NLRP1 Haplotypes Associated with Vitiligo and Autoimmunity Increase Interleukin-1Β Processing Via the NLRP1 Inflammasome

NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1β processing via the NLRP1 inﬂammasome

Cecilia B. Levandowskia, Christina M. Maillouxa, Tracey M. Ferraraa, Katherine Gowana, Songtao Bena, Ying Jina,b, Kimberly K. McFannc, Paulene J. Hollanda, Pamela R. Faina,b,d, Charles A. Dinarelloe,1, and Richard A. Spritza,b,1

aHuman Medical Genetics and Genomics Program and bDepartment of Pediatrics, University of Colorado School of Medicine, Aurora, CO 80045; cColorado Biostatistics Consortium, Colorado School of Public Health, Aurora, CO 80045; and dBarbara Davis Center for Childhood Diabetes, and eDepartment of Medicine, University of Colorado School of Medicine, Aurora, CO 80045

Contributed by Charles A. Dinarello, January 8, 2013 (sent for review December 26, 2012) Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key associated molecular patterns stimulate surface Toll-like recep- regulator of the innate immune system, particularly in the skin tors (TLRs) with subsequent assembly of the NLRP1 inflam- where, in response to molecular triggers such as pathogen- masome and activation of caspase-1. Active caspase-1 then associated or damage-associated molecular patterns, the NLRP1 cleaves the inactive IL-1β precursor (pro–IL-1β) to the mature inflammasome promotes caspase-1–dependent processing of bio- bioactive IL-1β (23), thereby stimulating downstream inflam- active interleukin-1β (IL-1β), resulting in IL-1β secretion and down- matory responses (23, 24). Because of its highly inflammatory stream inflammatory responses. NLRP1 is genetically associated and immunostimulating properties, the processing and release of with risk of several autoimmune diseases including generalized bioactive IL-1β are tightly controlled. However, in cryopyrin- vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, associated periodic syndromes (CAPSs), which include familial and others. Here we identify a repertoire of variation in NLRP1 cold autoinflammatory disease, Muckle–Wells syndrome, and by deep DNA resequencing. Predicted functional variations in neonatal onset multi-inflammatory disease, amino acid sub- NLRP1 reside in several common high-risk haplotypes that differ stitutions in the NACHT domain of NLRP3 result in constitutive IMMUNOLOGY from the reference by multiple nonsynonymous substitutions. The activation of caspase-1 and processing and secretion of bioactive haplotypes that are high risk for disease share two substitutions, IL-1β, accompanied by marked systemic and local inflammation L155H and M1184V, and are inherited largely intact due to exten- (21, 25–28). Blood monocytes from affected patients release sive linkage disequilibrium across the region. Functionally, we significantly more mature bioactive IL-1β than cells from healthy found that peripheral blood monocytes from healthy subjects ho- individuals (28). Similarly, a dominant activating mutation in mozygous for the predominant high-risk haplotype 2A processed mouse Nlrp1a has recently been shown to result in a severe sys- significantly greater (P < 0.0001) amounts of the IL-1β precursor temic inflammatory phenotype associated with greatly elevated to mature bioactive IL-1β under basal (resting) conditions and in release of active IL-1β (29). response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) com- In the present study we have investigated the hypothesis that pared with monocytes from subjects homozygous for the refer- common inherited variation in human NLRP1, which is associ- ence haplotype 1. The increase in basal release was 1.8-fold ated with genetic risk of autoimmune disease, similarly results in greater in haplotype 2A monocytes, and these differences be- constitutively increased processing and release of bioactive IL-1β. tween the two haplotypes were consistently observed three times By next-generation DNA sequencing, we identified common over a 3-mo period; no differences were observed for IL-1α or NLRP1 haplotypes containing multiple predicted functional var- TNFα. NLRP1 RNA and protein levels were