However, MDP only stimulated cytokine secretion in cultured neutrophils of patients with a wildtype NOD2/CARD15 genotype. Table 2 Proinflammatory Cytokine Levels in Cultured Cells of BactDNA-negative Patients According to NOD2/CARD15 Genotype     View Large Table 2 Proinflammatory Cytokine Levels in Cultured Cells of BactDNA-negative Patients According to NOD2/CARD15 Genotype     View Large Discussion Systemic inflammatory response is a crucial part of the host's immune reaction to the recently described episodes of bactDNA translocation in CD patients.23 In the present study we show that this inflammatory reaction is associated with bactDNA in a concentration-dependent manner and that a wildtype NOD2/CARD15 genotype is required for an adequate antimicrobial peptide response against these bacterial challenges. Defensins and cathelicidins are families of small peptides with a wide antimicrobial activity.19,33 In humans, defensins are distributed into two main forms: α-defensins, primarily located and constitutively expressed at neutrophils and Paneth cells, and β-defensins, most widely distributed at epithelial cells,34,35 whereas only a 37-residue peptide from cathelicidins can be found in blood.19 α-Defensins have been shown to fail induction in healthy controls after blood in vitro culture with LPS, whereas DEFB1 and DEFB2 responded to stimulation.16 Therefore, as a result of bactDNA translocation episodes in CD patients,23 we intended to test whether antimicrobial peptides expression was induced in vivo in peripheral blood neutrophils in this setting. In our study, DEFA1-3 and DEFB1 mRNA was constitutively expressed in blood neutrophils from controls and patients, irrespective of the presence of bactDNA or its concentration, whereas DEFB3 could not be detected in either patients or healthy controls. Relative expression of DEFB2 and of LL-37, however, showed differences according to the presence of bactDNA. Moreover, two clear-cut tendencies were distinguished according to bactDNA concentration (Fig. 1). Several studies have reported an association between the expression of these antimicrobial peptides and the status of NOD2/CARD15.8,14,22 When bactDNA(+) patients were distributed according to the presence of NOD2/CARD15 mutations a positive correlation could be established between DEFB2 and LL-37 and bactDNA concentration in patients with a NOD2/CARD15 wildtype genotype both at mRNA and protein level, whereas this correlation was not found in the presence of mutated NOD2/CARD15 (Figs. 2, 3). These clear results suggest the mediation of NOD2/CARD15 in the DEFB2 signaling pathway through nuclear factor (NF)-κB, as previously reported in cultured epithelial cells after stimulation with MDP.14 In fact, due to the powerful effects of peptidoglycans on DEFB2 and LL-37 expression and in order to assess the independent effect of bactDNA on release of these antimicrobial peptides, in vitro studies on blood neutrophils of bactDNA(−) patients were performed. The data reveal that bactDNA, at the same concentrations as present in patients, maintains a positive correlation both with DEFB2 and LL37 mRNA relative expression (Fig. 4) and protein levels (Table 2) suggesting that, although the influence of MDP cannot be ruled out in wildtype NOD2/CARD15 bactDNA(+) patients, gene expression and protein levels of DEFB2 and LL-37 can be independently affected by the presence of bacterial genomic fragments in a concentration-dependent manner. From a clinical standpoint, and as in previous studies,23 a lack of correlation has been observed between the presence of bactDNA and a further compromised clinical status despite the associated increased inflammatory response. Similar findings were described in the past between disease activity and clinical endotoxemia.36,37 Correlations with specific treatments were not observed in this as well as in previous studies, either, probably due to the fact that inflammation may persist, while therapies only control the symptoms.38 However, its effect on the inflammatory status must be considered relevant by itself. It has been shown that increased levels of TNF-α modify the composition of colonic flora and modulate certain species gene expression that account for the clinical course of the disease.39 This would suggest that an increased Th1 response, as a consequence of bactDNA translocation, may perpetuate the progression of the disease in a subgroup of patients. Actually, during the 6-month follow-up seven patients in remission relapsed. Interestingly, six of them presented bactDNA and mutated NOD2/CARD15, suggesting that patients with NOD2/CARD15 mutations are more exposed to relapse under a hypothetical event of bacterial challenge. Besides, a bactDNA presence has been shown to statistically affect prognosis in other clinical settings, even though the antigenic presence was not correlated with development of infections.40,41 A correlation between ileal CD and NOD2/CARD15 mutations has been suggested in the past.3 In our series, 58% of patients with pure ileal