Variants in NLRP3 and NLRC4 inflammasome associate with susceptibility and severity of multiple sclerosis

Highlights

• Low serum level of IL-18 represents a protective factor against MS development.

• Genetic variants associated to constitutive inflammasome activation contribute to MS severity.

• Loss-of-function NLRC4 SNP was associated with better treatment response in MS patients.

• MS monocytes presented high expression of inflammasome genes and produced more IL-1ß than controls.

Abstract

Background

Multiple sclerosis (MS) is a neurodegenerative disease of central nervous system (CNS) with autoimmune and inflammatory characteristics, and a still uncertain pathogenesis. Early events as well as evolution of MS are heterogeneous (three main clinical forms) and multifactorial. Genome-wide association studies indicate that MS pathogenesis shares features with both autoimmune and inflammatory diseases. Innate immunity has been recently proved to be an important factor in MS. Genetic variants in inflammasome components have been associated with both autoimmune and neurodegenerative diseases, letting us hypothesize that inflammasome, and related cytokines IL-1ß and IL-18, could represent important contributors in MS pathogenesis, and eventually explain, at least in part, the heterogeneity observed in MS patients.

Aim

To evaluate the contribution of inflammasome in MS, in term of (a) genetic effect on development, severity and/or prognosis, and (b) complex activation in peripheral blood as a measure of systemic inflammation.

Methods

Functional genetic variants in inflammasome components were analyzed in a cohort of MS patients, by the use of allele-specific assays and qPCR. Multivariate analysis was performed based on clinical form (recurrent remittent/RR, primary progressive/PP or secondary progressive/SP), severity index (EDSS) and progression index (PI), response to IFN-ß treatment. Peripheral blood monocytes (PBM) of patients were examined for inflammasome activation and expression profile.

Results and discussion

Variants associated with low serum levels of IL-18 were significantly less frequent in MS patients than in controls, suggesting a protective role of diminished IL-18-mediate inflammation in MS development. On the other hands, gain-of-function variants in NLRP3 (Q705K) and IL1B (-511 C >T) associated with severity and progression of MS, suggesting that a constitutive activation of NLRP3 inflammasome could represent a risk factor for MS clinical presentation. Accordingly, -511C >T SNP resulted more frequent in progressive forms than in RR MS, reinforcing the idea that increased inflammasome activation characterized bad prognosis of MS. Altogether these findings corroborate previous data about the harmful role of NLRP3 inflammasome in experimental autoimmune encephalitis (EAE). Moreover, we reported for the first time the beneficial effect of NLRC4 rs479333 G >C variant in MS progression and in the response to IFN-ß treatment. This intronic polymorphism have been previously associated to decreased NLRC4 transcription and low IL-18 serum level, indicated once more that less activation of inflammasome and IL-18 production are beneficial for MS patients.

PBM analysis showed that MS cells express higher level of inflammasome genes than HD ones, and are more prone to respond to a classical NLRP3 stimulus than HD.

Introduction

Multiple sclerosis (MS) is a neurodegenerative disease of central nervous system (CNS) with both autoimmune and inflammatory characteristics. It affects more than 2 million individuals worldwide, typically young people (30–40 years old) and prominently women (women/men: 2/1) (Hauser and Oksenberg, 2006, Harbo et al., 2013, Compston and Coles, 2008, Annapurna et al., 2002). Although the exact cause of MS still remains obscure, its pathogenesis is considered a result of interactions between genetic, infectious (i.e. Epstein Barr Virus/EBV) or other environmental factors, which lead to the loss of immune self-tolerance, and to the consequent CNS infiltration of peripheral blood mononuclear cells activated toward myelin and neurons components. Progressive myelin loss and local inflammation determine the neurologic dysfunction and the neurodegeneration (Hauser and Oksenberg, 2006, Compston and Coles, 2008).

Early events as well as evolution of MS are heterogeneous, making both diagnosis and prognosis a difficult matter. Magnetic resonance imaging (MRI), spinal fluid analysis, and/or evoked potential recordings are sensitive tests for MS but they are not specific. Other inflammatory demyelinating diseases, such as optic neuritis, neuromyelitis optica (NMO), idiopathic transverse myelitis, acute encephalomyelitis, Marburg disease, and metabolic diseases (i.e., adrenoleukodystrophy and metachromatic leukodystrophy) or infectious disease (i.e. HTLV-1, Lyme disease) could be confused with MS (Scolding, 2001).

About 85% of patients present a biphasic disease course, with a first phase of episodes of neurologic disability and benign periods (relapsing-remitting/RR MS), and a second one, occurring in 90% of RR-MS patients, of neurological decline (secondary progressive/SP MS). A rarer clinical presentation (about 10% of total patients) is characterized by a steady neurodegeneration without any recovery (primary progressive/PP MS) (Noseworthy, 1999 and Noseworthy et al., 2000, Hauser and Oksenberg, 2006, Trapp and Nave, 2008).

Genetic contribution in MS is strongly supported by epidemiologic studies in distinct ancestral groups (Caucasian versus African and Asian), and by family-based analysis (MS patients tend to cluster in families) (widely revised in Baranzini, 2017). The first locus to be associated to MS was the HLA region (6p21), and nowadays more than 200 susceptibility variants were reported in genome wide association studies (GWAS) and candidate-gene studies. As expected, several variants affect immune system genes, and, of note, are shared with other autoimmune diseases, such as type 1 diabetes (T1D) and systemic lupus erythematosus (SLE) (Baranzini, 2017). Moreover, several associated variants resulted to be expression quantitative trait loci (eQTL) in immune cells, such as T CD4+ lymphocytes and monocytes, suggesting the involvement of both adaptive and innate immune system dysregulation in MS (Baranzini, 2017).

Clinical studies recently showed an increased expression of proinflammatory cytokines IL-1ß and IL-18 in MS patients according to their presentation and severity (Balashov et al., 1999, Losy and Niezgoda, 2001, Shibatomi et al., 2001, Karni et al., 2002, Mann et al., 2002). IL-1ß, besides inducing the proliferation of macrophages and other neuroinflammatory cells (i.e., microglia, astrocytes) within CNS (Perry, 2013), mediates the differentiation of naïve T CD4+ cells into Th17 subset (Sato et al., 2011, Shaw et al., 2010), while IL-18, together with IL-12, induces a Th1 polarization (Shaw et al., 2010). Both Th17 and Th1 have been implicated in the pathogenesis of MS in an experimental model of the disease (Constantinescu et al., 2011).

IL-1ß and IL-18 are released by monocytes and other cells after the cleavage of their inactive pro-forms by the caspase-1, which is activated within a cytosolic high molecular complex named inflammasome (Martinon et al., 2002). Upon sensing pathogen-associated or damage-associated molecular patterns (PAMPs or DAMPs, respectively), several cytoplasmic innate immune receptors, such as NLRP1, NLRP3, or NLRC4, are able to mount an inflammasome and to mediate the cleavage of the pro-caspase-1 into mature and active form caspase-1. The specificity of each inflammasome receptor has yet to be fully understood. Some receptors, such as NAIP and NLRC4, have been shown to activate the complex in response to bacterial flagellin; while several PAMPs and DAMPs through cytosolic modifications such as ATP-dependent K⁺efflux, mitochondrial damage and consequent release of reactive oxygen species (ROS) and mtDNA, or cathepsins release from damaged lysosomes, activate NLRP3. Proteasome disturbing have been hypothesized to stimulate NLRP1 (Lamkanfi, 2014).

In murine model of MS, the absence of NLRP3 inflammasome components have been shown to protect against the demyelination and the development of experimental autoimmune encephalitis (EAE) (Furlan et al., 1999, Shaw et al., 2010, Lalor et al., 2011), strongly indicating the contribution of this inflammasome in the pathogenesis of EAE, even if the trigger for NLRP3 activation is still debated.

In humans, patients carrying gain-of-function mutations in NLRP3 showed a high comorbidity with MS (Schuh et al., 2015), once more suggesting that an increased activation of NLRP3 inflammasome could contribute to MS pathogenesis. Other inflammasome-forming receptors may play a role in MS, including NLRP1, as suggested by recent literature about the association of single nucleotide polymorphisms (SNPs) in these molecules and familiar form of MS (Alpayci et al., 2012). Moreover, variants in genes coding for IL-1ß and IL-18 also have been found to associate with MS, even if often with discordant findings (Isik et al., 2013, Orhan et al., 2016).

Aim of the current study has been the evaluation of the contribution of inflammasome variants in MS development, severity and/or prognosis in a 10-year longitudinal cohort of Brazilian patients.

Taking in account the difficulty in diagnosis and prognosis, we therefore evaluated the inflammasome profile in peripheral blood mononuclear cells (PBMC) of a subset of MS patients, trying to define novel clinical markers.