not altered by the pre- iants that are associated with disease. We next studied the in vitro dominant high-risk haplotype, indicating that altered function of production of cytokines from monocytes of healthy individuals the corresponding multivariant NLRP1 polypeptide predisposes to homozygous for the most prevalent high-risk NLRP1 haplotype autoimmune diseases by activation of the NLRP1 inflammasome. and compared the responses to monocytes from subjects with the low-risk reference haplotype. Our findings indicate that these cytokine | DNA sequencing | interleukin-1beta multivariant NLRP1 haplotypes predispose to common autoimmune diseases by activation of the NLRP1 inflammasome. utoimmune diseases are a diverse group of more than 80 Achronic disorders in which the immune system attacks “self” Results tissues and cells (1), altogether affecting 3–5% of the U.S. pop- NLRP1 Sequencing Identifies Conserved Disease-Associated Haplotypes ulation (2). Concomitant occurrence of multiple autoimmune That Differ by Multiple Nonsynonymous Substitutions. To identify diseases in some patients is well known (3–5) and results from genetic variation in NLRP1 that might contribute to autoimmune shared genetic and perhaps environmental risk factors among susceptibility, we resequenced the complete NLRP1 gene region these different diseases (6–8). Polymorphisms of the nuclear lo- in probands of the 114 families with multiple cases of vitiligo and calization leucine-rich-repeat protein 1 (NLRP1) gene encoding other autoimmune diseases in which we originally established the NACHT (NAIP, CIITA, HET-E, TP1) domain of NLRP1 genetic linkage and association with NLRP1 (9, 10). The 161.5 kb have been associated with a number of autoimmune diseases, including vitiligo (9, 10), Addison disease (11, 12), type 1 diabetes (11), celiac disease (13), systemic lupus erythematosus Author contributions: P.R.F., C.A.D., and R.A.S. designed research; C.B.L., C.M.M., T.M.F., (14), rheumatoid arthritis (15), systemic sclerosis (16), Kawasaki S.B., and Y.J. performed research; C.M.M., T.M.F., and P.J.H. contributed new reagents/ fl analytic tools; C.B.L., C.M.M., T.M.F., K.G., S.B., Y.J., K.K.M., P.R.F., C.A.D., and R.A.S. disease (17), as well as steroid responsiveness in in ammatory analyzed data; and C.B.L., K.K.M., C.A.D., and R.A.S. wrote the paper. fi bowel disease (18), although the speci c biological mechanism The authors declare no conflict of interest. underlying these genetic associations remains unknown. 1To whom correspondence may be addressed. E-mail: [email protected] or richard. NLRP1 is a regulator of the innate immune response (19–21) [email protected]. and is expressed in many immunocompetent cell types, particu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. larly the Langerhans cells of the skin (22). Pathogen- or damage- 1073/pnas.1222808110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1222808110 PNAS Early Edition | 1of5 Downloaded by guest on October 1, 2021 region sequenced (chr17:5386564–5548062) included the entire otherwise specific to haplotype 3. In addition to these major 70.4 kb NLRP1 structural gene, as well as 60.2 kb upstream and multivariant haplotypes, we also observed four singleton non- 30.9 kb downstream. We observed a total of 656 sequence variants synonymous substitutions, G354D, E869K, T1113M, and R1289H, (Dataset S1), including 261 not annotated in the Short Genetic of which only T1113M cosegregated with autoimmunity in the Variations database (dbSNP), Build 135. Within the exons, these corresponding multiplex family. Of the 16 observed non- included 16 nonsynonymous variants, with no observed nonsense synonymous substitutions, bioinformatic analyses predict that variants, splice junction variants, or coding region indels. We six are probably or possibly deleterious (Fig. 1), although in- verified all previously unannotated nonsynonymous variants by dividual in silico functional predictions are of uncertain val- Sanger sequencing in the corresponding patients, and we in- idity in the context of such multivariant haplotypes. Of these, tegrated the observed sequence variants with