Evidence of NLRP3-inflammasome activation in ...

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Basic and translational research

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Evidence of NLRP3-inflammasome activation in rheumatoid arthritis (RA); genetic variants within the NLRP3-inflammasome complex in relation to susceptibility to RA and response to anti-TNF treatment Rebeccah J Mathews,1 James I Robinson,1,2 Michele Battellino,1,3 Chi Wong,1,2 John C Taylor,2,4 Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS),5 Steve Eyre,6 Sarah M Churchman,1,2 Anthony G Wilson,7 John D Isaacs,8,9 Kimme Hyrich,6 Anne Barton,6,10 Darren Plant,10 Sinisa Savic,2,11 Graham P Cook,2,4 Piercarlo Sarzi-Puttini,3 Paul Emery,1,2 Jennifer H Barrett,2,4 Ann W Morgan,1,2 Michael F McDermott1,2 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2013-203276). For numbered affiliations see end of article. Correspondence to Professor Michael McDermott, Leeds Institute of Rheumatic and Musculoskeletal Medicine, Wellcome Trust Brenner Building, St. James’s University Hospital, Leeds LS9 7TF, UK; [email protected] RJM, JIR and MB contributed equally. Accepted 14 April 2013 Published Online First 17 May 2013

To cite: Mathews RJ, Robinson JI, Battellino M, et al. Ann Rheum Dis 2014;73:1202–1210.

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ABSTRACT Background The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). Methods Relative gene expression of NLRP3inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6 months, with secondary expression quantitative trait loci (eQTL) analyses. Results At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. Conclusions This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.

INTRODUCTION The pathogenesis of rheumatoid arthritis (RA) involves aberrant innate and adaptive immune responses.1 Several cytokines have been implicated in RA pathogenesis and persistence. In particular, tumour necrosis factor (TNF) and interleukin-1β (IL-1β), secreted by monocytes/macrophages help to induce and perpetuate inflammatory processes in affected joints.2 3 The NLRP3-inflammasome (also known as the caspase-1 inflammasome) is an intracellular complex involved in proteolytic activation of IL-1β and IL-18 (see online supplementary figure S1). Inflammasome activation, by stimuli such as K+ flux, adenosine triphosphate (ATP) and/or reactive oxygen species4 5 results in the caspase-1 mediated cleavage of pro-IL-1β into biologically active IL-1β. A role for the NLRP3-inflammasome in recurrent and chronic inflammation was initially described in a group of rare autoinflammatory conditions, termed cryopyrin-associated periodic syndromes (CAPS).6 7 Subsequently, the NLRP3-inflammasome has been implicated in many common diseases, including cancer, gout and diabetes.8 There are at least five different protein components integral to this complex, including NLRP3 itself, caspase recruitment domaincontaining protein 8 (CARD8), pyrin, apoptosisassociated speck-like protein containing CARD (ASC) and pro-caspase-1.9 10 The NLRP3-inflammasome senses damage-associated molecular pathogens and subsequent secretion of IL-1β and IL-18 may be destructive to tissues11 and play an important role in bone resorption and cartilage destruction in RA.12 In the resting state, NLRP3 expression in innate immune cells such as monocytes, splenic neutrophils and dendritic cells is low and activation requires transcription of NLRP3, induced by signals such as lipopolysaccharide, TNF and IL-1β.13 NLRP3 expression is not induced in lymphoid subsets and eosinophils.13 A number of alternatively spliced transcript variants encoding distinct isoforms have been identified for the NLRP3 gene. The short isoform, encoded by

Mathews RJ, et al. Ann Rheum Dis 2014;73:1202–1210. doi:10.1136/annrheumdis-2013-203276

