Structural localization of pathogenic mutations in the central nucleotide-binding domain (NBD) of nucleotide-binding oligomerization domain-2 (NOD2) protein and their inference in inflammatory disorders.

The human NBD domain which is centrally located in the NOD2 protein displays an essential role in oligomerization and initiates the immune response via CARD-RIPK2 interaction. The mutations associated with the NBD domain have been largely implicated in inflammatory disorders such as Blau syndrome and sarcoidosis. This study aims to determine the structural and phenotypic effect of a lethal mutation that occurs in the NBD domain which has an axiomatic impact on protein dysfunction. Initially, the most deleterious missense mutations were screened through various in silico analysis. Out of 33 variants, I-Mutant 3.0, SIFT, PolyPhen 2, Align GVGD, PHD SNP and SNP&GO have statistically identified 5 variants (R42W, D90E, E91K, G189D & W198L) as less stable, deleterious and damaging. Our predicted models have paved the way to understand the various structural properties such as physiochemical, secondary structural arrangements and stabilizing residues in folding associated with the native and mutant NBD domain especially of the functionally important regions. From the aforementioned results, R42W and G189D were found to be the more predominant among the mutants. Precisely, through molecular simulation, we have strongly justified the significant conformational disruption of R42W and G189D through the stabilization factors, folding and essential dynamics. Conclusively, these regions (α341-44, α13185-191 and ß6133-143ß7) seem to adopt such structures that are not conducive to wild-type-like functionality. Our prediction and validation of lethal mutations based on structural stability may be useful for conducting experimental studies in detail to uncover the protein deregulation leading to inflammatory disorders.

Design of pyrido[2,3-d]pyrimidin-7-one inhibitors of receptor interacting protein kinase-2 (RIPK2) and nucleotide-binding oligomerization domain (NOD) cell signaling.

Receptor interacting protein kinase-2 (RIPK2) is an enzyme involved in the transduction of pro-inflammatory nucleotide-binding oligomerization domain (NOD) cell signaling, a pathway implicated in numerous chronic inflammatory conditions. Herein, a pyrido[2,3-d]pyrimidin-7-one based class of RIPK2 kinase and NOD2 cell signaling inhibitors is described. For example, 33 (e.g. UH15-15) inhibited RIPK2 kinase (IC50 = 8 ± 4 nM) and displayed > 300-fold selectivity versus structurally related activin receptor-like kinase 2 (ALK2). This molecule blocked NOD2-dependent HEKBlue NF-κB activation (IC50 = 20 ± 5 nM) and CXCL8 production (at concentrations > 10 nM). Molecular docking suggests that engagement of Ser25 in the glycine-rich loop may provide increased selectivity versus ALK2 and optimal occupancy of the region between the gatekeeper and the αC-helix may contribute to potent NOD2 cell signaling inhibition. Finally, this compound also demonstrated favorable in vitro ADME and pharmacokinetic properties (e.g. Cmax = 5.7 µM, Tmax = 15 min, t1/2 = 3.4 h and Cl = 45 mL/min/kg following single 10 mg/kg intraperitoneal administration) further supporting the use of pyrido[2,3-d]pyrimidin-7-ones as a new structure class of RIPK2 kinase and NOD cell signaling inhibitors.

Identification of benzofused five-membered sultams, potent dual NOD1/NOD2 antagonists in vitro and in vivo.

Nucleotide-binding oligomerization domain-containing proteins 1 and 2 play important roles in immune system activation. Recently, a shift has occurred due to the emerging knowledge that preventing nucleotide-binding oligomerization domains (NODs) signaling could facilitate the treatment of some cancers, which warrants the search for dual antagonists of NOD1 and NOD2. Herein, we undertook the synthesis and identification of a new class of derivatives of dual NOD1/NOD2 antagonists with novel benzofused five-membered sultams. Compound 14k was finally demonstrated to be the most potent molecule that inhibits both NOD1-and NOD2-stimulated NF-κB and MAPK signaling in vitro and in vivo.

Palmitoylation of NOD1 and NOD2 is required for bacterial sensing.

The nucleotide oligomerization domain (NOD)-like receptors 1 and 2 (NOD1/2) are intracellular pattern-recognition proteins that activate immune signaling pathways in response to peptidoglycans associated with microorganisms. Recruitment to bacteria-containing endosomes and other intracellular membranes is required for NOD1/2 signaling, and NOD1/2 mutations that disrupt membrane localization are associated with inflammatory bowel disease and other inflammatory conditions. However, little is known about this recruitment process. We found that NOD1/2 S-palmitoylation is required for membrane recruitment and immune signaling. ZDHHC5 was identified as the palmitoyltransferase responsible for this critical posttranslational modification, and several disease-associated mutations in NOD2 were found to be associated with defective S-palmitoylation. Thus, ZDHHC5-mediated S-palmitoylation of NOD1/2 is critical for their ability to respond to peptidoglycans and to mount an effective immune response.

A Novel Rare Missense Variation of the NOD2 Gene: Evidencesof Implication in Crohn's Disease.

The NOD2 gene, involved in innate immune responses to bacterial peptidoglycan, has been found to be closely associated with Crohn's Disease (CD), with an Odds Ratio ranging from 3⁻36. Families with three or more CD-affected members were related to a high frequency of NOD2 gene variations, such as R702W, G908R, and 1007fs, and were reported in the EPIMAD Registry. However, some rare CD multiplex families were described without identification of common NOD2 linked-to-disease variations. In order to identify new genetic variation(s) closely linked with CD, whole exome sequencing was performed on available subjects, comprising four patients in two generations affected with Crohn's disease without R702W and G908R variation and three unaffected related subjects. A rare and, not yet, reported missense variation of the NOD2 gene, N1010K, was detected and co-segregated across affected patients. In silico evaluation and modelling highlighted evidence for an adverse effect of the N1010K variation with regard to CD. Moreover, cumulative characterization of N1010K and 1007fs as a compound heterozygous state in two, more severe CD family members strongly suggests that N1010K could well be a new risk factor involved in Crohn's disease genetic susceptibility.

Harnessing the untapped potential of nucleotide-binding oligomerization domain ligands for cancer immunotherapy.

In the last decade, cancer immunotherapy has emerged as an effective alternative to traditional therapies such as chemotherapy and radiation. In contrast to the latter, cancer immunotherapy has the potential to distinguish between cancer and healthy cells, and thus to avoid severe and intolerable side-effects, since the cancer cells are effectively eliminated by stimulated immune cells. The cytosolic nucleotide-binding oligomerization domains 1 and 2 receptors (NOD1 and NOD2) are important components of the innate immune system and constitute interesting targets in terms of strengthening the immune response against cancer cells. Many NOD ligands have been synthesized, in particular NOD2 agonists that exhibit favorable immunostimulatory and anticancer activity. Among them, mifamurtide has already been approved in Europe by the European Medicine Agency for treating patients with osteosarcoma in combination with chemotherapy after complete surgical removal of the primary tumor. This review is focused on NOD receptors as promising targets in cancer immunotherapy as well as summarizing current knowledge of the various NOD ligands exhibiting antitumor and even antimetastatic activity in vitro and in vivo.

Unique Variant of NOD2 Pediatric Granulomatous Arthritis With Severe 1,25-Dihydroxyvitamin D-Mediated Hypercalcemia and Generalized Osteosclerosis.

Pediatric granulomatous arthritis (PGA) refers to two formerly separate entities: autosomal dominant Blau syndrome (BS) and its sporadic phenocopy early-onset sarcoidosis (EOS). In 2001 BS and in 2005 EOS became explained by heterozygous mutations within the gene that encodes nucleotide-binding oligomerization domain-containing protein 2 (NOD2), also called caspase recruitment domain-containing protein 15 (CARD15). NOD2 is a microbe sensor in leukocyte cytosol that activates and regulates inflammation. PGA is characterized by a triad of autoinflammatory problems (dermatitis, uveitis, and arthritis) in early childhood, which suggests the causal NOD2/CARD15 mutations are activating defects. Additional complications of PGA were recognized especially when NOD2 mutation analysis became generally available. However, in PGA, hypercalcemia is only briefly mentioned, and generalized osteosclerosis is not reported, although NOD2 regulates NF-κB signaling essential for osteoclastogenesis and osteoclast function. Herein, we report a 4-year-old girl with PGA uniquely complicated by severe 1,25(OH)2 D-mediated hypercalcemia, nephrocalcinosis, and compromised renal function together with radiological and histopathological features of osteopetrosis (OPT). The classic triad of PGA complications was absent, although joint pain and an antalgic gait accompanied wrist, knee, and ankle swelling and soft non-tender masses over her hands, knees, and feet. MRI revealed tenosynovitis in her hands and suprapatellar effusions. Synovial biopsy demonstrated reactive synovitis without granulomas. Spontaneous resolution of metaphyseal osteosclerosis occurred while biochemical markers indicated active bone turnover. Anti-inflammatory medications suppressed circulating 1,25(OH)2 D, corrected the hypercalcemia, and improved her renal function, joint pain and swelling, and gait. Mutation analysis excluded idiopathic infantile hypercalcemia, type 1, and known forms of OPT, and identified a heterozygous germline missense mutation in NOD2 common in PGA (c.1001G>A, p.Arg334Gln). Thus, radiological and histological findings of OPT and severe hypercalcemia from apparent extrarenal production of 1,25(OH)2 D can complicate NOD2-associated PGA. Although the skeletal findings seem inconsequential, treatment of the hypercalcemia is crucial to protect the kidneys. © 2018 American Society for Bone and Mineral Research.

Crohn's Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70.

Nod2 is a cytosolic, innate immune receptor responsible for binding to bacterial cell wall fragments such as muramyl dipeptide (MDP). Upon binding, subsequent downstream activation of the NF-κB pathway leads to an immune response. Nod2 mutations are correlated with an increased susceptibility to Crohn's disease (CD) and ultimately result in a misregulated immune response. Previous work had demonstrated that Nod2 interacts with and is stabilized by the molecular chaperone Hsp70. In this work, it is shown using purified protein and in vitro biochemical assays that the critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the ability to bind bacterial ligands. A limited proteolysis assay and luciferase reporter assay reveal regions of Hsp70 that are capable of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino acid subset of Hsp70 that stabilizes the CD-associated variants of Nod2 and restores a proper immune response upon activation with MDP was identified. This work suggests that CD-associated Nod2 variants could be stabilized in vivo with a molecular chaperone.

Molecular modeling in the age of clinical genomics, the enterprise of the next generation.

Protein modeling and molecular dynamics hold a unique toolset to aide in the characterization of clinical variants that may result in disease. Not only do these techniques offer the ability to study under characterized proteins, but they do this with the speed that is needed for time-sensitive clinical cases. In this paper we retrospectively study a clinical variant in the XIAP protein, C203Y, while addressing additional variants seen in patients with similar gastrointestinal phenotypes as the C203Y mutation. In agreement with the clinical tests performed on the C203Y patient, protein modeling and molecular dynamics suggest that direct interactions with RIPK2 and Caspase3 are altered by the C203Y mutation and subsequent loss of Zn coordination in the second BIR domain of XIAP. Interestingly, the variant does not appear to alter interactions with SMAC, resulting in further damage to the caspase and NOD2 pathways. To expand the computational strategy designed when studying XIAP, we have applied the molecular modeling tools to a list of 140 variants seen in CFTR associated with cystic fibrosis, and a list of undiagnosed variants in 17 different genes. This paper shows the exciting applications of molecular modeling in the classification and characterization of genetic variants identified in next generation sequencing. Graphical abstract XIAP in Caspase 3 and NOD2 signaling pathways.

Enhancement of Muramyl Dipeptide-Dependent NOD2 Activity by a Self-Derived Peptide.

Nucleotide-binding and oligomerization domain like receptors (NLR) are pattern recognition receptors used to provide rapid immune response by detecting intracellular pathogen-associated molecules. Loss of NLR activity is implicated in genetic disorders, disruption of adaptive immunity, and chronic inflammation. One NLR protein, NOD2, is frequently mutated in Crohn's disease (CD), which is an inflammatory disease of the gastrointestinal tract. Three commonly occurring CD-associated NOD2 mutations, R702W, G908R, and L1007fs, are clustered near the regulatory domain, leucine rich region (LRR), and lowers the activity of NOD2 in response to muramyl dipeptide (MDP). As LRR is also the ligand binding domain, this suggests that the mutations either affect the binding of MDP or how the molecule responds to ligand binding. To model the role of R702 in ligand-dependent activation of NOD2, we used homology modeling to map the residue R702 to the interface between the oligomerization domain and LRR. We show that a peptide derived from NOD2(697-718) binds LRR in vitro, and upon co-expressing or importing the peptide into HEK293 expressing NOD2, there is an increase in the MDP-dependent NOD2 activity. The study thus suggests that the R702W mutation interferes with the conformational changes needed for MDP binding and activation. J. Cell. Biochem. 118: 1227-1238, 2017. © 2016 Wiley Periodicals, Inc.

SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling.

The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.

Crystal structure of NOD2 and its implications in human disease.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a member of the NOD-like receptors family, are crucial for innate immune responses. Mutations of NOD2 have been associated with chronic inflammatory disorders such as Crohn's disease (CD), Blau syndrome (BS) and early-onset sarcoidosis (EOS), but little is known about its signalling mechanism and the role it plays in these diseases. Here, we report the crystal structure of rabbit NOD2 in an ADP-bound state. The structure reveals an inactive closed conformation in which the subdomains in the NOD domain are closely packed by ADP-mediated and inter-domain interactions. Mapping of the BS- or EOS-associated gain-of-function mutations reveals that most of these mutations are located in the NOD subdomain interfaces, and are likely to disrupt the inner domain interactions, facilitating a conformational change to the active form. Conversely, mutations associated with CD are distributed throughout the protein, some of which may affect oligomer formation and ligand binding.

Structural models of zebrafish (Danio rerio) NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Insights into the molecular basis of the NOD2 signalling pathway.

The cytosolic pattern recognition receptor NOD2 is activated by the peptidoglycan fragment muramyl dipeptide to generate a proinflammatory immune response. Downstream effects include the secretion of cytokines such as interleukin 8, the upregulation of pro-interleukin 1ß, the induction of autophagy, the production of antimicrobial peptides and defensins, and contributions to the maintenance of the composition of the intestinal microbiota. Polymorphisms in NOD2 are the cause of the inflammatory disorder Blau syndrome and act as susceptibility factors for the inflammatory bowel condition Crohn's disease. The complexity of NOD2 signalling is highlighted by the observation that over 30 cellular proteins interact with NOD2 directly and influence or regulate its functional activity. Previously, the majority of reviews on NOD2 function have focused upon the role of NOD2 in inflammatory disease or in its interaction with and response to microbes. However, the functionality of NOD2 is underpinned by its biochemical interactions. Consequently, in this review, we have taken the opportunity to address the more 'basic' elements of NOD2 signalling. In particular, we have focused upon the core interactions of NOD2 with protein factors that influence and modulate the signal transduction pathways involved in NOD2 signalling. Further, where information exists, such as in relation to the role of RIP2, we have drawn comparison with the closely related, but functionally discrete, pattern recognition receptor NOD1. Overall, we provide a comprehensive resource targeted at understanding the complexities of NOD2 signalling.

The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease.

Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.

p62/SQSTM1 enhances NOD2-mediated signaling and cytokine production through stabilizing NOD2 oligomerization.

NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1ß and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.

RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.

Crohn's disease risk alleles on the NOD2 locus have been maintained by natural selection on standing variation.

