A functional inflammasome activation assay differentiates patients with pathogenic NLRP3 mutations and symptomatic patients with low penetrance variants

doi:10.1016/j.clim.2015.01.003

Clinical Immunology

Volume 157, Issue 1, March 2015, Pages 56-64

https://doi.org/10.1016/j.clim.2015.01.003 Get rights and content

Highlights

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NLRP3 inflammasome products are increased after stimulation in CAPS patients.
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No increase was found in patients with low penetrance NLRP3 variants.
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IL-1β secretion in patients with CAPS mutations correlated with disease severity.
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This inflammasome activation assay points towards alternative pathophysiological mechanisms in low penetrance NLRP3 variants.

Abstract

Cryopyrin-associated periodic syndromes (CAPS) are characterized by recurrent episodes of systemic inflammation caused by mutations in the NLRP3 gene. Besides confirmed pathogenic NLRP3 mutations, patients with CAPS-like symptoms frequently show low penetrance variants in NLRP3. The disease relevance of these variants is inconsistent. In this study, we investigated if an inflammasome activation assay differentiates between patients with confirmed pathogenic CAPS mutations, patients with low penetrance NLRP3 variants (V198M and Q703K) and healthy controls. The release of mature IL-1β, IL-18, and caspase-1 into cell culture supernatants after 4 h of inflammasome stimulation was significantly increased in patients with confirmed pathogenic CAPS mutations compared to low penetrance NLRP3 variants and controls. IL-1β secretion in CAPS patients correlated with disease severity. This inflammasome activation assay differentiates between autoinflammation patients with confirmed pathogenic CAPS mutations and patients with low penetrance NLRP3 variants, and points towards alternative pathophysiological mechanisms in low penetrance NLRP3 variants.

Introduction

The cryopyrin-associated periodic syndromes (CAPS) are characterized by recurrent episodes of systemic inflammation marked by fever, tissue inflammation, particularly of the joints and skin, urticarial rash and sensorineural hearing loss [1]. CAPS is caused by mutations in the NLRP3/CIAS gene encoding the cryopyrin protein, an important component of the NLRP3 inflammasome that activates caspase-1 resulting in inflammation driven by excessive secretion of the cytokine IL-1β [2]. Typical disease causing mutations in the NLRP3 gene combined with the characteristic symptoms are an indication for a continuous anti-IL-1β therapy to prevent long term sequelae, such as kidney failure or hearing loss [3], [4]. A diagnostic and therapeutic dilemma is often generated in patients with unspecific inflammatory symptoms such as fatigue, muscle pain, arthralgia, and genetic variations or polymorphisms in NLRP3/CIAS with an inconsistent clinical phenotype and response to anti-IL-1β therapy [5]. The analysis of IL-1β in the serum did not distinguish between CAPS patients and healthy controls. This is probably due to its low and volatile concentration in the blood and rapid neutralization by its endogenous IL-1 receptor antagonist (IL-1RA) in vivo [6]. Therefore, the effectiveness of an anti-IL-1 therapy cannot be predicted in these patients.

To increase diagnostic and therapeutic security, we propose the establishment of a functional ex vivo assay capable of differentiating the inflammasome activation between patients with confirmed disease-causing CAPS mutations and healthy controls. Interestingly, patients with clinical symptoms compatible with the diagnosis of CAPS and low penetrance NLRP3 variants showed an inflammasome activation similar to healthy controls. This points towards a different pathophysiological mechanism for the autoinflammation in these patients compared to patients with confirmed pathogenic CAPS mutations. As a perspective, this assay may help to differentiate patients with NLRP3 inflammasome hyperactivation from patients with other causes for hyperinflammatory symptoms, and may thus influence further diagnostic and therapeutic approaches.

Section snippets

Study population

The study was conducted at the University Children's Hospital Tuebingen (Germany). Informed written consent was obtained from all subjects included in the study or their legal representatives. All study methods were approved by the local ethics committee.

The study population consisted of 17 patients with genetically proven Muckle–Wells syndrome and 11 patients with low penetrance NLRP3 variants (Table 1). Additionally, we acquired frozen (according to standard freezing protocols) PBMC samples

Different IL-1β secretion in patients with CAPS, patients with low penetrance NLRP3 variants and healthy controls

An overactive NLRP3 inflammasome leads to enhanced IL-1β secretion. Consequently, we hypothesized that cells of CAPS patients would secrete higher amounts of mature IL-1β when compared to healthy controls. We isolated the PBMCs of 7 CAPS patients and 7 control donors and analyzed their IL-1β secretion under following conditions: unstimulated, LPS-, ATP- or LPS + ATP-stimulation in time intervals of 4 h, 8 h, 16 h, 24 h, 48 h, and 72 h.

The IL-1β levels of unstimulated PBMCs in all control samples and

Discussion

This study demonstrates that after LPS stimulation secretion of NLRP3 inflammasome products (IL-1β, IL-18, and caspase-1) was significantly increased in CAPS patients when compared to low penetrance NLRP3 variants or healthy controls. Our inflammasome activation assay differentiates between genetically proven CAPS patients and patients with low penetrance NLRP3 variants. The IL-1β secretion in the inflammasome activation assay correlated with the disease severity of CAPS patients.