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Basic and translational research exons 1–3, is referred to as NLRP3-SL14 and the full length isoform (NLRP3-FL) is encoded by exons 1–9.14 The role of NLRP3 in CAPS is established, with mutations in the NACHT (NAIP, CIITA, HET-E and TP1) domain leading to excessive IL-1β release. The ultimate goal in treating RA is to induce remission or very low disease activity. Biological therapies have improved the outcome for many patients with RA, with marked clinical and radiographic benefit.15 However, the incidence of remission in RA, as defined by DAS28 (disease activity score across 28 joints) 0.1, Hardy-Weinberg equilibrium p value>0.05).34 The total number of SNPs was reduced by prioritising markers based on their potential for modulating parent gene expression by analysing expression quantitative trait loci (eQTL) data from two publicly available resources (SNPexp v1.235 and Sanger Genevar).36 A total of 37 SNPs were successfully genotyped with a call rate of >90%, using Sequenom’s MassARRAY iPLEX system (Sequenom, Cambridge, UK), giving (71%, 66% and 58% gene coverage for NLRP3, MEFV and CARD8, respectively, data not shown). Three assays failed quality control due to Hardy-Weinberg equilibrium 0.15 for each comparison). Furthermore, one SNP (rs11672725) in CARD8 was nominally associated with EULAR response (p=0.032, see table 1), with improved response for those carrying the T allele (table 2). This variant was not in LD with the rs10403848 variant in CARD8 that was associated with change in DAS28 (r2=0.06). When genotyped in the PBMCs of the 29 patients studied, carriage of the rs11672725-T allele (green) was associated with lower baseline CARD8 levels (p=0.011, figure 2). However, carriage of the rs2043211-A variant was not associated with baseline CARD8 levels.

Caspase-1 and IL-18 levels Patients with RA and Muckle-Wells syndrome had significantly higher levels of caspase-1 than HCs (p=0.016 and p=0.0001,

respectively, see figure 3). IL-18 release in patients with RA was also significantly raised in comparison with HCs (p=0.034, figure 3).

eQTL analyses Secondary eQTL analyses of the 283 healthy individuals demonstrated a relatively weak association signal between SNPs in the NLRP3 region in monocytes, but not in B cells, and the probe ILMN-1696933 (Illumina), the strongest being rs56083970, p=4.5×10–5 (figure 4A). The SNPs rs4925659 and rs10925026 were also associated with this probe ( p=0.0010 and p=0.0039 in monocytes, respectively, see online supplementary table S4). However, rs56083970 was in strong LD with rs12565738 (r2=0.98), which was directly genotyped in the BRAGGSS cohort and not associated with response, suggesting the observed association may not be mediated by this eQTL. Several SNPs in the CARD8 region are clearly identifiable as eQTL SNPs with stronger associations in monocytes than B cells (figure 4B). The SNP rs11672725 demonstrated an association with the CARD8 probe ILMN-2192281 ( p=0.00057 in monocytes, see online supplementary table S4). However, other SNPs in the region were much more strongly associated, the highest being rs56012561, p=8.9×10–48 in monocytes, which was in modest LD with rs10500299 and rs2043211/rs16981853 (r2=0.63 and r2=0.68, respectively), neither of which were associated with response.

DISCUSSION This study was undertaken to examine the contribution of NLRP3-inflammasome components to active RA and the effects of anti-TNF therapy. NLRP3-inflammasome-related gene expression (ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1) in PBMCs were upregulated and CARD8 was lower in active RA prior to receiving TNF blockade, but were not significantly modulated by infliximab therapy at 14 weeks. This upregulation of NLRP3-inflammasome-related transcripts and downregulation of CARD8 in patients with active RA is likely to reflect increased activity of the inflammasome. The assembly of the NLRP3-inflammasome involves homotypic interactions of pyrin, NLRP3 and the adapter protein, ASC, through their respective N-terminal PYD (PYRIN domain) domains.5 ASC oligomerises via its CARD with pro-caspase-1 to mediate the proteolytic activation of caspase-1 and IL-1β release

Table 2 SNPs in NLRP3 and CARD8 associated with improvement in DAS28 and EULAR response. N N rs4925648 (NLRP3)

rs4925659 (NLRP3)

rs10925026 (NLRP3)

rs11672725 (CARD8)

rs10403848 (CARD8)

CC CT TT GG GA AA AA AC CC CC CT TT GG GA AA

949 261 15 415 607 196 518 580 127 787 378 40 757 416 46

Improvement in DAS28

EULAR response

Coeff

None

Moderate

81.3 17.8 0.8 42.4 42.8 14.8 37.0 49.4 13.6 69.0 29.7 1.3 66.1 31.4 2.5

77.1 21.7 1.3 30.2 51.9 17.9 42.2 48.7 9.1 61.6 34.5 3.9 62.4 33.8 3.9

1 0.10 −0.14 1 0.09 0.11 1 −0.10 −0.14 1 −0.06 0.36 1 0.11 0.59

(95% CI)

(−0.10 to 0.30) (−0.86 to 0.57) (−0.10 to 0.27) (−0.14 to 0.36) (−0.28 to 0.07) (−0.42 to 0.15) (−0.24 to 0.12) (−0.09 to 0.82) (−0.07 to 0.28) (0.14 to 1.03)

Response vs None

Good vs None

Good

IRR

IRR

75.3 23.2 1.5 35.2 51.1 13.8 46.7 42.7 10.7 69.3 26.7 4.0 57.9 37.9 4.2

1 1.06 1.07 1 1.12 1.11 1 0.96 0.88 1 1.03 1.19 1 1.02 1.09

(95% CI)

(0.99 to 1.13) (0.88 to 1.31) (1.04 to 1.20) (1.01 to 1.21) (0.90 to 1.02) (0.78 to 0.99) (0.97 to 1.10) (1.06 to 1.33) (0.96 to 1.09) (0.95 to 1.25)

1 1.19 1.10 1 1.16 1.09 1 0.84 0.83 1 1.01 1.43 1 1.08 1.22

(95% CI)

(1.02 to 1.39) (0.73 to 1.65) (0.99 to 1.36) (0.87 to 1.37) (0.73 to 0.98) (0.64 to 1.06) (0.86 to 1.18) (1.04 to 1.96) (0.94 to 1.25) (0.87 to 1.72)

DAS28, disease activity score across 28 joints; SNP, single nucleotide polymorphism.

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Mathews RJ, et al. Ann Rheum Dis 2014;73:1202–1210. doi:10.1136/annrheumdis-2013-203276

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Basic and translational research

Figure 2 Expression of NLRP3 and CARD8 in PBMC of patients at baseline for carriage (circle) or non-carriage (cross) of minor allele. Minor alleles are as follows: rs11672725-T, rs2043211-A, rs1092502-C, rs4925659-A). (see online supplementary figure S1). However, there is limited understanding of the regulatory mechanisms of NLRP3inflammasome activation and, in particular, the negative feedback mechanisms which limit the extent of IL-1β secretion, and 41 the roles of individual proteins in this process. Our genotyping data show nominal associations between genotype and disease susceptibility and response to anti-TNF therapy, suggesting that genetic variation in this complex may influence response to anti-TNF therapy in RA, which corroborates some of the previously reported data from the Swedish studies.27 28 None of the observed associations with treatment response met a significance level of 0.001. The study is well powered to detect effects of reasonable size at this level, so the fact that no associations reach this level of significance suggests that the effect of any individual SNP must be small. The observed effect sizes were all small, and a larger study would be required to demonstrate their significance at a stringent significance level. For example, the only SNP showing a nominally significant effect on improvement

in DAS28 (rs10403848 in CARD8) had a regression coefficient of around 0.2 and a MAF of 0.2, which would explain about 0.6% of the variance in change in DAS28. The study had 28% power to detect an effect of this size at a significance level of 0.001. Similarly the study has low power to detect effects on EULAR response of the sizes observed. Our analyses identified eQTL SNPs at the NLRP3 and CARD8 loci.38 However, the SNPs associated with susceptibility/response appear to be separate to the eQTL SNPs in the NLRP3 and CARD8 loci, with the strongest eQTL variants, or variants in strong LD with these, not displaying association with disease susceptibility or response. Likewise, carriage of the rs2043211-A variant, which encodes a premature stop codon, was not associated with baseline CARD8 levels. Further work is therefore required to confirm these associations and unravel the functional variants that are driving the observed association with RA and response to TNF blockade at these loci. It is notable that SNPs within the NLRP3 and CARD8 loci have been

Figure 3 Comparison of Caspase-1 and interleukin (IL)-18 levels in serum of patients with rheumatoid arthritis (RA) (n=39), osteoarthritis (OA) (n=22), Muckle-Wells syndrome (MWS) (n=2) and healthy controls (HCs) (n=8). A Kruskal-Wallis test with Dunn’s multiple comparison correction was carried out to analyse significant differences between the groups (*p