disease and 44% of patients with a penetrating behavior showed mutated NOD2/CARD15. The association of this receptor's mutations with a more complicated disease course, and the related implications in the therapeutic strategy, has been widely studied, with controversial results, probably related to environmental factors,42,–45 and remains under discussion. What seems more evident is the important role of a NOD2 genotype in mediating the inflammatory response. In the present study a positive correlation could be established between DEFB2 expression and bactDNA concentration in CD patients only when the wildtype NOD2/CARD15 genotype was present. Similarly, significantly increased TNF-α and IFN-γ levels were present in active CD patients versus those in remission when NOD2/CARD15 showed the wildtype genotype. The mutated NOD2/CARD15 genotype failed to control this situation, probably due to the fact that the disease in 62% of our patients was of ileal origin and it has been suggested that NOD2/CARD15 mutations may differently affect immune tolerance mechanisms of the ileum and of the colon.44,46 In summary, DEFB2 and LL-37 expression is increased in peripheral blood neutrophils of CD patients with bactDNA. This association is established in a bactDNA concentration-dependent manner and mediated through wildtype NOD2/CARD15 genotype. The proinflammatory cytokine cascade is also affected by bactDNA concentration even in patients in remission. References 1 Hou JK, El-Serag H, Thirumurthi S. Distribution and manifestations of inflammatory bowel disease in Asians, Hispanics, and African Americans: a systematic review. Am J Gastroenterol . 2009; 104: 2100- 2109. CrossRef Search ADS PubMed  2 Shanahan F, Bernstein CN. The evolving epidemiology of inflammatory bowel disease. Curr Opin Gastroenterol . 2009; 25: 301- 305. CrossRef Search ADS PubMed  3 Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature . 2001; 411: 603- 606. CrossRef Search ADS PubMed  4 Sartor RB. Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am J Gastroenterol . 1997; 92: 5S- 11S. PubMed  5 O'Neil DA, Porter EM, Elewaut D, et al. Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol . 1999; 163: 6718- 6724. PubMed  6 Ramasundara M, Leach ST, Lemberg DA, et al. Defensins and inflammation: the role of defensins in inflammatory bowel disease. J Gastroenterol Hepatol . 2009; 24: 202- 208. CrossRef Search ADS PubMed  7 Wehkamp J, Fellermann K, Herrlinger KR, et al. Human beta-defensin 2 but not beta-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease. Eur J Gastroenterol Hepatol . 2002; 14: 745- 752. CrossRef Search ADS PubMed  8 Wehkamp J, Harder J, Weichenthal M, et al. NOD2 (CARD15) mutations in Crohn's disease are associated with diminished mucosal alpha-defensin expression. Gut . 2004; 53: 1658- 1664. CrossRef Search ADS PubMed  9 Simms LA, Doecke JD, Walsh MD, et al. Reduced alpha-defensin expression is associated with inflammation and not NOD2 mutation status in ileal Crohn's disease. Gut . 2008; 57: 903- 910. CrossRef Search ADS PubMed  10 Fishbein T, Novitskiy G, Mishra L, et al. NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity of the transplanted human small intestine. Gut . 2008; 57: 323- 330. CrossRef Search ADS PubMed  11 Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science . 2005; 307: 731- 734. CrossRef Search ADS PubMed  12 Wehkamp J, Salzman NH, Porter E, et al. Reduced Paneth cell alpha-defensins in ileal Crohn's disease. Proc Natl Acad Sci U S A . 2005; 102: 18129- 18134. CrossRef Search ADS PubMed  13 Petnicki-Ocwieja T, Hrncir T, Liu YJ, et al. Nod2 is required for the regulation of commensal microbiota in the intestine. Proc Natl Acad Sci U S A . 2009; 106: 15813- 15818. CrossRef Search ADS PubMed  14 Voss E, Wehkamp J, Wehkamp K, et al. NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2. J Biol Chem . 2006; 281: 2005- 2011. CrossRef Search ADS PubMed  15 Yamaguchi N, Isomoto H, Mukae H, et al. Concentrations of alpha- and beta-defensins in plasma of patients with inflammatory bowel disease. Inflamm Res . 2009; 58: 192- 197. CrossRef Search ADS PubMed  16 Fang XM, Shu Q, Chen QX, et al. Differential expression of alpha- and beta-defensins in human peripheral blood. Eur J Clin Invest . 2003; 33: 82- 87. CrossRef Search ADS PubMed  17 De Y, Chen Q, Schmidt AP, et al. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med . 2000; 192: 1069- 1074. CrossRef Search ADS PubMed  18 Sorensen OE, Follin P, Johnsen AH, et al. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood . 2001; 97: 3951- 3959. CrossRef Search ADS PubMed  19 Henzler-Wildman KA, Martinez GV, Brown MF, et al. Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37. Biochemistry . 2004; 43: 8459- 8469. CrossRef Search ADS PubMed  20 Fellermann K, Stange DE, Schaeffeler E, et al. A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon. Am J Hum Genet . 2006; 79: 439- 448. CrossRef Search ADS PubMed  21 Hugot JP, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature . 2001; 411: 599- 603. CrossRef Search ADS PubMed  22 Wang TT, Nestel FP, Bourdeau V, et al. Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immunol . 2004; 173: 2909- 2912. CrossRef Search ADS PubMed  23 Gutierrez A, Frances R, Amoros A, et al. Cytokine association with bacterial DNA in serum of patients with inflammatory bowel disease. Inflamm Bowel Dis . 2009; 15: 508- 514. CrossRef Search ADS PubMed  24 Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature . 2000; 408: 740- 745. CrossRef Search ADS PubMed  25 Wagner H. Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity. Curr Opin Microbiol . 2002; 5: 62- 69. CrossRef Search ADS PubMed  26 Eckmann L, Karin M. NOD2 and Crohn's disease: loss or gain of function? Immunity . 2005; 22: 661- 667. CrossRef Search ADS PubMed  27 Lala S, Ogura Y, Osborne C, et al. Crohn's disease and the NOD2 gene: a role for paneth cells. Gastroenterology . 2003; 125: 47- 57. CrossRef Search ADS PubMed  28 Ogura Y, Inohara N, Benito A, et al. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem . 2001; 276: 4812- 4818. CrossRef Search ADS PubMed  29 Sands BE. From symptom to diagnosis: clinical distinctions among various forms of intestinal inflammation. Gastroenterology . 2004; 126: 1518- 1532. CrossRef Search ADS PubMed  30 Silverberg MS, Satsangi J, Ahmad T, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol . 2005; 19(suppl A): 5- 36. CrossRef Search ADS   31 Hampe J, Grebe J, Nikolaus S, et al. Association of NOD2 (CARD 15) genotype with clinical course of Crohn's disease: a cohort study. Lancet . 2002; 359: 1661- 1665. CrossRef Search ADS PubMed  32 Such J, Frances R, Munoz C, et al. Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites. Hepatology . 2002; 36: 135- 141. CrossRef Search ADS PubMed  33 Wehkamp J, Schmid M, Fellermann K, et al. Defensin deficiency, intestinal microbes, and the clinical phenotypes of Crohn's disease. J Leukoc Biol . 2005; 77: 460- 465. CrossRef Search ADS PubMed  34 Rehaume LM, Hancock RE. Neutrophil-derived defensins as modulators of innate immune function. Crit Rev Immunol . 2008; 28: 185- 200. CrossRef Search ADS PubMed  35 Pazgier M, Hoover DM, Yang D, et al. Human beta-defensins. Cell Mol Life Sci . 2006; 63: 1294- 1313. CrossRef Search ADS PubMed  36 Caradonna L, Amati L, Magrone T, et al. Enteric bacteria, lipopolysaccharides and related cytokines in inflammatory bowel disease: biological and clinical significance. J Endotoxin Res . 2000; 6: 205- 214. PubMed  37 Caradonna L, Amati L, Lella P, et al. Phagocytosis, killing, lymphocyte-mediated antibacterial activity, serum autoantibodies, and plasma endotoxins in inflammatory bowel disease. Am J Gastroenterol . 2000; 95: 1495- 1502. CrossRef Search ADS PubMed  38 Modigliani R, Mary JY, Simon JF, et al. Clinical, biological, and endoscopic picture of attacks of Crohn's disease. Evolution on prednisolone. Groupe d'Etude Therapeutique des Affections Inflammatoires Digestives. Gastroenterology . 1990; 98: 811- 818. CrossRef Search ADS PubMed  39 Hecht GA. Inflammatory bowel disease—live transmission. N Engl J Med . 2008; 358: 528- 530. CrossRef Search ADS PubMed  40 Frances R, Zapater P, Gonzalez-Navajas JM, et al. Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis. Hepatology . 2008; 47: 978- 985. CrossRef Search ADS PubMed  41 Zapater P, Frances R, Gonzalez-Navajas JM, et al. Presence of bacterial DNA is a new survival indicator in patients with cirrhosis and non-infected ascitic fluid. Hepatology . 2008; 48: 1924- 31. CrossRef Search ADS PubMed  42 Ahmad T, Armuzzi A, Bunce M, et al. The molecular classification of the clinical manifestations of Crohn's disease. Gastroenterology . 2002; 122: 854- 866. CrossRef Search ADS PubMed  43 Cosnes J, Cattan S, Blain A, et al. Long-term evolution of disease behavior of Crohn's disease. Inflamm Bowel Dis . 2002; 8: 244- 250. CrossRef Search ADS PubMed  44 Lesage S, Zouali H, Cezard JP, et al. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet . 2002; 70: 845- 857. CrossRef Search ADS PubMed  45 Louis E, Collard A, Oger AF, et al. Behaviour of Crohn's disease according to the Vienna classification: changing pattern over the course of the disease. Gut . 2001; 49: 777- 782. CrossRef Search ADS PubMed  46 Colombel JF. The CARD15 (also known as NOD2) gene in Crohn's disease: are there implications for current clinical practice? Clin Gastroenterol Hepatol . 2003; 1: 5- 9. CrossRef Search ADS PubMed  Supporting Information Additional supporting information may be found in the online version of this article: Additional supporting information may be found in the online version of this article. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.
TI - Antimicrobial peptide response to blood translocation of bacterial DNA in Crohn's disease is affected by NOD2/CARD15 genotype
JO - Inflammatory Bowel Diseases
DO - 10.1002/ibd.21537
DA - 2011-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/antimicrobial-peptide-response-to-blood-translocation-of-bacterial-dna-krrr2B8gsA
SP - 1641
EP - 1650
VL - 17
IS - 8
DP - DeepDyve
ER -