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Genetic Variations in the NLRP3 Inflammasome: Susceptibility Factor for Chronic Inflammation
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

NLRP3 has been recognized as one of the key components of innate immunity. Upon activation, NLRP3 forms a multiprotein complex called as the ‘inflammasome’ which leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the NLRP3 gene can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under Cryopyrin associated periodic syndromes (CAPS, cryopyrin being an alternative name for NLRP3).

Paper I in this thesis presents the case of a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was a heterozygous carrier of two common polymorphisms, Q705K in NLRP3 and C10X in CARD-8. Experimental studies indicated elevated activity of caspase-1 and IL-1β levels in the patient and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms simultaneously occur in almost 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. We, therefore, investigated a cohort of rheumatoid arthritis (RA) patients in paper II, and found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup might benefit from IL-1β blockade. Paper III presents two patients: siblings, who did not fit into a typical CAPS phenotype. The inflammatory symptoms in both the patients appeared in adult life. A novel and functional M299V mutation in NLRP3 was detected in the siblings who neither had common symptoms nor the same disease severity. Consequent with inflammasome activation, abnormally elevated caspase-1 activity and IL-1β levels were seen. Patients in papers I and III highlight the risk of missing out such patients if attempting a very conventional diagnosis. Paper IV dissects the functional role of Q705K in NLRP3 using THP-1 cells in an in vitro model. Moderately elevated IL-1β and IL-18 levels could be observed in the THP-1 cells expressing Q705K, as compared to the wild type expressing cells, indicating a gain-of-function. Due to the presence of this alteration in healthy individuals it can be classified as a low-penetrance alteration. Additional studies are warranted to elucidate the mechanistic details of this polymorphism.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2011. , 69 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1250
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-70077ISBN: 978-91-7393-116-8 (print)OAI: oai:DiVA.org:liu-70077DiVA: diva2:435304
Public defence
2011-09-09, Linden, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2011-08-18 Created: 2011-08-18 Last updated: 2011-10-03Bibliographically approved
List of papers
1. Gene polymorphisms in the NALP3 inflammasome are associated with interleukin-1 production and severe inflammation: Relation to Common Inflammatory Diseases?
Open this publication in new window or tab >>Gene polymorphisms in the NALP3 inflammasome are associated with interleukin-1 production and severe inflammation: Relation to Common Inflammatory Diseases?
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2008 (English)In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 58, no 3, 888-894 p.Article in journal (Refereed) Published
Abstract [en]

Objective: NALP3, ASC, and TUCAN are components of the NALP3 inflammasome, which triggers caspase 1-mediated interleukin-1β (IL-1β) 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-1β 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 patient's 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-1β measured in samples from the patient returned to normal levels after treatment with anakinra.

Conclusion: Our results indicate that the patient's symptoms were due to elevated levels of IL-1β, since treatment with anakinra effectively abolished the symptoms. The compound SNPs may explain the increased IL-1β 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19048 (URN)10.1002/art.23286 (DOI)
Available from: 2009-06-11 Created: 2009-06-09 Last updated: 2017-12-13Bibliographically approved
2. Genetic variation in proteins of the cryopyrin inflammasome influences susceptibility and severity of rheumatoid arthritis (the Swedish TIRA project)
Open this publication in new window or tab >>Genetic variation in proteins of the cryopyrin inflammasome influences susceptibility and severity of rheumatoid arthritis (the Swedish TIRA project)
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2008 (English)In: Rheumatology, ISSN 1462-0324, Vol. 47, no 4, 415-417 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: The genetic background to RA is incompletely understood.As new cytokine-targeted therapies emerge, early predictorsof disease severity are becoming increasingly important. Theinflammasomes are essential regulators of cytokine production.We investigated whether two polymorphisms in the genes encodingcryopyrin (CIAS1) and TUCAN (CARD8) influence susceptibilityand disease course in RA.

Methods: Genotype frequencies were assessed in 174 Swedish patientswith early RA and 360 population-based controls without rheumaticdisease. Genotypes were categorized according to the presence(+) or absence (–) of two wild-type alleles and comparedbetween patients and controls. In the RA patients, antibodiestowards cyclic citrullinated peptides (anti-CCP) and the ‘sharedepitope’ (SE) were assessed, and medication and measuresof disease activity were monitored regularly during 3 yrs.

Results: The combination of CIAS1/TUCAN/–, ascompared with CIAS1/TUCAN +/+, was significantly more commonamong patients than in controls [odds ratio (OR) 2.2, 95% CI1.03–4.6]. This association was strengthened when patientswere divided into anti-CCP+ [OR 2.8 (1.1–6.7)] or presenceof 1 SE copy [OR 2.8 (1.3–6.2)]. At most time-points duringthe 3-yr follow-up, patients with CIAS1/TUCAN/–showed significantly higher disease activity. Furthermore, CIAS1/TUCAN/– patients proved to be much more likely to receiveTNF-blocking therapy [relative risk 20 (2.6–149)].

Conclusions: Compound polymorphisms in CIAS1 and TUCAN associatewith RA susceptibility and severity. The cryopyrin inflammasomeneeds further attention regarding a possible aetiopathogeneticconnection with RA.

Keyword
Disease course, Genetics, Inflammasome, Rheumatoid arthritis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14380 (URN)10.1093/rheumatology/kem372 (DOI)
Available from: 2007-04-20 Created: 2007-04-20 Last updated: 2015-08-31Bibliographically approved
3. Two adult siblings with atypical cryopyrin-associated periodic syndrome due to a novel M299V mutation in NLRP3
Open this publication in new window or tab >>Two adult siblings with atypical cryopyrin-associated periodic syndrome due to a novel M299V mutation in NLRP3
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2010 (English)In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 62, no 7, 2138-2143 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-58807 (URN)10.1002/art.27489 (DOI)000282758500034 ()20506209 (PubMedID)
Available from: 2010-08-27 Created: 2010-08-27 Last updated: 2017-12-12Bibliographically approved
4. The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production
Open this publication in new window or tab >>The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4Article in journal (Refereed) Published
Abstract [en]

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.

Methods / Principle 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 production 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.

Place, publisher, year, edition, pages
Public Library of Science, 2012
Keyword
NALP3, CIAS-1, polymorphism, anakinra, TNF-α, inflammasome
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-70076 (URN)10.1371/journal.pone.0034977 (DOI)000305347400028 ()
Available from: 2011-08-18 Created: 2011-08-18 Last updated: 2017-12-08Bibliographically approved

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