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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

The NALP3 inflammasome is a multiprotein complex that triggers caspase 1–mediated interleukin-1β (IL-1β) 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-1β, 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-1β 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-1β levels at basal conditions as compared with healthy control subjects. THP-1 cells expressing mutated M299V revealed almost 10-fold higher IL-1β production compared with the wild-type construct.

Conclusion

M299V is an activating mutation in NLRP3 resulting in elevated spontaneous caspase 1 activity and IL-1β 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-1β 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.

NALP3 is a member of the NOD-like receptor protein family, which associates with the adaptor proteins ASC and caspase activation and recruitment domain 8 (CARD-8) to form a complex termed the inflammasome (1). This interaction activates caspase 1, which then cleaves and activates interleukin-1β (IL-1β) and interleukin-18 (IL-18) from their inactive pro forms.

Heterozygous missense mutations in the gene encoding NALP3 (NLRP3) lead to symptoms that are broadly classified as cryopyrin-associated periodic syndromes (CAPS), which include familial cold-induced autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID) (2). Recurrent fever, joint pain, skin rashes, and systemic inflammation are common symptoms in patients with these disorders. Blockade of IL-1β using anakinra has been shown to be tremendously beneficial in treating such patients (3). Furthermore, IL-1β blockade has recently been shown to be useful also in familial Mediterranean fever and tumor necrosis factor receptor–associated periodic syndrome, suggesting that IL-1β is a central mediator in recurrent fever syndromes (4). Common polymorphisms in NLRP3 (Q705K) and CARD8 (C10X) have previously been suggested to be involved in the chronic inflammatory symptoms of a patient (5). The C10X polymorphism in CARD8 has also been associated with Crohn's disease, rheumatoid arthritis, and Alzheimer's disease (6–8).

We report a novel genetic variation in NLRP3 in 2 siblings of Swedish descent. The diffuse symptoms in these siblings, such as fever and muscle pain, presented at ages 44 years and 53 years, respectively, but the siblings did not possess the typical symptoms of FCAS, MWS, or NOMID, thereby adding to the spectrum of clinical symptoms of CAPS.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Study subjects.

Two siblings, each of whom presented with diffuse symptoms of inflammation, were examined 1 week after the withdrawal of all immunosuppressive drugs. Two healthy, age- and sex-matched control subjects with normal results of routine laboratory tests, as well as 5 randomly selected healthy individuals representing the general Swedish population, were selected for the ex vivo studies (additional information is available from the corresponding author). Informed consent was obtained from the patients and control subjects, and the ethical principles of the Declaration of Helsinki were followed. The study was approved by the regional ethics committee in Linköping (M177-07).

Case reports.

Case 1.

The first patient (PM), a previously healthy Caucasian man, presented in 1997, at the age of 44 years, with low-grade fever, nasal congestion, and throat pain, 1 week after cessation of smoking. Symptoms of fatigue, night sweats, and antibiotic-resistant persistent low-grade fever followed. These symptoms were complicated by febrile episodes reaching 41°C occurring at least once weekly for 12–72 hours. The fever episodes were accompanied by facial and thoracic flush, red eyes, thoracic discomfort, dyspnea, and general itching, as well as flu-like symptoms with diffuse muscle and joint pain. At the time of presentation, the patient's C-reactive protein (CRP) level was 41 mg/liter, the erythrocyte sedimentation rate (ESR) was 96 mm/hour, and the blood leukocyte count was 11.9 × 109/liter, with neutrophil granulocytes predominating. The phenotype did not fulfill criteria for systemic lupus erythematosus (SLE), vasculitis, adult Still's disease, or any periodic fever syndrome.

Nine months after presentation, treatment with prednisolone at a dosage of 40 mg/day was initiated; however, upon reducing the dosage (12.5–15 mg/day), fever relapsed, the CRP level increased, and thoracic discomfort and musculoskeletal pain persisted. During this time, cetirizin (an antihistamine) was given to the patient to relieve itching. Besides corticosteroids, azathioprine (100 mg/day during 4 months), intravenous cyclophosphamide (1,000 mg every third week during 4 months), methotrexate (12.5–20 mg/week during 8 months), and cyclosporin A (200 mg/day during 1.5 months) were tested. Methylprednisolone (1,000 mg/pulse) was given concomitantly with cyclophosphamide. Colchicine treatment (1 mg/day) was started in January 2003, and the dosage was later increased to 2 mg/day. Despite a slight benefit, colchicine treatment had to be terminated after 4 months due to gastrointestinal side effects. In October 2007, 10 years after presentation, anakinra treatment (100 mg subcutaneously once daily) was started. Within 4 weeks, muscle pain improved substantially, and fever did not relapse in spite of a decreased dose of prednisolone from 30 mg to 7.5 mg. After initially causing local skin reactions, treatment with anakinra together with prednisolone (10 mg/day) is now successful. In spite of having achieved normal CRP values, the patient is still hesitant to decrease/terminate prednisolone treatment.

