Brief Article Open Access
Copyright ©2011 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jun 21, 2011; 17(23): 2829-2837
Published online Jun 21, 2011. doi: 10.3748/wjg.v17.i23.2829
NOD2 and ATG16L1 polymorphisms affect monocyte responses in Crohn’s disease
Dylan M Glubb, Richard B Gearry, Murray L Barclay, Rebecca L Roberts, John Pearson, Jacqui I Keenan, Judy McKenzie, Robert W Bentley
Dylan M Glubb, Department of Medicine, University of Chicago, IL 60637-1470, United States
Richard B Gearry, Murray L Barclay, Rebecca L Roberts, Department of Medicine, University of Otago, Christchurch, 8140, New Zealand
Richard B Gearry, Murray L Barclay, Department of Gastroenterology, Christchurch Hospital, Christchurch, 8140, New Zealand
Rebecca L Roberts, Department of Biochemistry, University of Otago, Dunedin, 9054, New Zealand
John Pearson, Department of Pathology, University of Otago, Christchurch, 8140, New Zealand
Jacqui I Keenan, Department of Surgery, University of Otago, Christchurch, 8140, New Zealand
Judy McKenzie, Department of Haematology, University of Otago, Christchurch, 8140, New Zealand
Robert W Bentley, Department of Paediatrics, University of Otago, Christchurch, 8140, New Zealand
Author contributions: Glubb DM, McKenzie J and Bentley RW performed the research; Glubb DM, Roberts RL, Bentley RW and Gearry RB designed the research; Pearson J, Glubb DM, Roberts RL and Bentley RW analyzed the data; Barclay ML and Gearry RB provided patient samples; all authors contributed to the writing of the manuscript.
Supported by Broad Medical Research Program of The Broad Foundation, Inflammatory Bowel Disease Grant IBD-0236. RLR is the recipient of a Sir Charles Hercus Health Research Fellowship from the Health Research Council of New Zealand
Correspondence to: Robert W Bentley, PhD, Department of Paediatrics, University of Otago, PO Box 4345, Christchurch 8140, New Zealand. robert.bentley@otago.ac.nz
Telephone: +64-3-3641558 Fax: +64-3-3640009
Received: July 1, 2010
Revised: September 30, 2010
Accepted: October 7, 2010
Published online: June 21, 2011

Abstract

AIM: To assess whether polymorphisms in NOD2 and ATG16L1 affect cytokine responses and mycobacterium avium subspecies paratuberculosis (MAP) survival in monocytes from Crohn’s disease (CD) patients.

METHODS: Monocytes were isolated from peripheral blood of CD patients of known genotype for common single nucleotide polymorphisms of NOD2 and ATG16L1. Monocytes were challenged with MAP and bacterial persistence assessed at subsequent time-points. Cytokine responses were assayed using a Milliplex multi-analyte profiling assay for 13 cytokines.

RESULTS: Monocytes heterozygous for a NOD2 polymorphism (R702W, P268S, or 1007fs) were more permissive for growth of MAP (P = 0.045) than those without. There was no effect of NOD2 genotype on subsequent cytokine expression. The T300A polymorphism of ATG16L1 did not affect growth of MAP in our model (P = 0.175), but did increase expression of cytokines interleukin (IL)-10 (P = 0.047) and IL-6 (P = 0.019).

CONCLUSION: CD-associated polymorphisms affected the elimination of MAP from ex vivo monocytes (NOD2), or expression of certain cytokines (ATG16L1), implying independent but contributory roles in the pathogenesis of CD.

Key Words: Inflammatory bowel disease, Mycobacterium avium subspecies paratuberculosis, Cytokine, CARD15, Autophagy



INTRODUCTION

Crohn’s disease (CD) has been proposed as being the product of chronic inflammation caused by a dysfunctional interaction between the intestinal immune system and commensal gut microbiota[1]. The inflammation seen in CD is characterized by pronounced Th1 and Th17/23 responses involving the cytokines interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, IFN-γ[1-3], IL-23 and IL-17[4-6].

Although the commensal intestinal microflora appear to play an important role in the etiology of CD, certain bacterial species have also been implicated as putative causal agents of CD. These include Pseudomonas maltophilia[7], Chlamydia trachomatis[8], Bacteroides fragilis[9] Yersinia species[10], adherent invasive Escherichia coli (E. coli)[11] and Mycobacterium avium subspecies paratuberculosis (MAP)[12]. The role of MAP in the etiology of CD is unclear. However, MAP causes chronic intestinal inflammation in ruminants (Johne’s disease) with similar pathophysiology to CD[13] and it has been reported as a causative agent of regional enteritis with similarities to CD in a number of monogastrics, including two primate species[14,15]. MAP has also been cultured from both resected tissue[16] and peripheral blood of CD patients[17], and has been visualized inside macrophages from CD patients[18]. Meta-analyses of epidemiological studies confirm an association of MAP with CD[19,20], although it remains unknown whether MAP is pathogenic in humans, or whether this association reflects a defective host immune system permissive for the survival of MAP.

