Deepti Verma

Combined Polymorphisms in Genes Encoding the Inflammasome Components NALP3 and CARD8 Confer Susceptibility to Crohn's Disease in Swedish Men

OBJECTIVES:Crohns disease (CD) is characterized by overproduction of proinflammatory cytokines like interleukin (IL)-1β. Production of mature IL-1β is dependent on a caspase-1-activatingprotein complex called the NALP3 inflammasome, composed of NALP3, ASC, and CARD8. NALP3 shares structural similarities with Nod2, and both of these proteins are required forbacteria-induced IL-1β secretion. The combination of the polymorphisms CARD8 (C10X)and NALP3 (Q705K) was recently shown to be associated with rheumatoid arthritis.Our aim was to investigate whether these combined polymorphisms play a role in the susceptibility to CD.METHODS:The study included 498 CD patients in two cohorts from different regions and 742 control individuals from a Swedish population. DNA was isolated from whole blood. Polymorphisms of (Q705K) NALP3 and (C10X) CARD8, as well as the Nod2 variants, R702W and G908R, were genotyped using the Taqman single nucleotide polymorphism assay. The Nod2 frameshift mutation, L1007fs, was detected by Megabace SNuPe genotyping.RESULTS:Our results show that men who have both the C10X and Q705K alleles in CARD8 and NALP3, and who express wild-type alleles of Nod2 are at an increased risk of developing CD (odds ratio, OR: 3.40 range: 1.32–8.76); P=0.011). No association with these polymorphisms was found in women (OR: 0.89 (range: 0.44–1.77); P=0.74).CONCLUSIONS:We suggest a role for combined polymorphisms in CARD8 and NALP3 in the development of CD in men, with obvious sex differences in the genetic susceptibility pattern. These findings give further support to the importance of innate immune responses in CD.

Gene Polymorphisms in the NALP3 Inflammasome Are Associated With Interleukin-1 Production and Severe Inflammation : Relation to Common Inflammatory Diseases?

OBJECTIVE NALP3, ASC, and TUCAN are components of the NALP3 inflammasome, which triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Activating mutations in the gene encoding NALP3 (NLRP3) have recently been linked to familial periodic fever syndromes. We undertook this study to determine whether a patient with arthritis and antibiotic-resistant fever carried mutations in the genes encoding the NALP3 inflammasome. METHODS Genetic analysis of NLRP3 and the gene encoding TUCAN (CARD-8) was performed on genomic DNA from the patient and from a population-based collection of DNA (806 subjects). For in vitro studies of IL-1beta production and caspase 1 activity, blood was obtained from the patient at different time points after administration of anakinra, an IL-1 receptor antagonist, as well as from 5 healthy age- and sex-matched control subjects. RESULTS Mutation analysis of the patients genes encoding NALP3, ASC, and TUCAN revealed variations in the NLRP3 (Q705K) and CARD-8 (C10X) genes. The allele frequencies of these single-nucleotide polymorphisms (SNPs) in the population were 6.5% and 34%, respectively. The elevated activity of caspase 1 and the high levels of IL-1beta measured in samples from the patient returned to normal levels after treatment with anakinra. CONCLUSION Our results indicate that the patients symptoms were due to elevated levels of IL-1beta, since treatment with anakinra effectively abolished the symptoms. The compound SNPs may explain the increased IL-1beta levels and inflammatory symptoms observed, but further studies are needed to reveal a functional relationship. The prevalence of the polymorphisms (4% of the population carry both SNPs) in the general population may suggest a genetic predisposition for common inflammatory disorders.

The Q705K Polymorphism in NLRP3 Is a Gain-of-Function Alteration Leading to Excessive Interleukin-1β and IL-18 Production

Background The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system. Principal Findings NLRP3-WT and NLRP3-Q705K were retrovirally transduced into the human monocytic cell line THP-1, followed by the assessment of IL-1β and IL-18 levels in the cell culture supernatant. THP-1 cells expressing the above NLRP3 variants were sorted based upon Green Fluorescent Protein (GFP) expression. Cytokine response to alum (one of the most widely used adjuvants in vaccines) in the cells stably expressing NLRP3-WT and NLRP3-Q705K were determined. IL-1β and IL-18 levels were found to be elevated in THP-1 cells transduced with NLRP3-Q705K compared to the NLRP3-WT. Upon exposure to alum, THP-1 cells stably expressing NLRP3-Q705K displayed an increased release of IL-1β, IL-18 and TNF-α, in a caspase-1 and IL-1 receptor-dependent manner. Conclusions Collectively, these findings show that the Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to an overactive NLRP3 inflammasome. The option of IL-1β blockade may be considered in patients with chronic inflammatory disorders that are unresponsive to conventional treatments.

