Epistatic interaction between TLR4 and NOD2 in patients with Crohn’s Disease: relation with risk and phenotype in a Spanish cohort
Introduction
Crohn’s Disease (CD) is an inflammatory, chronic and relapsing disorder of the gastrointestinal tract and, together with Ulcerative Colitis (UC), is part of the Inflammatory Bowel Disease (IBD) (Baumgart and Sandborn, 2012). Particularly, CD is characterized by a chronic and discontinuous transmural inflammation, involving any portion of the gastrointestinal tract, from the mouth to the anus (Goyette et al., 2007). The etiology of CD remains still unknown but the main hypothesis is that this disease is caused by a dysregulation of the mucosal immune system that triggers a pathogenic immune response against the commensal gut microbiota (Ananthakrishnan, 2015).
Thousands of non pathogenic bacterial species colonize the human gut (normal flora or commensal microbiota). They interact with the host and participate in several functions, as metabolism or defense. Healthy intestinal microbiota composition and concentration are not continuous throughout the gastrointestinal tract, and several factors can affect the diversity, as age, genetics, diet, hygiene, drug treatment, etc. (Cho and Blaser, 2012). Many studies have revealed that the gut microbiota is an essential factor in IBD development (Kostic et al., 2014). The mucosa-associated lymphoid tissue has to distinguish commensal to pathogenic microorganism, thus the interaction between microbiota and immune system is tightly regulated. Several cellular types, as epithelial cells and immune cells, can recognize some conserved Microbe-Associated Molecular Patterns (MAMPs) through the Patterns Recognition Receptors (PRRs), and this crosstalk between the microbiota and the immune system is a key mechanism for the tolerance against normal flora and the maintenance of intestinal homeostasis (Cario, 2010, Wells et al., 2011).
The Toll-like Receptors (TLRs) are a family of signaling receptors that belong to PRRs. To date, 13 TLRs have been described in mammals, but only 10 of these are functional in humans (Takeda et al., 2003, Skevaki et al., 2015). The ectodomain of TLRs contains multiple Leucine-Rich Repeats (LRR) motifs involved directly in the recognition of the ligand (Akira and Takeda, 2004). Despite the conservation among LRR domain, each TLR is involved in the recognition of different ligands: e.g. TLR3 binds double-stranded DNA, TLR5 flagelline or TLR9 CpG DNA (Takeda et al., 2003).
TLR4 is a transmembrane receptor that mainly recognizes lipopolysaccharide (LPS) derived from Gram (−) bacteria (Franchimont et al., 2004). After LPS binding, several signaling pathways are activated, as NFkB’s which leads to the production of inflammatory cytokines (Akira and Takeda, 2004). The LPS from commensal microbiota produces a basal activation of TLR4, triggering a mixed Th1/Th2 response with a Th1 polarization (Magalhaes et al., 2008), but the expression of TLR4 in healthy mucosa is minimally detectable in intestinal epithelial cells and this is a key mechanism to the maintenance of the tolerance against microbiota (Cario and Podolsky, 2000). However, in the CD-mucosa a considerable increase of the expression of TLR4 has been observed, in part due to high levels of TNF-α and IFN-γ (Cario and Podolsky, 2000, Brand et al., 2005), and this contributes to the observed increase of Th1 inflammatory response in CD patients. There are two common TLR4 variants (D299 G and T399I), related to LPS hyporesponsiveness (Arbour et al., 2000), that have been associated with CD risk (Franchimont et al., 2004, Török et al., 2004, Gazouli et al., 2005, Brand et al., 2005, Ouburg et al., 2005, Braat et al., 2005, Pierik et al., 2006, Jager et al., 2007, de Ridder et al., 2007, Hume et al., 2008) and phenotype, as colonic location (Ouburg et al., 2005, Hume et al., 2008) or early age of onset (Oostenbrug et al., 2005).
On the other hand, Nucleotid-binding Oligomerization Domain-containing protein 2 (NOD2) is another PRR that interacts with Muramyl DiPeptide (MDP), a peptidoglycan degradation product (Philpott et al., 2014). NOD2 is a key molecular regulator at gut mucosa and its tonic activation by peptidoglycan from commensal microbiota actively contributes to the maintenance of intestinal homeostasis (Kaser et al., 2010). NOD2 gene, also known as CARD15 (Caspase Recruitment Domain 5), is one of the strongest genetic associations described with CD (Cavanaugh, 2001, Ahmad et al., 2002, Hampe et al., 2002, Economou et al., 2004, Karban et al., 2004, Hampe et al., 2007, Franke et al., 2007, Barrett et al., 2008, Adler et al., 2011, Jung et al., 2012, Jostins et al., 2012). Three main NOD2 variants have been identified (R702W, G908R and 1007insC) that account for >80% of the identified germline variants (Limbergen et al., 2014). NOD2 activation drives a NFkB-dependent signaling and a predominant Th2 cell polarization profile (Magalhaes et al., 2008). The role of NOD2 in TLR4-mediated production of inflammatory cytokines is not fully understood. There is an evidence for a synergism between TLR4 and NOD2 signaling (Magalhaes et al., 2008), since NOD2 enhances, paradoxically, Th1 immune response elicits by TLR4 (Philpott et al., 2014). However, in vitro studies showed that NOD2 prestimulation of human dendritic cells causes a diminished capacity of TLR4 ligand to induce production of several cytokines, as IL-12, and it serves to maintain the intestinal immune balance and tissue homeostasis (Watanabe et al., 2008, Kim et al., 2015). Multiple evidences suggest that in patients with CD-related NOD2 variants, the regulatory effect of NOD2 is impaired, and the altered Th2 response results in a TLR-driven Th1 exaggerated response toward gut microbiota (Watanabe et al., 2008, Magalhaes et al., 2008). Thus, the relation of NOD2 and TLR4 pathways is an important modulatory mechanism for the innate and adaptative responses to gut microbiota, and the disturbance in these pathways may alter the gut homeostasis in a microbial context-dependent manner.
Although several studies were performed, the underlying mechanisms of CD pathogenesis are not yet fully understood and further research is needed to unravel the role of TLR4 variants in CD pathogenesis, and their relation to the phenotype. In this study, we aim to analyze the association of TLR4, as well as NOD2 and the interaction of both, with CD risk and the phenotype in a large Spanish cohort.
Section snippets
Cohort
A total of 371 CD patients from “Section of Digestive, Hospital Virgen de las Nieves” of Granada were included in the study. Blood samples were obtained at the time of Crohn’s Disease diagnosis which was performed upon standard clinical, radiological, endoscopic and histological criteria (Lennard-Jones, 1989). Moreover, a total of 636 blood samples from Regional blood bank donors of Granada were included as healthy controls. Controls and patients were checked in order to eliminate subjects
Cohort
A total of 371 patients with CD were included in the study. The patients were classified according to Montreal’s classification (Silverberg et al., 2005); the clinical features of the patients are shown in Table 2. The median age at diagnosis was 28 years old and there are significantly more men than women [p < 0.001; OR(CI95%)♂ = 1.652 (1.276–2.138)]. Most CD patients suffer inflammatory disease behavior with intermittent evolution. 41.7% of CD patients had ileal location, 20.3% colonic and 38%
Discussion
In our study we investigated the association of the two TLR4 common variants with CD risk and clinical features. Also, the patients were stratified according to NOD2 status to check the relationship between TLR4 and the three common NOD2 variants.
The high concentration of microbes in human gut and the proved importance of dysbiosis in CD pathogenesis make the study of PRRs and their relation with susceptibility and phenotype interesting. Due to this reason, besides NOD2, the TLRs have also been
Conclusions
In conclusion, we found that D299G and T399I SNPs were associated with CD susceptibility in patients carrying NOD2 variants. The patients carrying any mutant TLR4 variant were predisposed to develop a stricturing disease, especially in the presence of NOD2 mutation. We also described a strong association of mutant NOD2 with CD risk and ileal disease, and it was increased in presence of TLR4 variants. TLR4 and NOD2 genotyping in patients at diagnosis could help to the disease management and to
Conflict of interest
The authors declare no conflict of interest.
Funding source declaration
This work was supported partially by a grant from the Servicio Andaluz de Salud (S.A.S.), Reference Number PI0671-2012.
Author agreement/declaration
This work has not been published and it is not under consideration with any other journal. All authors contributed to the preparation of the manuscript, and approved the final version to be published.
Acknowledgments
We would like to thank the patients, researchers and laboratory technicians, especially to Antonia Martín, who have made possible the realization of this work. This work was supported partially by a grant from the Servicio Andaluz de Salud (S.A.S.), Reference Number PI0671-2012.
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