Twin and family studies have demonstrated a significant genetic component to the development and progression of CD[21-23]. Linkage analysis, candidate gene approaches and, most recently, genome-wide association studies (GWAS) have identified over 30 risk genes for CD[24-26], many of which are involved with bacterial recognition (e.g. NOD2) or processing and elimination of bacteria through the autophagy pathway (e.g. IRGM[27] and ATG16L1[25]).

In this study, we developed an ex vivo monocyte model to assess the impact of the CD-associated single nucleotide polymorphisms (SNPs) in NOD2 (rs2066842, P268S; rs2066844, R702W; and 1007fs, rs2066847) and ATG16L1 (rs2241880, T300A) on cytokine responses to the putative pathogen MAP. MAP can survive and replicate within phagocytic cells, and consequently we also evaluated the impact of these polymorphisms on the intracellular persistence of MAP. The use of ex vivo monocytes allows functional evaluation of SNPs associated with CD, and may provide a more realistic insight into the impact that genotype has on CD compared to studies which involve abrogation of protein expression or whole gene deletion.

MATERIALS AND METHODS
Patient recruitment

Patients for the current study were selected from a New Zealand population-based Caucasian inflammatory bowel disease (IBD) cohort recruited to investigate genetic and environmental factors that contribute to IBD etiology[28-32]. Inclusion criteria for the current study were a confirmed diagnosis of CD and negative MAP status as ascertained by IS900 PCR in peripheral blood[28].

The genotype combinations and patient phenotype information are detailed in Table 1. Briefly, the potential impact of ATG16L1 and NOD2 polymorphisms were assessed separately. For analysis of ATG16L1, monocytes were collected from CD patients who had a wild-type NOD2 genotype and were homozygous for either the major (G) allele (n = 6) or minor (A) allele (n = 6) of ATG16L1 1138G > A (rs2241880). Conversely, for experiments evaluating the effect of NOD2 genotype, monocytes were collected from patients who were ATG16L1 1138G homozygotes and were heterozygous for one of the three NOD2 SNPs previously associated with CD; 2104C > T (R702W, rs2066844), 2722G > C (G908R, rs2066845), or 3020insC (1007fs, rs2066847), and were heterozygous for the background variant 802C > T (P268S, rs2066842). A total of 12 patients were recruited, 6 carried the polymorphisms described, and 6 were homozygous at these NOD2 loci (Table 1). None of the patients included in this study had the SNPs rs13361189 and rs4958847. These SNPs are in complete linkage disequilibrium with a 20 kb insertion/deletion polymorphism which has been shown to alter expression of the autophagy gene IRGM[33].

Table 1 IRGM, ATG16L1, and NOD2 genotypes of Crohn’s disease patients from whom monocytes were collected for ex vivo experiments.
IRGM
ATG16L1
NOD2
Clinical characteristics
Patientrs133611891rs49588471T300A 1138G > Ars2241880R702W 2104C > Trs2066844P268S 802C > Trs2066842G908R 2722G > Crs20668451007fs 3020insCrs2066847GenderTime since diagnosis (yr)Disease locationHarvey bradshaw index2
1TTGGAACCCCGG00M53Ileal0
2TTGGAACCCCGG00M5Ileo-colonic0
3TTGGAACCCCGG00F5Colonic0
4TTGGGGCCCCGG00M25Ileo-colonic2
5TTGGAACCCCGG00F6Ileo-colonic1
6TTGGAACCCCGG00F9Ileal3
7TTGGAACCCCGG00M1Ileal0
8TTGGGGCCCCGG00M6Colonic2
9TTGGGGCCCCGG00F10Colonic5
10TTGGGGCCCCGG00M24Ileo-colonic1
11TTGGGGCCCCGG00F11Colonic0
12TTGGGGCCCCGG00F7Ileo-colonic4
13TTGGGGCTCTGG00F5Colonic4
14TTGGGGCTCTGG00F9Ileo-colonic0
15TTGGGGCTCTGG00M17Ileal0
16TTGGGGCTCTGG00F18Colonic4
17TTGGGGCCCTGG0CM7Ileal1
18TTGGGGCTCTGG00F16Ileo-colonic4
Preparation of monocytes from peripheral blood

Blood (40 mL) was drawn into heparin tubes (Sigma-Aldrich), divided into 20 mL aliquots, and 15 mL of Phosphate Buffered Saline (PBS) was added to each. Ficoll-Paque™ PREMIUM (10 mL) (GE Healthcare Bio-Sciences Uppsala, Sweden) was layered under each aliquot and the samples centrifuged (1000 ×g, 20 min). Mononuclear cells collected from the interface were added to 30 mL of PBS, centrifuged (350 ×g, 10 min) and resuspended in 15 mL of PBS. In order to standardize the number of monocytes used in experiments, mononuclear cells were enumerated using a hemocytometer and a 200 μL aliquot was analyzed on a Beckman Coulter FC500 MPL flow cytometer to determine the percentage of monocytes based on forward and side scatter characteristics. After centrifugation (350 ×g, 10 min), cells were resuspended in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) to a concentration of 4 × 105 monocytes/mL. Then 500 μL of this preparation were plated per well in a 24-well tissue culture plate (Nunc, Roskilde, Denmark). Monocytes were left to adhere for 1 h in a humidified incubator at 37°C with 5% CO2 and non-adherent cells were removed by washing three times with 1 mL of room temperature (RT) PBS. Adherent monocytes were incubated overnight in 500 μL of RPMI 1640/10% FBS.

Culture of MAP

MAP strain Dominic (ATCC 43545) was inoculated into 10 mL volumes of Difco™ Middlebrook 7H9 broth (BD Biosciences, Sparks, MD, USA) supplemented with 10% v/v BBL™ Middlebrook OADC Enrichment (BD Biosciences), 0.05% v/v Tween 80 (Sigma-Aldrich, St Louis, MO, USA) and 2 mg/mL Mycobactin J (Allied Monitor, Fayette, MO, USA) and grown at 37°C. A standard growth curve of MAP was obtained by measuring the optical density at 600 nm (OD600) of an aliquot of bacterial suspension during the log phase, which had been passed repeatedly through a 25-gauge needle, and enumerating the bacteria by plating on the same medium supplemented with 1.5% agar (Invitrogen, Carlsbad, CA, USA).

Monocyte challenge experiments

Bacteria were grown to mid log phase (approximately 1 × 108 cells/mL, with reference to previously obtained growth curve data) and harvested by centrifugation (13 000 ×g, 5 min). Bacteria were resuspended in 500 μL of PBS and passed ten times through a 25-gauge needle to break up clumps of cells. One 500 μL aliquot of MAP was heat-inactivated at 90°C for 5 min and then both aliquots were diluted to 4 × 106 cells/mL in RPMI 1640/10% FBS. The optimal temperature and incubation time for heat-inactivation had been previously confirmed by plating heat-treated MAP cells onto agar. Growth medium was removed from the monocytes and 500 μL of the MAP suspension was added to each well. Challenge experiments were performed in triplicate for each time-point/genotype combination. After incubation for 4 h at 37°C, 5% CO2, the supernatant was removed and stored at -80°C as a zero time-point sample for cytokine assays. Monolayers were washed three times with 1 mL of PBS to remove extracellular bacteria. A zero time-point lysate to assess bacterial uptake and persistence was removed by incubating 500 μL of a 0.1% sodium deoxycholate (Sigma-Aldrich, St Louis, MO, USA) solution with the monolayer for 5 min at RT. Lysates were centrifuged (13 000 ×g, 5 min) and resuspended in 200 μL of PBS. Serial dilutions of the lysates were made with PBS and 50 μL were spread on to a Middlebrook agar plate. The agar plates were left to incubate for four weeks at 37°C before counting MAP colonies. Subsequent samples at time-points of 24, 48, 72 and 96 h were similarly processed.

Confirmation of genotypes of study participants

ATG16L1 genotypes were confirmed at recruitment by direct DNA sequencing of PCR products. Briefly, genomic DNA was extracted from fresh peripheral blood using GenElute™ (Sigma-Aldrich, St Louis, MO, USA) spin columns according to manufacturer’s protocols. A 480 bp fragment containing the ATG16L1 1138G > A SNP was amplified for DNA sequencing from patient genomic DNA using the following primers: 5'-CCACAGGTTAGTGTGCAGGA-3' (forward primer) and 5'-CACAGCTGACAGAGCCAAAA-3' (reverse primer). PCR was carried out in a 20 μL volume containing 1 μL of genomic DNA, 0.3 μmol/L of each primer, 200 μmol/L dNTPs, 0.75 mmol/L MgCl2, 1 × TAQ-Ti reaction buffer (Thermo Fisher Scientific, Pittsburgh, PA, USA) and 0.25 U of TAQ-Ti DNA polymerase (Thermo Fisher Scientific). After an initial denaturation step of 94°C for 2 min, 35 cycles were performed at 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. Five microlitres of each PCR product was checked on 1% agarose. Another 5 μL aliquot was purified with Exo-SAP-IT (USB Corporation, Ohio, USA) and sequenced using BigDye chemistry (Applied Biosystems, California, USA) on an ABI 3730 Genetic Analyzer (Foster City, California, USA). The NOD2 and IRGM genotypes of study participants were established as previously described using allele-specific PCR and pre-designed TaqMan SNP genotyping assays, respectively[34,35].

Multiplex cytokine analysis

Cytokine analysis was performed using a 13-plex MILLIPLEX™ MAP human cytokine kit according to manufacturer’s recommendations (Millipore) for the following: IFNγ, IL-10, IL-12p40, IL-12p70, IL-17, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, TNFα, TNFβ.

Statistical analysis

Data from the five time-points were analyzed by repeated measures ANOVA, with fixed effects for time (as categorical variable) and random effects for subject i.e. genotype. For cytokine analyses, where readings were below the threshold for detection (3.2 pg/mL) they were included as 3.2 pg/mL. Results were considered significant at P≤ 0.05.

Ethical considerations

Informed written consent was obtained from all participants in this study and ethical approval for this work was granted by the Upper South B Regional Ethics Committee of New Zealand.

RESULTS
Bacterial persistence ATG16L1

Numbers of MAP increased from a mean log10 colony forming units (cfu) of 4.24 at 0 h to 5.08 at 96 h for the AA genotype, and from log10 cfu of 4.47 to 5.29 for the GG genotype (Figure 1). There was no evidence (T-testing) for the effect of the ATG16L1 T300A polymorphism on MAP numbers at any individual time-point, or overall (pooled time-points ANOVA, P = 0.175) (Figure 1).

Figure 1
Figure 1 Effect of ATG16L1 T300A variant on mycobacterium avium subspecies paratuberculosis numbers. Time course of mycobacterium avium subspecies paratuberculosis (MAP) growth in the context of the ATG16L1 1138 G > A variant. Monocytes were derived from subjects homozygous for either ATG16L1 allele and carrying NOD2 wild-type alleles (n = 6 for each group). MAP growth is expressed as colony forming units (cfu).
NOD2

MAP also grew in the monocytes with different NOD2 genotypes, from a mean log10cfu of 4.30 at 0 h to 5.12 for the cells homozygous for the major alleles of the NOD2 variants, and from log10cfu of 4.58 to 5.42 for cells heterozygous for any NOD2 variant (Figure 2). There was no significant effect (T-testing) of NOD2 genotype on MAP numbers at any individual time-point. However, analysis of all time-points indicated that monocytes heterozygous for a NOD2 polymorphism were more permissive for growth of MAP (ANOVA, P = 0.045) (Figure 2).

Figure 2
Figure 2 Effect of NOD2 variants on mycobacterium avium subspecies paratuberculosis numbers. Time course of mycobacterium avium subspecies paratuberculosis (MAP) growth in the context of NOD2 genetic variation. Monocytes were derived from subjects homozygous for the ATG16L1 1138G allele and with/without NOD2 genetic variants (n = 6 for each group). MAP growth is expressed as colony forming units (cfu).
Multiplex cytokine panels

Thirteen cytokines were evaluated from ex vivo monocyte supernatants using a MILLIPLEX™ MAP human cytokine kit. Each time-point/genotype combination was assayed in triplicate. All assays passed quality controls and r2 values for the standard curves were ≥ 0.99.

Of the thirteen cytokines analyzed in the multiplex format, only four, IL-10, IL-6, IL-8 and TNFα, had measurable responses above the detection threshold (> 3.2 pg/mL) on the multiplex ELISA platform used. Where samples had values of < 3.2 pg/mL, they were considered to be 3.2 pg/mL for statistical purposes.

Effect of ATG16L1 genotype

Cytokine expression results are shown in Figure 3. The AA genotype of ATG16L1 was associated with greater expression of cytokines IL-10 and IL-6 in response to challenge with MAP (P = 0.047 and P = 0.019, respectively). No significant difference was seen between ATG16L1 genotypes AA and GG for expression of either IL-8 or TNFα (P = 0.758 and P = 0.289, respectively) (Figure 3).

Figure 3
Figure 3 Effect of the ATG16L1 T300A polymorphism on expression of cytokines following challenge with mycobacterium avium subspecies paratuberculosis. Cytokine concentrations are in pg/mL. Results are the means (and standard error of the mean) of triplicate samples from patients in Table 1. P-values are derived from repeated measures ANOVA testing for the effect of genotype on cytokine expression. Fixed effects for time (categorical variable) and ATG16L1 genotype, random effect for subject. IL: Interleukin; TNF: Tumor necrosis factor.
Effect of NOD2 genotype

Cytokine expression changes for IL-10, IL-6, IL-8 and TNFα by cells of different NOD2 genotypes are shown in Figure 4. In general, the presence of a NOD2 polymorphism resulted in a trend of lower expression of these four cytokines throughout the time-course, compared to monocytes without NOD2 variants.

Figure 4
Figure 4 Effect of NOD2 polymorphisms on expression of cytokines following challenge with mycobacterium avium subspecies paratuberculosis. Cytokine concentrations are in pg/mL. Results are the means (and standard error of the mean) of triplicate samples from patients in Table 1. P-values are derived from repeated measures ANOVA testing for the effect of genotype on cytokine expression. Fixed effects for time (categorical variable) and NOD2 genotype, random effect for subject. IL: Interleukin; TNF: Tumor necrosis factor.

However, this effect was not significant, with P-values of 0.56, 0.32, 0.41 and 0.97 for cytokines IL-10, IL-6, IL-8 and TNFα, respectively (Figure 4).

DISCUSSION

Numerous genetic association studies have indicated a strong role for NOD2 and ATG16L1 in the etiology of CD. However, functional studies have yet to fully elucidate whether, and to what extent, polymorphic variation in these genes affects bacterial clearance and inflammation in CD. Models of gene/environment interaction have mostly used epithelial or monocyte cell lines in vitro with bacterial pathogens such as Salmonella typhimurium that are not generally associated with CD. Also, the use of gene silencing or ablation techniques in these models may assist our understanding of the function of these genes, but may not be representative of the effects of SNPs which have been associated with CD. In this study we developed an ex vivo cellular model using monocytes from CD patients and a putative CD pathogen, MAP, in order to assess the effect of SNPs in NOD2 and ATG16L1 on bacterial survival and subsequent inflammatory response.

NOD2 is located in the cytoplasm, and plays an important role in cellular responses to bacterial infection[36] through recognition of muramyl dipeptide (MDP), a subunit of bacterial peptidoglycan (PGN)[37]. Furthermore, the R702W, G908R and 1007fs NOD2 variants, which confer susceptibility to CD, have been shown to impair responses to the bacterial antigen lipopolysaccharide[38].

We found the effect of NOD2 polymorphisms appeared to be primarily on bacterial persistence/growth, with heterozygosity at R702W or 1007fs making monocytes from CD patients significantly more permissive to growth of MAP. Indeed, a recent study has observed that these variants are associated with an impairment of monocyte phagocytosis and the development of bacteremia in intensive care unit patients[39]. Despite the increased bacterial load, no differences were seen in subsequent cytokine responses for the different host NOD2 genotypes. Whilst there has been no prior published research with respect to MAP in this type of model of CD, the functional effects of NOD2 SNPs have been examined for adherent invasive E. coli (AIEC). No differences were found in the persistence or growth of AIEC in monocytes from patients who were heterozygous or homozygous for the minor allele of NOD2 variants R702W, G908R and 1007fs compared to NOD2 major allele homozygotes[40]. It has also been shown that in peripheral blood mononuclear cells from CD patients, homozygosity for R702W does not affect the cytokine response to Gram-negative (Helicobacter pylori) bacterial peptidoglycan[41], although homozygosity for the 1007 fs mutation did. In our study we recruited no 1007fs homozygotes and only a single 1007 fs heterozygote so were unable to make any direct comparisons with this previous study. Comparative studies for a broader range of bacterial species in ex vivo cells from patients who are homozygous for these NOD2 SNPs will help clarify the role of this genotype in disease pathogenesis.

Both MAP and AIEC are capable of intracellular survival and growth in monocytes and macrophages, and it appears that the presence of NOD2 polymorphisms may influence their respective intracellular survival and growth in different ways[42].

Autophagy has been identified as a mechanism for clearing intracellular pathogens, and two autophagy genes, ATG16L1 and IRGM, have been associated with CD[25,27]. In our study, we investigated the effect of the ATG16L1 T300A polymorphism, and controlled for genotypic variation in IRGM (Table 1). ATG16L1 T300A (rs2241880; 1138G > A) is a common non-synonymous SNP where the G major allele confers greater disease risk and results in a threonine-to-alanine substitution at amino acid position 300 of the ATG16L1 protein (T300A). This SNP appears to account for all of the disease risk conferred by this locus[43], and functionally, this polymorphism has been proposed to contribute to defective macrophage killing of bacteria[44]. This assertion is supported by the results of two in vitro studies. Kuballa et al[45] found that the T300A variant impaired handling and autophagy of Salmonella within human epithelial cells, and Lapaquette et al[46] showed that siRNA knockdown of ATG16L1 led to loss of autophagy of intracellular AIEC bacteria by HeLa cells. Transfection of affected HeLa cells with wild-type ATG16L1 restored autophagic function, whereas transfection with the T300A polymorphic form did not. In contrast, ATG16L1 T300A had no effect on the survival of either S. typhimurium or group A Streptococcus in mouse embryonic fibroblasts[47]. Although direct comparison of different model systems is complex and potentially misleading, our results with MAP in ex vivo monocytes generate the question as to whether the T300A polymorphism of ATG16L1 affects autophagic clearance of certain intracellular bacteria as profoundly as indicated by knockdown or silencing models of gene function.

In our study, the T300A polymorphism was associated with significant changes in production of the cytokines IL-6 and IL-10 in response to bacterial challenge with MAP. These two cytokines are components of the Th1 (pro-inflammatory) and Th2 (modulatory) pathways of inflammation, respectively, and it is likely that CD results from an imbalance between these two pathways. It is tempting to speculate that the relative levels of the two cytokines that were induced are indicative of an imbalance, but extrapolating from a very specific model to describe a complex disease state would be misleading and clearly further comparative work is required in this area in disease-relevant models.

Our study is the first to investigate the effect of NOD2 and ATG16L1 genotype on the response of ex vivo human monocytes to the putative CD pathogen MAP. Although our results are preliminary and need to be replicated in a larger sample, they provide novel insights into the effect of disease-associated SNPs in innate immunity genes on detection, handling, and elimination of bacteria, and ultimately CD pathogenesis. Our observations indicate that NOD2 SNPs R702W, P268S, or 1007 fs impair the elimination of MAP yet do not impact on cytokine production. They may, therefore, increase susceptibility to prolonged intracellular bacterial infection. Conversely, the ATG16L1 T300A polymorphism significantly alters the expression of certain Th1 and Th2 cytokines after MAP challenge, but does not seem to affect the autophagic clearance of this putative CD pathogen.