Enhanced IL-1β

Authorship contributions

Conception and design: NR, DH, and JKD; analysis and interpretation: NR, AG; TE, RH, DH, and JKD; experiments: NR, AG; AS, HO, LH, and IS; and drafting the manuscript for important intellectual content: NR, AG, RH, DH, and JKD.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgment

The study was supported by an unrestricted research grant from Novartis to NR, DH and JKD.

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Cited by (34)

In-vitro NLRP3 functional test assists the diagnosis of cryopyrin-associated periodic syndrome (CAPS) patients: A Brazilian cooperation
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Citation Excerpt :
CAPS diagnosis relies on clinical and genetic evaluation and due to heterogeneity of clinical presentation, overlapping of signs and symptoms with other AIDs, CAPS diagnosis is still challenging. Several groups from high-income countries have developed functional assays to discriminate AIDs patients, sustain diagnosis and a personalized therapy, but none is still worldwide nor commercially available nor in routine practices [7]. In this work we report our five-years´ experience on the use of a functional assay for the differential diagnosis of Brazilian patients with a clinical suspicion of CAPS.
To report our five-years experience on the use of NLRP3 inflammasome functional assays in the differential diagnosis of Brazilian patients with a clinical suspicion of CAPS.
The study included 9 patients belonging to 2 families (I, II) and 7 unrelated patients with a clinical suspicion of AID according to Eurofever/PRINTO classification, recruited between 2017 and 2022. The control group for the NLRP3 functional assay consisted of 10 healthy donors and for the CBA cytokines measurement of 19 healthy controls. Patients underwent clinical evaluation, genetic and functional analysis.
All members of the family I received the diagnosis of Muckle-Wells Syndrome (MWS), carried the NLRP3 Thr348Met variant and resulted positive for the functional assay. The 2 patients of the family II resulted negative for the mutational screening but positive for the functional assay compatible with a MWS clinical phenotype. In 2 unrelated patients with NLRP3 mutations, including a novel mutation (Gly309Val, Asp303His), a positive functional test confirmed the clinical diagnosis of NOMID. 3 unrelated MWS and 1 FCAS patients resulted negative to the genetic screening and positive for the functional test. One patient with a FCAS-like phenotype harbored the NLRP12 His304Tyr variant confirming the diagnosis of FCAS2.
The NLRP3 inflammasome functional assay can assist the clinical diagnosis of CAPS even in patients with unknown genetic defects.
MCC950 blocks enhanced interleukin-1β production in patients with NLRP3 low penetrance variants
2019, Clinical Immunology
Citation Excerpt :
Nevertheless, in clinical routine the usage of frozen material in this assay might facilitate applicability. In contrast to others [24,42,43], we also found that compared to HC, secretion of IL-6 from PBMC of low penetrance mutation variants was enhanced spontaneously in several patients, while the release of both IL-6 and TNF-α were increased after stimulation with LPS alone and after combined treatment with LPS and inflammasome stimuli. However, the secretion of IL-6 and TNF-α was NLRP3-independent, as it was not suppressed by MCC950.
To determine the role of the NLRP3 inflammasome by using the selective NLRP3 inhibitor MCC950 in patients with NLRP3 low penetrance variants and clinical symptoms suggestive for an autoinflammatory syndrome including central nervous system (CNS) involvement.
Nineteen symptomatic patients with low penetrance NLRP3 variants (Q703K n = 17, V198M n = 2) recruited between 2011 and 2017 were included in this monocentric study. A functional inflammasome activation assay was performed in patients in comparison to healthy controls (HC), including the determination of interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-α) secretion in the presence of the NLRP3 selective small-molecule inhibitor MCC950. Detailed clinical features were assessed and anti-IL-1 treatment response was determined.
Peripheral blood mononuclear cells (PBMC) from patients with low penetrance NLRP3 variants displayed enhanced IL-1β levels following inflammasome activation compared to HC. Furthermore, IL-1β release was NLRP3-dependent as it was blocked by MCC950. The production of IL-6 and TNF-α was also increased in patients with low penetrance NLRP3 variants. Clinically, they presented with a heterogenous spectrum of neurological manifestations, while cranial nerve inflammation was the most common feature. Overall inflammasome activation did not correlate with disease severity. Eight of ten treated patients responded to anti IL-1 treatment, however a complete response was only documented in four patients.
PBMC of several patients with NLRP3 low penetrance variants and CNS manifestation showed increased NLRP3-specific IL-1β release upon stimulation and elevated NLRP3-independent IL-6 and TNF-α levels as those were not suppressed by MCC950. Our data suggest that beside the possible causal involvement of the NLRP3 inflammasome additional, yet unidentified genetic or environmental factors may contribute to the multi-organ inflammation in our patients and explain the partial response to IL-1 targeting therapies.
An optimized whole blood assay measuring expression and activity of NLRP3, NLRC4 and AIM2 inflammasomes
2018, Clinical Immunology
Citation Excerpt :