Case 2.

The second patient (PF), a Caucasian female who is the sister of PM, was admitted to our clinic in 2008 in order to explore the possibility of a hereditary autoinflammatory syndrome. No other kindred had experienced symptoms of inflammation. PF, who was previously healthy, presented in 2003, at the age of 53 years, with relapsing low-grade fever, headache, muscle pain, fatigue, dyspnea, and, subsequently, small pleural and pericardial effusions. A maximal blood leukocyte count of 19.3 × 109/liter was detected in September 2004 and was associated with low-grade fever, dyspnea, and fatigue. In November 2004, pneumonia was verified radiographically, whereas pulmonary radiographs were normal on several other occasions. The CRP level was 391 mg/liter, and the leukocyte count was 11.5 × 109/liter at the time when pneumonia was verified. However, the leukocyte counts have mostly been in the normal range. SLE, vasculitis, adult Still's disease, or any known recurrent fever syndromes could not be detected according to established criteria. During 3 years, the diffuse inflammatory symptoms and low-grade fever recurred several times. However, the number of fever episodes decreased, and the continuous muscle pain localized to proximal parts of the arms and legs, shoulders, scapulae, and feet partially responded to nonsteroidal antiinflammatory drugs (NSAIDs). Swollen joints were never reported. The ESR was usually in the range of 20–30 mm/hour, with a maximum value of 50 mm/hour. During recent years, the patient's CRP levels have mostly been lower than 10 mg/liter. Hydroxychloroquine and methotrexate (15 mg/week) were tested, without success. Currently, PF is receiving prednisolone (5 mg/day) and the NSAID ketoprofen (200 mg/day). Due to a milder inflammatory phenotype, the patient has not required anakinra treatment.

Mutation analysis.

Genetic screening of NLRP3 and CARD8 was performed as previously described (5). Exons 1, 2, 3, 5, and 10 of MEVF and exons 2, 3, and 4 of TNFRSF1A were screened (the primer sequences used are available from the corresponding author). Three single-nucleotide polymorphisms (SNPs) in P2X7 (H155Y, T357S, and E496A) were investigated in all study subjects using TaqMan assays (identification no. C__3019032_1_, C__15853705_20, and C__27495274_10, respectively) according to the manufacturer's instructions (Applied Biosystems). One hundred DNA samples, from a DNA bank of healthy individuals randomly collected from the regional population register, were analyzed in order to assess the mutation frequencies.

Analysis of caspase 1 activity and IL-1β production.

Peripheral blood samples were obtained from the patients and healthy control subjects. Caspase 1 activity was determined using the Caspase 1 FLICA kit, following the manufacturer's protocol (Immunochemistry Technologies). Treatment with lipopolysaccharide (LPS; 50 ng/ml) (Sigma) for 3 hours at 37°C and/or ATP (1 mM; Sigma), added for the last half-hour of the incubation, was used to stimulate whole blood. Erythrocytes were lysed, and leukocytes were collected. R-phycoerythrin (R-PE)–Cy5–conjugated mouse anti-human CD45 (Dako) and R-PE–conjugated mouse antihuman CD14 (Dako) were used to distinguish monocytes from other leukocytes. Flow cytometry analysis (Altra; Beckman Coulter) was performed by measuring FLICA fluorescence in 30,000 events/sample, which provided the mean fluorescence intensity. For cytokine determination, monocytes were isolated from blood as previously described (5) and cultured overnight (for 16 hours at 37°C) with LPS (50 ng/ml) followed by a 15-minute treatment with ATP (1 mM). Cytokine levels (IL-1β and tumor necrosis factor α [TNFα]) were determined in cell culture supernatant, using an enzyme-linked immunosorbent assay (ELISA) (Diaclone; for IL-1β and TNFα, the lower detection limits are 15 pg/ml and 10 pg/ml, respectively).

Functional characterization of M299V.