COMMENTS
Background

Polymorphisms of the genes NOD2 and ATG16L1 have been associated with susceptibility to Crohn’s disease (CD). These genes are important for an effective innate immune response against potential bacterial pathogens [such as Mycobacterium avium subspecies paratuberculosis (MAP)] which may trigger or exacerbate inflammation. Monocytes from CD patients of known genotype were used to determine whether polymorphisms in NOD2 and ATG16L1 alter cytokine responses and bacterial survival following challenge with MAP.

Research frontiers

Previous research has investigated the role of polymorphisms in NOD2 and ATG16L1 in various model systems. In general, these have used gene silencing strategies that may not realistically reflect the biological consequences of single nucleotide changes in these genes, or model bacterial pathogens that have little relevance to CD. None have reported the functional consequences of the naturally occurring single nucleotide polymorphisms using patient-derived cells and bacteria such as MAP that have been implicated in CD etiology.

Innovations and breakthroughs

The authors findings indicate that monocytes heterozygous for a NOD2 polymorphism were more permissive for the intracellular growth of MAP than those without. However, these polymorphisms did not affect subsequent cytokine expression. The T300A polymorphism of ATG16L1 did not affect growth of MAP in our monocyte model but did result in increased expression of certain cytokines - interleukin (IL)-10 and IL-6.

Applications

By understanding how naturally occurring disease-related polymorphisms of NOD2 and ATG16L1 influence bacterial survival and also the production of inflammatory mediators, the authors may gain insight into the contribution of these genetic changes to the function of the host innate immune system. Development of this model system that utilizes patient cells with known single nucleotide changes in key CD-susceptibility genes will provide another research tool to assist better understanding of disease pathogenesis related to bacterial handling.

Terminology

NOD2 (CARD15) - nucleotide oligomerization domain 2 - is a cytosolic pattern recognition receptor that recognizes muramyl dipeptide, a component of bacterial peptidoglycan. Polymorphisms in NOD2 have been associated with altered susceptibility to CD in many genetic studies. ATG16L1 - autophagy-related 16-like 1 - is a key component of the autophagic apparatus that is involved with uptake and digestion of intracellular bacteria. The T300A polymorphism of ATG16L1 has also been associated with altered susceptibility to CD. MAP is an intracellular bacterium that has been cited in several studies as a putative causal agent of CD.

Peer review

This is a very well-designed and well-written study, with interesting and important scientific merit. Not just simple polymorphism descriptions, but their effect on human monocyte cytokine production and intracellular pathogen survival were examined with a very functional methodology. Their ex vivo model is much closer to the real pathogenesis of CD than any earlier one. Using the author’s concept, more descriptive polymorphism analysis of CD and other diseases may be placed into functional analysis.

Footnotes

Peer reviewers: Shashi Bala, PhD, Post Doctoral Associate, Department of Medicine, LRB 270L, 364 Plantation street, UMass Medical School, Worcester, MA 01605, United States; Giuliana Decorti, MD, PhD, Department of Life Sciences, University of Trieste, Via L. Giorgieri n° 7, Trieste 34127, Italy

S- Editor Sun H L- Editor Logan S E- Editor Ma WH

References
1.  Packey CD, Sartor RB. Interplay of commensal and pathogenic bacteria, genetic mutations, and immunoregulatory defects in the pathogenesis of inflammatory bowel diseases. J Intern Med. 2008;263:597-606.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Reimund JM, Wittersheim C, Dumont S, Muller CD, Kenney JS, Baumann R, Poindron P, Duclos B. Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn's disease. Gut. 1996;39:684-689.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Hisamatsu T, Suzuki M, Reinecker HC, Nadeau WJ, McCormick BA, Podolsky DK. CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells. Gastroenterology. 2003;124:993-1000.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Filì L, Ferri S, Frosali F. Phenotypic and functional features of human Th17 cells. J Exp Med. 2007;204:1849-1861.  [PubMed]  [DOI]  [Cited in This Article: ]
5.  Kamada N, Hisamatsu T, Okamoto S, Chinen H, Kobayashi T, Sato T, Sakuraba A, Kitazume MT, Sugita A, Koganei K. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis. J Clin Invest. 2008;118:2269-2280.  [PubMed]  [DOI]  [Cited in This Article: ]
6.  Kobayashi K, Blaser MJ, Brown WR. Immunohistochemical examination for mycobacteria in intestinal tissues from patients with Crohn's disease. Gastroenterology. 1989;96:1009-1015.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Graham DY, Yoshimura HH, Estes MK. DNA hybridization studies of the association of Pseudomonas maltophilia with inflammatory bowel diseases. J Lab Clin Med. 1983;101:940-954.  [PubMed]  [DOI]  [Cited in This Article: ]
8.  Orda R, Samra Z, Levy Y, Shperber Y, Scapa E. Chlamydia trachomatis and inflammatory bowel disease--a coincidence? J R Soc Med. 1990;83:15-17.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser AL, Barnich N, Bringer MA, Swidsinski A, Beaugerie L, Colombel JF. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease. Gastroenterology. 2004;127:412-421.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  Kallinowski F, Wassmer A, Hofmann MA, Harmsen D, Heesemann J, Karch H, Herfarth C, Buhr HJ. Prevalence of enteropathogenic bacteria in surgically treated chronic inflammatory bowel disease. Hepatogastroenterology. 1998;45:1552-1558.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Rolhion N, Darfeuille-Michaud A. Adherent-invasive Escherichia coli in inflammatory bowel disease. Inflamm Bowel Dis. 2007;13:1277-1283.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  McFadden JJ, Butcher PD, Chiodini R, Hermon-Taylor J. Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J Clin Microbiol. 1987;25:796-801.  [PubMed]  [DOI]  [Cited in This Article: ]
13.  Greenstein RJ. Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease. Lancet Infect Dis. 2003;3:507-514.  [PubMed]  [DOI]  [Cited in This Article: ]
14.  McClure HM, Chiodini RJ, Anderson DC, Swenson RB, Thayer WR, Coutu JA. Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides). J Infect Dis. 1987;155:1011-1019.  [PubMed]  [DOI]  [Cited in This Article: ]
15.  Zwick LS, Walsh TF, Barbiers R, Collins MT, Kinsel MJ, Murnane RD. Paratuberculosis in a mandrill (Papio sphinx). J Vet Diagn Invest. 2002;14:326-328.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Chiodini RJ, Van Kruiningen HJ, Merkal RS, Thayer WR Jr, Coutu JA. Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease. J Clin Microbiol. 1984;20:966-971.  [PubMed]  [DOI]  [Cited in This Article: ]
17.  Naser SA, Ghobrial G, Romero C, Valentine JF. Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease. Lancet. 2004;364:1039-1044.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Gearry RB, Aitken JM, Roberts RL, Ismail S, Keenan J, Barclay ML. Images of interest. Gastrointestinal: Mycobacterium avium paratuberculosis and Crohn's disease. J Gastroenterol Hepatol. 2005;20:1943.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Abubakar I, Myhill D, Aliyu SH, Hunter PR. Detection of Mycobacterium avium subspecies paratuberculosis from patients with Crohn's disease using nucleic acid-based techniques: a systematic review and meta-analysis. Inflamm Bowel Dis. 2008;14:401-410.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Feller M, Huwiler K, Stephan R, Altpeter E, Shang A, Furrer H, Pfyffer GE, Jemmi T, Baumgartner A, Egger M. Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis. Lancet Infect Dis. 2007;7:607-613.  [PubMed]  [DOI]  [Cited in This Article: ]
21.  Orholm M, Binder V, Sørensen TI, Rasmussen LP, Kyvik KO. Concordance of inflammatory bowel disease among Danish twins. Results of a nationwide study. Scand J Gastroenterol. 2000;35:1075-1081.  [PubMed]  [DOI]  [Cited in This Article: ]
22.  Satsangi J, Grootscholten C, Holt H, Jewell DP. Clinical patterns of familial inflammatory bowel disease. Gut. 1996;38:738-741.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Spehlmann ME, Begun AZ, Burghardt J, Lepage P, Raedler A, Schreiber S. Epidemiology of inflammatory bowel disease in a German twin cohort: results of a nationwide study. Inflamm. Bowel Dis. 2008;14:968-976.  [PubMed]  [DOI]  [Cited in This Article: ]
24.  Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, Brant SR, Silverberg MS, Taylor KD, Barmada MM. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet. 2008;40:955-962.  [PubMed]  [DOI]  [Cited in This Article: ]
25.  Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, Green T, Kuballa P, Barmada MM, Datta LW. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39:596-604.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, Albrecht M, Mayr G, De La Vega FM, Briggs J. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet. 2007;39:207-211.  [PubMed]  [DOI]  [Cited in This Article: ]
27.  Singh SB, Davis AS, Taylor GA, Deretic V. Human IRGM induces autophagy to eliminate intracellular mycobacteria. Science. 2006;313:1438-1441.  [PubMed]  [DOI]  [Cited in This Article: ]
28.  Bentley RW, Keenan JI, Gearry RB, Kennedy MA, Barclay ML, Roberts RL. Incidence of Mycobacterium avium subspecies paratuberculosis in a population-based cohort of patients with Crohn's disease and control subjects. Am J Gastroenterol. 2008;103:1168-1172.  [PubMed]  [DOI]  [Cited in This Article: ]
29.  Bentley RW, Pearson J, Gearry RB, Barclay ML, McKinney C, Merriman TR, Roberts RL. Association of higher DEFB4 genomic copy number with Crohn's disease. Am J Gastroenterol. 2010;105:354-359.  [PubMed]  [DOI]  [Cited in This Article: ]
30.  Gearry RB, Richardson A, Frampton CM, Collett JA, Burt MJ, Chapman BA, Barclay ML. High incidence of Crohn's disease in Canterbury, New Zealand: results of an epidemiologic study. Inflamm Bowel Dis. 2006;12:936-943.  [PubMed]  [DOI]  [Cited in This Article: ]
31.  Roberts RL, Gearry RB, Hollis-Moffatt JE, Miller AL, Reid J, Abkevich V, Timms KM, Gutin A, Lanchbury JS, Merriman TR. IL23R R381Q and ATG16L1 T300A are strongly associated with Crohn's disease in a study of New Zealand Caucasians with inflammatory bowel disease. Am J Gastroenterol. 2007;102:2754-2761.  [PubMed]  [DOI]  [Cited in This Article: ]
32.  Roberts RL, Hollis-Moffatt JE, Gearry RB, Kennedy MA, Barclay ML, Merriman TR. Confirmation of association of IRGM and NCF4 with ileal Crohn's disease in a population-based cohort. Genes Immun. 2008;9:561-565.  [PubMed]  [DOI]  [Cited in This Article: ]
33.  McCarroll SA, Huett A, Kuballa P, Chilewski SD, Landry A, Goyette P, Zody MC, Hall JL, Brant SR, Cho JH. Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease. Nat Genet. 2008;40:1107-1112.  [PubMed]  [DOI]  [Cited in This Article: ]
34.  Roberts RL, Gearry RB, Hollis-Moffatt JE, Miller AL, Reid J, Abkevich V, Timms KM, Gutin A, Lanchbury JS, Merriman TR. IL23R R381Q and ATG16L1 T300A are strongly associated with Crohn's disease in a study of New Zealand Caucasians with inflammatory bowel disease. Am J Gastroenterol. 2007;102:2754-2761.  [PubMed]  [DOI]  [Cited in This Article: ]
35.  Roberts RL, Gearry RB, Barclay ML, Kennedy MA. Rapid detection of common CARD15 variants in patients with inflammatory bowel disease. Mol Diagn. 2004;8:101-105.  [PubMed]  [DOI]  [Cited in This Article: ]
36.  Opitz B, Püschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, Schumann RR, Suttorp N, Hippenstiel S. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae. J Biol Chem. 2004;279:36426-36432.  [PubMed]  [DOI]  [Cited in This Article: ]
37.  Rosenstiel P, Sina C, End C, Renner M, Lyer S, Till A, Hellmig S, Nikolaus S, Fölsch UR, Helmke B. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion. J Immunol. 2007;178:8203-8211.  [PubMed]  [DOI]  [Cited in This Article: ]
38.  Bonen DK, Cho JH. The genetics of inflammatory bowel disease. Gastroenterology. 2003;124:521-536.  [PubMed]  [DOI]  [Cited in This Article: ]
39.  Henckaerts L, Nielsen KR, Steffensen R, Van Steen K, Mathieu C, Giulietti A, Wouters PJ, Milants I, Vanhorebeek I, Langouche L. Polymorphisms in innate immunity genes predispose to bacteremia and death in the medical intensive care unit. Crit Care Med. 2009;37:192-201, e1-e3.  [PubMed]  [DOI]  [Cited in This Article: ]
40.  Rutgeerts P, Goboes K, Peeters M, Hiele M, Penninckx F, Aerts R, Kerremans R, Vantrappen G. Effect of faecal stream diversion on recurrence of Crohn's disease in the neoterminal ileum. Lancet. 1991;338:771-774.  [PubMed]  [DOI]  [Cited in This Article: ]
41.  Ferwerda G, Kullberg BJ, de Jong DJ, Girardin SE, Langenberg DM, van Crevel R, Ottenhoff TH, Van der Meer JW, Netea MG. Mycobacterium paratuberculosis is recognized by Toll-like receptors and NOD2. J Leukoc Biol. 2007;82:1011-1018.  [PubMed]  [DOI]  [Cited in This Article: ]
42.  Chamaillard M, Philpott D, Girardin SE, Zouali H, Lesage S, Chareyre F, Bui TH, Giovannini M, Zaehringer U, Penard-Lacronique V. Gene-environment interaction modulated by allelic heterogeneity in inflammatory diseases. Proc Natl Acad Sci USA. 2003;100:3455-3460.  [PubMed]  [DOI]  [Cited in This Article: ]
43.  Grant SF, Baldassano RN, Hakonarson H. Classification of genetic profiles of Crohn's disease: a focus on the ATG16L1 gene. Expert Rev Mol Diagn. 2008;8:199-207.  [PubMed]  [DOI]  [Cited in This Article: ]
44.  Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, Brant SR, Caprilli R, Colombel JF, Gasche C, Geboes K. 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.  [PubMed]  [DOI]  [Cited in This Article: ]
45.  Kuballa P, Huett A, Rioux JD, Daly MJ, Xavier RJ. Impaired autophagy of an intracellular pathogen induced by a Crohn's disease associated ATG16L1 variant. PLoS One. 2008;3:e3391.  [PubMed]  [DOI]  [Cited in This Article: ]
46.  Lapaquette P, Glasser AL, Huett A, Xavier RJ, Darfeuille-Michaud A. Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. Cell Microbiol. 2010;12:99-113.  [PubMed]  [DOI]  [Cited in This Article: ]
47.  Fujita H, Eishi Y, Ishige I, Saitoh K, Takizawa T, Arima T, Koike M. Quantitative analysis of bacterial DNA from Mycobacteria spp., Bacteroides vulgatus, and Escherichia coli in tissue samples from patients with inflammatory bowel diseases. J Gastroenterol. 2002;37:509-516.  [PubMed]  [DOI]  [Cited in This Article: ]