CARD8 p.C10X polymorphism is associated with inflammatory activity in early rheumatoid arthritis

Objectives CARD8 and NLRP3 are constituents of the inflammasome which regulates interleukin 1β production. The influence of polymorphisms in CARD8 and NLRP3 on rheumatoid arthritis (RA) susceptibility and severity were evaluated. Methods CARD8 p.C10X and NLRP3 p.Q705K genotypes were assessed in >500 controls and patients with early RA from northern Sweden. The patients were monitored regularly over a 2–year period. The 28-joint disease activity score (DAS28) and its separate components were compared across genotypes. Results Patients with one or more variant alleles in CARD8 (CARD8-X) had increased DAS28, tender joint count and erythrocyte sedimentation rate during the 2-year follow-up period despite receiving disease-modifying antirheumatic drugs to a greater extent. CARD8-X was significantly over-represented among patients who received anti-tumour necrosis factor therapy during the first 2 years. CARD8 and NLRP3 genotypes did not influence radiological joint damage and were not associated with an increased susceptibility. Conclusions Carriage of CARD8-X is associated with a worse disease course in early RA.

Genetic variation and alterations of genes involved in NFκB/TNFAIP3- and NLRP3-inflammasome signaling affect susceptibility and outcome of colorectal cancer

Colorectal tumors are continuously exposed to an inflammatory environment, which together with mitogenic signals sustain several cancer hallmarks. Nuclear factor-kappa B (NFκB) is a major regulator of inflammation and variation in NFκB-associated genes could potentially be used as biomarkers to identify patients with increased risk of colorectal cancer (CRC) development, and/or a rapidly progressing disease. In this study, 348 CRC cases and 806 randomly selected healthy individuals from southeastern Sweden were examined with regard to seven polymorphisms in NFκB pathway-associated genes. Log-rank-tests and Cox proportional hazard regression analysis examined the association between the polymorphisms and CRC-specific survival, whereas chi-square tests and logistic regression analysis were used to test for associations between the polymorphisms and CRC susceptibility. Gene expression and loss of heterozygosity analyses of TNFAIP3 were carried out in a subset of tumors to assess its role as a tumor suppressor in CRC. Heterozygous and polymorphic TNFAIP3 (rs6920220), heterozygous NLRP3 (Q705K) and polymorphic NFκB -94 ATTG ins/del genotypes were found to be associated with poorer survival in patients diagnosed with invasive CRC (aHR = 5.2, 95% CI: 2.5-10.9, P < 0.001). TNFAIP3 mRNA levels were significantly decreased in tumors compared with adjacent non-neoplastic mucosa (P < 0.0001) and loss of heterozygosity of 6q23.3 (TNFAIP3) was detected in 17% of cases, whereas only 2.5% of the investigated specimens displayed TNFAIP3 gene mutations. We propose that TNFAIP3 (rs6920220), NLRP3 (Q705K) and NFκB -94 ATTG ins/del polymorphisms are associated with poor survival in patients with advanced CRC and may be used as prognostic markers. Experimental results indicate that TNFAIP3 may act as a tumor suppressor in CRC.

Inflammasome polymorphisms confer susceptibility to sporadic malignant melanoma

Genetic variants of NLRP3 and NLRP1 are known to modulate levels of pro‐inflammatory cytokine interleukin (IL)‐1β. The purpose of this study was to investigate the association of NLRP3/NLRP1 polymorphisms with susceptibility and clinical features of malignant melanoma in a Swedish case–control study. Common variants in NLRP3/NLRP1 were investigated in sporadic malignant melanoma patients and healthy controls followed by analysis using logistic regression. NLRP3 variant (rs35829419) was significantly more common in male patients than in controls (OR, 2.22; CI, 1.27–3.86). Upon stratification, significant association with nodular melanoma was observed (OR, 2.89; CI, 1.33–6.30), which intensified in male patients (OR 4.03, CI 1.40–11.59). The NLRP1 variant (rs12150220) was significantly more common in fair‐skinned female patients (OR, 1.85; CI, 1.04–3.33) and showed strong associations with nodular melanoma (OR, 6.03; CI, 1.33–25). Our data suggest that NLRP3/NLRP1 polymorphisms are associated with melanoma susceptibility; these findings warrant validation in other independent populations.

Human Gene Variants Linked to Enhanced NLRP3 Activity Limit Intramacrophage Growth of Mycobacterium tuberculosis

Activation of the NLRP3 inflammasome and subsequent generation of interleukin 1β is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequent infection of the cells with virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.

Genetic variants in CARD8 but not in NLRP3 are associated with ankylosing spondylitis.

Objectives: The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is important for interleukin-1beta (IL-1β) processing as part of an innate immune response. Caspase recruitment domain family, member 8 (CARD8) is an inhibitor of nuclear factor kappa B (NF-κB) and possibly also a part of the NLRP3 inflammasome. The objective of this study was to evaluate one single nucleotide polymorphism (SNP) in CARD8 and three SNPs in NLRP3 in ankylosing spondylitis (AS) susceptibility and disease phenotype. Method: We recruited 492 AS patients from Southern Sweden fulfilling the modified New York criteria for AS, and assessed phenotypic characteristics from medical records and questionnaires. Patients with psoriasis or clinically overt inflammatory bowel disease (IBD) were excluded, as were patients without human leucocyte antigen B27 (HLA-B27). Three NLRP3 SNPs (rs35829419, rs4353135, and rs10733113) and one SNP in CARD8 (rs2043211) were genotyped by commercially available TaqMan assays, and the results compared at genotype and allele levels to those of 793 population-based controls. In a subgroup of the patients (n = 169), faecal calprotectin was assessed as a marker of subclinical intestinal inflammation. Results: The minor allele (A) of CARD8-C10X (rs2043211) was associated with a decreased risk of AS in a dominant model [odds ratio (OR) 0.74, 95% confidence interval (CI) 0.54–0.94, p = 0.012] and at the allelic level (OR 0.81, 95% CI 0.68–0.97, p = 0.02), but was not associated with levels of faecal calprotectin. There was no association regarding NLRP3 SNPs and AS susceptibility, and none of the investigated SNPs were associated with iritis, anti-tumour necrosis factor (anti-TNF) therapy, or peripheral joint involvement. Conclusion: In a Swedish population, the minor allele of CARD8-C10X is associated with a decreased risk of AS, but not with levels of faecal calprotectin or disease phenotype.

Genetic variations of NLRP1 : susceptibility in psoriasis

NACHT, LRR and PYD domain‐containing protein (NLRP)1 is part of the inflammasome multiprotein complex involved in the production of interleukin (IL)‐1β and IL‐18, two cytokines strongly implicated in psoriasis pathogenesis. Genetic variations in NLRP1 are associated with a predisposition for chronic inflammatory conditions.

Two Adult Siblings With Atypical Cryopyrin-Associated Periodic Syndrome Due to a Novel M299V Mutation in NLRP3

OBJECTIVE The NALP3 inflammasome is a multiprotein complex that triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Mutations in the gene encoding NALP3 (NLRP3) underlie the cryopyrin-associated periodic syndrome (CAPS). The aim of this study was to report a novel NLRP3 mutation in 2 siblings of Swedish descent in whom symptoms first presented in adulthood. METHODS Mutation analysis of NLRP3 was performed on DNA from patients with CAPS and 100 control subjects. For assessment of caspase 1 and IL-1beta, blood was collected from patients and age- and sex-matched healthy control subjects. Genetic constructs containing mutant or wild-type NLRP3 were transduced into THP-1 cells, followed by assessment of IL-1beta levels in cell supernatant. RESULTS Both siblings carried a novel M299V mutation in NLRP3, which was not present in the control population. The samples obtained from the patients displayed increased caspase 1 activity and elevated IL-1beta levels at basal conditions as compared with healthy control subjects. THP-1 cells expressing mutated M299V revealed almost 10-fold higher IL-1beta production compared with the wild-type construct. CONCLUSION M299V is an activating mutation in NLRP3 resulting in elevated spontaneous caspase 1 activity and IL-1beta levels. The classic CAPS phenotype was lacking in these adult siblings. Whereas one sibling displayed a milder phenotype that has so far responded satisfactorily to oral nonsteroidal antiinflammatory drugs in combination with low-dose corticosteroids, the inflammatory symptoms in the sibling with the more severe case responded well to IL-1beta blockade. Understanding the pathogenic mechanism underlying such disorders can be helpful for the physician. Our study reinforces the importance of genetic testing and laboratory investigations in combination with careful phenotypic evaluation for the diagnosis of such patients.