The M299V and R260L mutations were generated by mutagenesis of wild-type NALP3–expressing recombinant retroviral vector pHSPG tagged with green fluorescent protein (GFP), using the QuikChange II protocol (Stratagene). Retroviral transduction using spinoculation was performed as follows: recombinant retrovirus was added to human monocytic THP-1 cells (0.5 × 106) in culture medium supplemented with 4 μg/ml polybrene. The virus/cell mixture was centrifuged at 1,500g for 1.5 hours at room temperature. The supernatant was replaced with fresh medium (RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, and streptomycin). After 48 hours, the cell supernatant was harvested and analyzed for IL-1β, using an ELISA (R&D Systems; for IL-1β, the lower detection limit using this kit is 0.16 pg/ml). The cells were subjected to flow cytometry to determine the efficiency of transduction based on GFP expression.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Both patients carried a heterozygous point mutation, M299V (895 A>G), in NLRP3, which could not be detected in 100 control individuals. Neither of the healthy control subjects involved in the ex vivo experiments carried the M299V mutation or any other NLRP3 mutation. Patient PM also carried a heterozygous C10X polymorphism in the CARD-8 gene. Neither of the patients displayed any alterations on MEVF and TNFRSFIA, respectively. Patient PF carried a heterozygous polymorphism, H155Y (489C>T) in P2X7, while patient PM was negative for all investigated SNPs in this gene. The minor allele frequency (T allele) in a control population comprising 801 individuals was determined to be 42% (Verma D, et al: unpublished observations).

Monocytes from the patients displayed significantly increased spontaneous caspase 1 activity (Figure 1A) and IL-1β levels (Figure 1C) compared with control subjects. Caspase 1 activity was greatly increased when cells from the control subjects were incubated with ATP; this was not observed for cells from the patients (Figure 1B). Upon LPS challenge, both patients and control subjects displayed increased caspase 1 activity and IL-1β levels, but the increase was more pronounced in monocytes from the patients (Figures 2A and B). ATP did not induce any significant further response in caspase 1 or IL-1β levels. Patient PM responded with markedly increased TNFα production after the LPS challenge, whereas this was not seen in patient PF (results not shown).

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Figure 1. A and C, Baseline levels of caspase 1 activity and interleukin-1β (IL-1β) production. Caspase 1 activity in monocytes (A) and levels of IL-1β in the supernatant from monocytes (C) of the 2 patients (PM and PF), their age- and sex-matched controls (CM and CF), and 5 healthy individuals from the general population (C1–5) are shown. IL-1β levels were measured in triplicate. B, ATP-induced increase in caspase 1 activity. Bars show the mean ± SD. MFI = mean fluorescence intensity; n.d. = not detectable (i.e., <15 pg/ml).

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Figure 2. Caspase 1 activity and IL-1β production upon challenge with lipopolysaccharide (LPS) with or without ATP. Caspase 1 activity in monocytes (A) and levels of IL-1β in the supernatant from monocytes (B) in the 2 patients (PM and PF), their age- and sex-matched controls (CM and CF), and 5 healthy individuals from the general population (C1–5) in unchallenged cells (−) and in cells challenged with LPS or LPS plus ATP are shown. Unchallenged cells (i.e., those not treated with LPS or ATP) were handled exactly as the challenged cells. IL-1β levels were measured in triplicate. The level of IL-1β in unchallenged cells was <0.04 ng/ml in all study subjects. Bars show the mean and SD. See Figure 1 for other definitions.

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Cells transduced with the M299V variant demonstrated an almost 3-fold increased production of IL-1β as compared with the cells expressing wild-type NALP3 (Figure 3A). Transduction with R260L resulted in almost 15-fold increased IL-1β levels compared with the wild-type construct. Upon correcting the IL-1β production for the percentage of cells expressing GFP, an ∼10-fold increase was observed (Figure 3C).

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Figure 3. M299V-mediated interleukin-1β (IL-1β) production in THP-1 cells. THP-1 cells were transduced with recombinant retrovirus–expressing empty vector (EV), wild-type (WT), or mutant forms (M229V and R260L) of NALP3. A, Level of IL-1β secretion, as determined 48 hours after transduction in cell supernatants. B, Retroviral transduction efficiency, as determined by monitoring green fluorescent protein (GFP) using flow cytometry. C, IL-1β values shown in A, corrected for the percentage of GFP-positive cells in culture. Error bars represent the SD between transductions performed in triplicate. Data are from 1 of 2 independent experiments.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

In this report, we describe a novel M299V mutation in NLRP3 in 2 siblings of Swedish descent. The siblings presented with diffuse symptoms of inflammation, including fever and muscle pain, but lacked symptoms of classic CAPS such as the typical skin lesions and deafness (4). Intriguingly, patient PM displayed more severe disease activity compared with his sister, and their clinical manifestations were also different from each other. However, different intrafamilial phenotypes resulting from the same NLRP3 mutation have been previously described (9). Our data suggest that the M299V mutation could be a familial or a rare polymorphic variant, because it could not be detected in our 100 control subjects from the general Swedish population.