Risk alleles for complex diseases are widely spread throughout human populations. However, little is known about the geographic distribution and frequencies of risk alleles, which may contribute to differences in disease susceptibility and prevalence among populations. Here, we focus on Crohn's disease (CD) as a model for the evolutionary study of complex disease alleles. Recent genome-wide association studies and classical linkage analyses have identified more than 70 susceptible genomic regions for CD in Europeans, but only a few have been confirmed in non-European populations. Our analysis of eight European-specific susceptibility genes using HapMap data shows that at the NOD2 locus the CD-risk alleles are linked with a haplotype specific to CEU at a frequency that is significantly higher compared with the entire genome. We subsequently examined nine global populations and found that the CD-risk alleles spread through hitchhiking with a high-frequency haplotype (H1) exclusive to Europeans. To examine the neutrality of NOD2, we performed phylogenetic network analyses, coalescent simulation, protein structural prediction, characterization of mutation patterns, and estimations of population growth and time to most recent common ancestor (TMRCA). We found that while H1 was significantly prevalent in European populations, the H1 TMRCA predated human migration out of Africa. H1 is likely to have undergone negative selection because 1) the root of H1 genealogy is defined by a preexisting amino acid substitution that causes serious conformational changes to the NOD2 protein, 2) the haplotype has almost become extinct in Africa, and 3) the haplotype has not been affected by the recent European expansion reflected in the other haplotypes. Nevertheless, H1 has survived in European populations, suggesting that the haplotype is advantageous to this group. We propose that several CD-risk alleles, which destabilize and disrupt the NOD2 protein, have been maintained by natural selection on standing variation because the deleterious haplotype of NOD2 is advantageous in diploid individuals due to heterozygote advantage and/or intergenic interactions.

Transcription of pattern recognition receptors and abortive agents induced chemokines in the bovine pregnant uterus.

Pattern recognition receptors (PRRs) are important components of the innate immune system whose ligands are specific pathogen associated molecular patterns (PAMPs). Considering the scarcity of studies on transcription of PRRs in the pregnant uterus of cows, and its response to PAMPs and microorganisms that cause abortion in cattle, this study aimed to characterize the transcription of TLR1-10, NOD1, NOD2 and MD2 in bovine uterus throughout gestation and to investigate the sensitivity of different uterine tissues at third trimester of pregnancy to purified TLR ligands or heat-killed Brucella abortus, Salmonella enterica serotype Dublin (S. Dublin), Listeria monocytogenes, and Aspergillus fumigatus, by assessing chemokine transcription. RNA extracted from endometrium, placentome and intercotiledonary region of cows at the first (n=6), second (n=6), and third (n=6) trimesters of pregnancy were subjected to real time RT-PCR. After stimulation of endometrium and intercotiledonary regions with purified TLR ligands or heat-killed microorganisms, gene transcription was assessed by real time RT-PCR. In the placentome, there was no significant variation in TLRs transcription throughout the three trimesters of pregnancy. In the endometrium, there was significant variation in TLR4 and TLR5 transcription during the three stages of gestation; i.e. TLR4 transcription was higher during the third trimester, whereas TLR5 transcription was higher during the last two trimesters. In the intercotiledonary region, there was significant variation in transcription of TLR1/6, TLR7, and TLR8, which were more strongly expressed during the first trimester of pregnancy. At the third trimester of gestation, significant transcription of CXCL6 and CXCL8 was detected mostly in endometrial tissues in response to purified TLR4 and TLR2 ligands. Transcription of these chemokines was induced in the endometrium and intercotiledonary region at the third trimester of pregnancy when stimulated with heat-killed B. abortus or S. Dublin. Therefore, this study demonstrates that some PRRs are expressed in the uterus during pregnancy, which coincides with its ability to respond to stimulation with TLRs ligands as well as heat-killed organisms known to cause abortion in cattle.

NOD2, an intracellular innate immune sensor involved in host defense and Crohn's disease.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular sensor for small peptides derived from the bacterial cell wall component, peptidoglycan. Recent studies have uncovered unexpected functions of NOD2 in innate immune responses such as induction of type I interferon and facilitation of autophagy; moreover, they have disclosed extensive cross-talk between NOD2 and Toll-like receptors, which has an indispensable role both in host defense against microbial infection and in the development of autoimmunity. Of particular interest, polymorphisms of CARD15 encoding NOD2 are associated with Crohn's disease and other autoimmune states such as graft vs. host disease. In this review, we summarize recent findings regarding normal functions of NOD2 and discuss the mechanisms by which NOD2 polymorphisms associated with Crohn's disease lead to intestinal inflammation.