Active caspase-1 induces the maturation and secretion of the proinflammatory cytokines IL-1β and IL-18 and mediates pyroptosis, a programmed proinflammatory cell death [1]. Typically, studies analyzing disease-specific inflammasome activity in patients are performed ex vivo using peripheral blood mononuclear cell (PBMC) based assays [4–7]. Soluble plasma-factors as well as cellular interactions, both known to modulate the innate immune response [8,9], are lost with the PBMC isolation.
The proinflammatory protease caspase-1 plays pivotal roles in central pathways of innate immunity, thereby contributing to pathogen clearance. Beside its physiological role, dysregulated activity of caspase-1 is known to contribute to an increasing number of diseases. In this study, we optimized and validated a low-volume human whole blood assay facilitating the measurement of caspase-1 activation and inflammasome-related gene expression upon stimulation of the NLRP3, NLRC4 or AIM2 inflammasome. Using the NLRP3 inflammasome specific inhibitor MCC950, we were able to measure the activity of canonical or alternative NLRP3 pathways, AIM2 and NLRC4 inflammasomes in whole blood. Based on our data we assume a superposition of NLRP3 and NLRC4 inflammasome activities in human whole blood following stimulation with S. typhimurium. The optimized whole blood assay may be suitable for diagnostic and research purposes for pediatric patients who can only donate small amounts of blood.
Human NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase
2017, Journal of Allergy and Clinical Immunology
Citation Excerpt :
MWS is an autoinflammatory disease caused by gain-of-function mutations in the NLRP3 gene CIAS and is characterized by excessive IL-1β release compared with that seen in healthy donors (see Fig E6 in this article's Online Repository at www.jacionline.org). To explore whether this phenotype could be ameliorated by BTK inhibition, we triggered IL-1β release by LPS (no second stimulus required because of NLRP3 autoactivation, see Prota et al34) in PBMCs from 4 patients with MWS in the absence or presence of ibrutinib. As shown in Fig 6, D, LPS-dependent IL-1β and caspase-1 release was strongly reduced for all assessed patients, and the level of inhibition corresponded to the dose of ibrutinib (Fig 6, E).
The Nod-like receptor NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive.
We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators.
After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation.
Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration–approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced.
Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome–linked inflammation could potentially be targeted pharmacologically through BTK.
The role of the inflammasome in patients with autoinflammatory diseases
2016, Journal of Allergy and Clinical Immunology
Citation Excerpt :
There are also cases of myeloid-restricted somatic mosaicism with disease onset in adulthood.14 In both patients with FMF and those with CAPS, there are well-documented examples of low-penetrance mutations associated with a range of phenotypes from typical clinical presentation, mild or atypical disease, to complete absence of signs or symptoms.15,16 Although these new genetic findings are intriguing for understanding disease pathophysiology, they similarly introduce new diagnostic dilemmas for the treating physician and further underscore the idea that autoinflammatory diseases continue to challenge our traditional clinical paradigms.
Autoinflammatory diseases are disorders of the innate immune system, characterized by systemic inflammation often driven by inflammasomes, and independent of infection and autoreactive antibodies or antigen-specific T cells. These diseases are increasingly recognized as disorders of immune dysregulation, presenting with a constellation of fevers, rashes, and mucosal symptoms in many cases, which suggests that the allergist/immunologist is the appropriate specialist for these patients. However, many practicing physicians are unaware of these disorders in their pediatric and adult patient populations, leading to substantial delays in diagnosis. Recognizing autoinflammatory disease symptom patterns, performing appropriate diagnostic tests, and instituting early effective therapy are essential to reduce morbidity and mortality in these patients. This review will focus on understanding the molecular basis of inflammasomes, recognizing the distinguishing features of the classic autoinflammatory disorders, and appreciating the treatment modalities available.
Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production
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¹: Equal contribution.

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A functional inflammasome activation assay differentiates patients with pathogenic NLRP3 mutations and symptomatic patients with low penetrance variants

Highlights

Abstract

Introduction

Section snippets

Study population

Different IL-1β secretion in patients with CAPS, patients with low penetrance NLRP3 variants and healthy controls

Discussion

Authorship contributions

Conflict of interest statement

Acknowledgment

Clin. Immunol.

Cell

Immunity

Immunity

Phenotypic and Genotypic Characteristics of Cryopyrin-associated Periodic Syndrome: A Series of 136 Patients From the Eurofever Registry, Annals of the Rheumatic Diseases

Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle–Wells syndrome

Nat. Genet.

Use of canakinumab in the cryopyrin-associated periodic syndrome

N. Engl. J. Med.

Long-term clinical course of patients carrying the Q703K mutation in the NLRP3 gene: a case series

Clin. Exp. Rheumatol.

In vivo regulation of interleukin 1beta in patients with cryopyrin-associated periodic syndromes

J. Exp. Med.

Efficacy and safety of anakinra therapy in pediatric and adult patients with the autoinflammatory Muckle–Wells syndrome

Arthritis Rheum.