M299V is located in exon 3, which corresponds to the NACHT domain of NLRP3. Its close proximity to other disease-causing mutations such as G301D, D303N, and D303H suggests the presence of potentially important functional sites in this region. Both siblings carrying the M299V mutation displayed increased caspase 1 activity and elevated IL-1β levels, indicating a gain-of-function mutation associated with inflammasome activation. Furthermore, whereas the caspase 1 activity of the control monocytes was low at baseline, it was greatly amplified by ATP. The caspase 1 activity in the patients' monocytes was, at the start, at maximum capacity, and this output was not enhanced by ATP. This phenomenon was recently shown to exist in patients with NOMID and those with MWS who have NLRP3 mutations (10). ATP indirectly activates the inflammasome via the purinergic receptor P2X7. Patient PF carried a H155Y alteration in P2X7, which is proposed to induce a gain-of-function (11). However, its prevalence in the population makes its functional relevance with respect to IL-1β production uncertain.

Upon LPS challenge, caspase 1 activity and IL-1β release were augmented in monocytes from both patients and control subjects, but the increase was more pronounced in the patient-derived monocytes. LPS participates in IL-1β production by stimulating the synthesis of proIL-1β (12). The substantially increased IL-1β observed in the patients' monocytes upon LPS stimulation might therefore be the result of the presence of a constitutively active inflammasome together with large amounts of LPS-induced proIL-1β. The differences in LPS-induced IL-1β responses in the 2 healthy control individuals probably reflect the wide interindividual variation, as has been demonstrated to exist in the healthy population (10). In the patients, no significant further response in LPS-mediated caspase 1 activity or IL-1β levels could be seen after ATP stimulation, once again showing that M299V monocytes have a constitutively active caspase 1 that bypasses the need for stimuli-induced inflammasome activation. The above ex vivo results were confirmed by functional studies using genetic constructs of mutated and wild-type NALP3. Consistent with previous reports, NALP3 mutants induced increased spontaneous production of IL-1β (10). A previously known MWS-causing mutation, R260L, was included as a positive control, and, as expected, generated the highest IL-1β levels. We obtained lower transduction efficiencies in disease-associated mutants as compared with the wild-type control. It is likely that these dominant mutants exhibit a toxic effect on the cells, thereby affecting the viral titer and uptake of retrovirus by THP-1 cells.

CARD-8 has been shown to inhibit the transcription factor NF-κB (13). The C10X polymorphism in CARD8, which was detected in patient PM, causes a severe truncation of the resulting protein, consequently leading to diminished inhibition of NF-κB (7). Because NF-κB is involved in the formation of proIL-1β, it is tempting to speculate that the above-described alteration in CARD-8 might contribute with increased levels of proIL-1β, which is then readily converted to IL-1β upon encountering the active caspase 1 from M299V-mutated NLRP3. Furthermore, the substantially increased LPS-induced TNFα levels detected in patient PM could possibly be a consequence of increased IL-1β release, as observed in monocytes from patients with MWS (1).

In summary, we report a novel mutation in NLRP3 in 2 siblings who first presented with symptoms of inflammation in older adult life. These diffuse symptoms could not be readily classified as typical CAPS. One of the most remarkable features was the late onset of symptoms, which is in contrast to other patients with CAPS, in whom CAPS has a neonatal-to–early childhood debut (4). Patient PM, who had a 12-year history of inflammation, showed remarkable clinical improvement upon receiving treatment with anakinra. Patient PF, who displayed a milder inflammatory phenotype, has so far responded well to NSAIDs and hence was never given anakinra. The more severe clinical picture of patient PM may be attributed to an additional genetic or environmental factor, and it is even tempting to speculate that the C10X polymorphism in CARD8 (which was absent in patient PF) could have enhanced the effect of M299V in NLRP3. However, truly functional studies elucidating the role of C10X are warranted. Interestingly, the symptoms of both siblings were distinct from those of typical FCAS, MWS, or NOMID, thereby also expanding the spectrum of clinical manifestations in CAPS. Other NLRP3 variants giving rise to nonclassic CAPS symptoms have previously been described, e.g., Q705K and V198M (5, 14); however, functional studies on these mutants are lacking.

In conclusion, patients presenting with diffuse symptoms of inflammation can pose a diagnostic challenge for the rheumatologist. Being aware of the pathophysiologic mechanisms underlying the genetic disorders may be helpful for the physician. We emphasize integrated use of genetic analysis and examination of the caspase 1 and IL-1β activation/secretion pattern, together with a careful phenotypic evaluation, to facilitate the diagnosis in such patients.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Ms Verma had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Verma, Eriksson, Särndahl, Söderkvist.

Acquisition of data. Verma, Eriksson, Sahdo, Persson, Ejdebäck, Särndahl, Söderkvist.

Analysis and interpretation of data. Verma, Eriksson, Sahdo, Persson, Ejdebäck, Särndahl, Söderkvist.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank all participants in this study. We acknowledge Dr. Jenny Ting (University of North Carolina) for providing us with the retroviral construct of NLRP3 and Dr. Jan-Ingvar Jönsson (Linköping University) for assistance with retroviral transductions. Elisabet Tina and Johnson Effoe Efome are acknowledged for excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES