Tammy M. Martin

The NOD2 defect in Blau syndrome does not result in excess interleukin 1 activity

OBJECTIVE Blau syndrome is a rare, autosomal-dominant, autoinflammatory disorder characterized by granulomatous arthritis, uveitis, and dermatitis. Genetics studies have shown that the disease is caused by single nonsynonymous substitutions in NOD-2, a member of the NOD-like receptor or NACHT-leucine-rich repeat (NLR) family of intracellular proteins. Several NLRs function in the innate immune system as sensors of pathogen components and participate in immune-mediated cellular responses via the caspase 1 inflammasome. Mutations in a gene related to NOD-2, NLRP3, are responsible for excess caspase 1-dependent interleukin-1beta (IL-1beta) in cryopyrinopathies such as Muckle-Wells syndrome. Furthermore, functional studies demonstrate that caspase 1-mediated release of IL-1beta also involves NOD-2. The aim of this study was to test the hypothesis that IL-1beta may mediate the inflammation seen in patients with Blau syndrome. METHODS IL-1beta release was measured in peripheral blood mononuclear cells cultured in vitro, obtained from 5 Blau syndrome individuals with a NOD2 (CARD15) mutation. RESULTS We observed no evidence for increased IL-1beta production in cells obtained from subjects with Blau syndrome compared with healthy control subjects. Furthermore, we presented 2 cases of Blau syndrome in which recombinant human IL-1 receptor antagonist (anakinra) was ineffective treatment. CONCLUSION Taken together, these data suggest that in contrast to related IL-1beta-dependent autoinflammatory cryopyrinopathies, Blau syndrome is not mediated by excess IL-1beta or other IL-1 activity.

Blau syndrome revisited.

Purpose of reviewBlau syndrome is a monogenic disease resulting from mutations in nucleotide oligomerization domain 2 (NOD2) and is phenotypically characterized by granulomatous polyarthritis and uveitis. Not only there has been significant progress in disease characterization but also the biological pathways associated with NOD2 and related proteins of the innate immunity are better understood. Recent findingsThe phenotype of Blau syndrome has proven to be more complex than initially thought. A discussion on those manifestations will be provided in the clinical sections of this review. As more patients and pedigrees are found new mutations in the NOD2 gene have emerged and we discuss them in some detail. Due to its importance in Crohns disease NOD2 has become the focus of intense research. A brief review of more recent advances in relevant pathways is presented and published reviews referenced for the interested reader. The granulomatous character of Blau syndrome provides an opportunity to look at possible pathogenic effects of NOD2 ‘gain of function’. New immunohistochemical data are briefly reviewed as well. SummaryElucidation of downstream effects of NOD2 mutations could provide valuable clues to mechanisms of arthritis and uveitis in general as well as granulomatous diseases in particular.

Hla-b27–associated uveitis: overview and current perspectives

Purpose of review To review current knowledge about the pathogenesis, clinical presentation, and treatment of HLA-B27–associated uveitis, which is the most commonly identified cause of uveitis in community-based practice and an important cause of ocular morbidity. Recent findings Significant advances have been made in understanding the pathogenesis of HLA-B27–associated ocular and systemic disease, especially with regard to the genetic underpinning of these diseases. Increasing attention has also been focused on the use of alternative therapies in the treatment of HLA-B27–associated uveitis, with special attention to sulfa class antibiotics, historically have been used to treat the articular manifestations of the spondyloarthritides, and newer drugs that inhibit tumor necrosis factor-&agr;. Summary The next several years promise to yield exciting new advances in understanding of the genetic epidemiology and treatment of HLA-B27–associated uveitis.

Uveitis in patients with sarcoidosis is not associated with mutations in NOD2 (CARD15)

PURPOSE Mutations in NOD2 are responsible for Blau syndrome, a systemic disease triad involving the uvea, joints, and skin. NOD2 mutations are also associated with Crohn disease. Both Blau syndrome and Crohn disease involve granulomatous inflammation and uveitis, as does sarcoidosis. We sought to determine if NOD2 mutations were present in patients with sarcoidosis, especially those with uveitis. METHODS NOD2 gene exons were sequenced from DNA obtained from sarcoidosis patients. The diagnoses of sarcoidosis and uveitis were verified from clinical records. RESULTS NOD2 polymorphisms were found in 26 patients with sarcoidosis (13 with uveitis). There was no significant difference in allele frequencies between patients with and without uveitis. CONCLUSIONS Despite the strikingly similar pathologies of Blau syndrome and sarcoidosis, no mutations were found to be associated with sarcoidosis in a group of patients, regardless of the presence of uveitis.

NOD2, the Gene Responsible for Familial Granulomatous Uveitis, in a Mouse Model of Uveitis

PURPOSE NOD2 plays an important role in the recognition of intracellular bacteria through its ability to sense the components of bacterial peptidoglycan (PGN), namely muramyl dipeptide (MDP) and muramyl tripeptide (MTP). Specific mutations in the human NOD2 gene cause Blau syndrome, an autosomal dominant form of uveitis, arthritis, and dermatitis. As a first step toward understanding the role of NOD2 in the pathogenesis of uveitis, the authors developed a mouse model of MDP-dependent uveitis. METHODS BALB/c mice and mice deficient in L-selectin or NOD2 received intravitreal injection of MDP, MTP, or PGN. The intravascular response within the iris and cellular infiltration was quantified by intravital microscopy and histologic assessment. RESULTS MDP induced an acute, ocular inflammatory response, wherein rolling and adhering leukocytes within the vasculature were significantly increased within 6 hours after MDP treatment. A minor increase in cellular infiltration occurred at 12 hours after MDP treatment. The adhesion molecule L-selectin participated in MDP-induced vascular inflammation because L-selectin knockout mice showed a significant decrease in the number of rolling cells. Importantly, NOD2 plays an essential role in ocular inflammation induced by MDP, as indicated by the fact that uveitis did not develop in Nod2 knockout mice in response to MDP. Nod2 knockout mice also showed abolished ocular inflammation in response to MTP but not to PGN treatment. CONCLUSIONS These findings demonstrate a novel mouse model of uveitis, wherein NOD2 plays an essential role in inflammation induced by the minimal components of PGN. Thus, innate immune responses mediated by NOD2 may participate in the development of uveitis in response to bacterial products.

HLA-DRB1*0102 is associated with TINU syndrome and bilateral, sudden-onset anterior uveitis but not with interstitial nephritis alone

Background Tubulointerstitial nephritis and uveitis (TINU) syndrome is a rare form of uveitis. Previously, the authors had demonstrated a strong association of human leukocyte antigen (HLA) DRB1*0102 with TINU. Here, the authors performed HLA analysis on subjects with isolated bilateral sudden-onset uveitis (as in the TINU subtype) or with isolated tubulointerstitial nephritis (TIN). Methods Patients with sudden onset, anterior, bilateral uveitis not fulfilling a diagnosis of TINU were identified. Pathology reports were reviewed to identify subjects with biopsy-proven TIN. Molecular typing of the HLA-DRB1 gene was performed by the Luminex technology-based sequence-specific oligonucleotide (SSO) hybridisation method (One Lambda, Canoga Park, California). HLA-DRB1 allele frequencies were compared with normal published controls (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/ihwg.cgi dbMHC Europe cohort) and the published TINU cohort (n=18). Results The authors included 28 subjects with uveitis and 14 with TIN. There was a significantly higher frequency of DRB1*0102 in the isolated uveitis cohort versus in normal controls (10.7% vs 0.6%, respectively, p<0.0001; RR 14.3 (6.9–29.8)). None of the nephritis patients showed this HLA subtype. Another association with HLA-DRB1*08 was seen in the isolated uveitis cohort with an allele frequency of 10.7% versus 2.7% in normal controls (p=0.0019; RR 4.0 (1.8–9.0)). In contrast, the HLA-DRB1*08 was not different from controls in the TINU cohort (allele frequency 2.8%, p=not significant). Conclusion The incidence of HLA-DRB1*0102 is increased in sudden-onset bilateral anterior uveitis, as seen in patients with TINU. The same allele does not appear to occur in increased frequency in patients with isolated TIN. HLA DRB1*0102 might predispose to this subset of uveitis.

Cloning, sequencing and expression analysis of the mouse NOD2/CARD15 gene

Abstract:Background: Mutations in the human NOD2/CARD15 gene have been associated with Crohns disease and Blau syndrome. The objective of the present study was to clone the murine form of NOD2 and characterize its tissue distribution, function and response to inflammatory stimuli. Methods: Murine NOD2 was isolated using anchored polymerize chain reaction (PCR). Sequence analysis confirmed the identification of full-length cDNA representing the murine NOD2 gene. Using this sequence to search a Mus musculus supercontig database, NOD2 genomic DNA was identified. NOD2 was transfected into human embryonic kidney (HEK) cells and nuclear factor kappa B (NF-κB) activation was measured using a reporter assay. Tissue distribution and changes in transcription in mouse monocytes in response to inflammatory stimuli was determined by real time PCR. Results: The NOD2 gene spans 39 KB and contains 12 coding exons on chromosome 8. Expression of mouse NOD2 into HEK cells resulted in NF-κB activation. NOD2 was found to be expressed in all mouse tissues analyzed except skin, with highest levels in lung, thymus and spleen. NOD2 mRNA levels increased greater than two-fold in a monocyte cell line in response to lipopolysaccharide, lipoteichoic acid, interferon-g and tumor necrosis factor-α. Conclusions: Common structural and functional features between human and mouse NOD2 were identified. This should allow for development of relevant animal models to evaluate the role of NOD2 in chronic inflammatory disorders.

Genetic Dissection of Acute Anterior Uveitis Reveals Similarities and Differences in Associations observed with Ankylosing Spondylitis

To use high‐density genotyping to investigate the genetic associations of acute anterior uveitis (AAU) in patients with and those without ankylosing spondylitis (AS).

An update on the genetics of HLA B27-associated acute anterior uveitis.

The discovery of the association of HLA B27 with spondyloarthropathy led to more questions than answers about the role of this gene in disease susceptibility. The realization that HLA B27 was not responsible for all of the genetic effects helped to lay a foundation for further investigation into the genetics of uveitis. Over several decades, genetic findings have provided clues to advance the understanding of mechanisms of uveitis and to catalyze new research on diagnostics, animal models, and therapies. From the early candidate gene studies on immune mediators to the recent genome-wide investigations, much has been discovered. However, these discoveries have come with the caveat that a genetic finding does not automatically reveal the disease-relevant functional effect of the associated variant.

Nucleotide Oligomerization Domain-2 (NOD2)-Induced Uveitis: Dependence on IFN-γ

PURPOSE Nucleotide oligomerization domain-2 (NOD2) plays an important role in innate immunity to sense muramyl dipeptide (MDP), a component of bacterial cell walls. Notably, NOD2 is linked to eye inflammation because mutations in NOD2 cause a granulomatous type of uveitis called Blau syndrome. A mouse model of NOD2-dependent ocular inflammation was employed to test the role of a cytokine strongly implicated in granuloma formation, IFN-gamma, in order to gain insight into downstream functional consequences of NOD2 activation within the eye triggering uveitis. METHODS Mice deficient in IFN-gamma, NOD2, or CD11b and their wild-type controls were treated with intravitreal injection of MDP in the presence or absence of IFN-gamma. IFN-gamma production in the eye was measured by ELISA. The intravascular inflammatory response within the iris was quantified by intravital microscopy. RESULTS NOD2 activation resulted in the production of IFN-gamma within the eye. Deficiency in IFN-gamma diminished the development of MDP-induced uveitis, indicating its crucial role in downstream inflammatory events triggered by NOD2. Moreover, exogenous IFN-gamma markedly exacerbated MDP-induced ocular inflammation in a NOD2-dependent mechanism. The potential of IFN-gamma to enhance inflammation required the adhesion molecule CD11b because CD11b-deficient mice failed to show the synergistic effects of IFN-gamma and MDP cotreatment on adhering and infiltrating cells. CONCLUSIONS IFN-gamma was identified as a downstream mediator of NOD2-driven inflammation and the capacity of IFN-gamma in vivo to enhance the inflammatory potential of NOD2 was demonstrated. Extrapolation of these findings in mice suggests that the dysregulation of IFN-gamma may occur in patients with Blau syndrome, thereby contributing to the granulomatous nature of the disease.