high-density NLRP1 L155H is the only predicted deleterious substitution shared by region SNP data in these same families (9). both high-risk haplotypes 2A and 3, which additionally share The NLRP1 region consists of three main haplotype blocks, the predicted benign variant M1184V. comprising the NLRP1 structural gene, the 5′-untranslated sequence and extended promoter region, and the 3′-untranslated Increased Processing of Mature IL-1β from Monocytes of Healthy and 3′- flanking region. Of the 16 nonsynonymous variants, 12 Subjects with the Prevalent High-Risk NLRP1 Haplotype. To de- were genotyped directly or were sufficiently frequent to permit termine whether the multivariant NLRP1 polypeptides encoded accurate genotype imputation (r2 > 0.3) in the corresponding by disease-associated NLRP1 haplotypes differentially affect families. Previous family-based association analyses showed that NLRP1 function, we screened healthy individuals to identify rs12150220 (L155H) was the single variant most significantly subjects alternatively homozygous for the NLRP1 reference associated with vitiligo (9). However, the L155H variant occurs haplotype 1 or for the prevalent high-risk NLRP1 haplotype 2A. on three main haplotypes, each differing by multiple additional We genotyped a total of 115 unrelated healthy non–Hispanic/ nonsynonymous substitutions (Fig. 1 and Dataset S1), with no Latino European-derived white (EUR) individuals without apparent ancestral recombinants observed through the region. evidence of autoimmune disease for 14 SNPs spanning the Thus, the corresponding NLRP1 polypeptides would likewise NLRP1 structural gene and extended promoter region, which differ by multiple amino acid substitutions, although all include together distinguish haplotype 1, haplotype 2A, haplotype 2B, L155H. The reference haplotype 1 was the most prevalent hap- and haplotype 3, although they do not distinguish the rare haplotype (frequency, 0.53). Haplotype 2 (overall frequency, 0.42) lotype 2C from the prevalent haplotype 2A (Table S1). Among comprised three subtypes: 2A, 2B, and 2C. Haplotype 2A was the these healthy subjects, the frequency of haplotypes 1, 2A+2C, most prevalent high-risk haplotype (OR, 1.6; frequency, 0.39), 2B, and 3 were 0.52, 0.43, 0.01, and 0.04, respectively, which containing three nonsynonymous substitutions (L155H–V1059M– was not significantly different from the distribution of NLRP1 M1184V) compared with reference haplotype 1. Haplotypes 2B haplotype frequencies observed in the 114 multiplex family and 2C were less frequent (frequency, 0.01); both appear to be probands (P = 0.91). derived from haplotype 2A and respectively contained three To determine whether the predominant high-risk NLRP1 (L155H–V1059M–M1184T) and four (L155H–V939M–V1059M– haplotype 2A affects function by increasing processing of pro– M1184V) nonsynonymous substitutions. Haplotype 3 was even IL-1β to mature bioactive IL-1β, as in CAPS patients with higher risk (OR, 3.7; frequency, 0.05) and contained a remarkable NLRP3 mutations, we studied healthy subjects homozygous for nine nonsynonymous substitutions (L155H–T246S–T782S–T878M– NLRP1 reference haplotype 1 (n = 6) as well as subjects ho- T995I–M1119V–M1184V–V1241L–R1366C), with only a single mozygous for the common high-risk haplotype 2A (n = 10). observed apparently intermediate form that contained L155H, From each subject we cultured freshly obtained peripheral blood M1184V, R1366C, and many synonymous and intronic variants monocytes on three separate occasions over a minimal period of

Fig. 1. NLRP1 nonsynonymous substitutions and haplotypes. The 1,473 amino acid NLRP1 protein and functional domains are indicated with nonsynonymous substitutions overline: red, predicted deleterious; yellow, possibly deleterious; green, nondeleterious (predicted by SeattleSeq Annotation). The vertical arrow indicates the posttranslational autolytic cleavage activation site at S1213 (31). Phase of the unique E869K, T1113M, and R1289H variants cannot be assigned. M1184T derives from M1184V via a second change. OR, odds ratio; p, reference haplotype frequency; q, variant haplotype frequency.

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1222808110 Levandowski et al. Downloaded by guest on October 1, 2021 3 mo. Adherent monocytes were cultured for 24 h either without was also observed for each stimulation condition (P < 0.0001 for stimulation (resting) or activated by the TLR4 ligand lipopoly- each condition), although the increase was proportionately less saccharide (LPS), the Nucleotide-binding oligomerization do- than in the unstimulated cells, suggesting that this mostly reflects main-containing protein 2 (NOD2) ligand muramyl dipeptide the basal difference in IL-1β processing. (MDP), the TLR2 ligand Staphylococcus epidermidis, or a com- To demonstrate the specificity of these findings for IL-1β,we bination of LPS plus MDP. After 24 h of culture, the percent also determined IL-1α (Fig. 3A) and TNFα (Fig. 3B) production cytotoxicity was assayed by measurement of release of lactate in the same samples. There was no significant difference in the dehydrogenase (LDH). As shown in Fig. S1, there was no sig- levels of these cytokines in cells from subjects homozygous for nificant increase in LDH release associated with each of the four the high-risk versus low-risk haplotypes. These data thus indicate stimulated culture conditions, indicating that these stimuli do not that the difference in percent processed IL-1β between the cause extensive cell lysis. haplotype 2A and haplotype 1 homozygotes is not due to dif- Next, for each subject’s monocyte culture, the levels of both ferences in the numbers of monocytes, to expression levels of IL-1β and pro–IL-1β were determined in the intracellular com- TLR, or to mutations in the gene encoding the intracellular re- NOD2 partment (cell lysates) and in the extracellular compartment ceptor for MDP, . (supernatant media) by use of specific, non–cross-reacting NLRP1 RNA and Protein Expression and Stability. High-risk NLRP1 ELISA, one measuring processed (mature) IL-1β and the other haplotype 2A contains three nonsynonymous substitutions, measuring the IL-1β precursor (pro–IL-1β). The values from L155H–V1059M–M1184V, of which only L155H is predicted to these two ELISAs for each compartment were added to calculate be individually deleterious. To exclude the possibility that total IL-1β. The total amount of IL-1β produced was then cal- greater IL-1β processing associated with haplotype 2A versus culated by adding the values of pro– and mature IL-1β in the two fl NLRP1 β haplotype 1 might re ect differentially elevated mRNA compartments. The percent of mature IL-1 processing from expression from haplotype 2A versus haplotype 1, we measured pro–IL-1β was then calculated. As shown in Fig. 2A, there was no NLRP1 β steady-state RNA levels in lymphoblastoid cells from difference in the total amount of IL-1 produced by monocytes normal subjects homozygous for haplotype 1 or for haplotype of the two alternative haplotypes 2A/2A versus 1/1 under each 2A, and found no significant difference (Fig. 4A). Similarly, to B condition of stimulation. In contrast, as shown in Fig. 2 , even in exclude the possibility that the observed haplotype-specific dif- unstimulated (resting) monocytes, the mean percent level of ference might reflect greater amounts of the NLRP1 protein or β processed IL-1 was 1.8-fold higher (16.3%) in cells from indi- protein stability, we measured steady-state NLRP1 protein in IMMUNOLOGY viduals homozygous for high-risk haplotype 2A compared with cells of individuals homozygous for haplotype 1 and haplotype cells from individuals homozygous for the reference haplotype 1 2A, again finding no significant difference (Fig. 4B and Fig. S2). (8.9%). This difference was highly significant (P < 0.0001). Sim- To specifically test the effect of the L155H substitution on ilarly, increased processing of IL-1β by monocytes from the hap- NLRP1 protein stability, we transfected HeLa cells with ex- lotype 2A homozygotes compared with haplotype 1 homozygotes pression constructs containing NLRP1 cDNA with the 155H versus 155L substitutions on the background of the reference haplotype, again observing no difference in the amount of steady-state NLRP1 protein (Fig. S3). Together, these data demonstrate that there is no differential NLRP1 transcription, translation, or protein stability associated with NLRP1 haplotype 2A versus haplotype 1. Thus, we conclude that increased IL-1β processing in cells from NRLP1 haplotype 2A homozygotes results from altered functional properties of the corresponding multivariant NLRP1 polypeptide carrying the L155H–V1059M– M1184V substitutions. Discussion This study identifies a series of common NLRP1 haplotypes that differ by multiple amino acid substitutions in the corresponding NLRP1 polypeptides. At least two of these haplotypes are associated with susceptibility to vitiligo and other autoimmune diseases, likely resulting in part from the L155H substitution shared by these haplotypes. Adherent monocytes cultured from the peripheral blood of healthy individuals homozygous for the common disease-associated haplotype 2A exhibited a 1.8-fold greater amount of IL-1β processing compared with cells from individuals homozygous for the reference haplotype 1. This difference was also observed in resting monocytes—that is, without exogenous stimulation. These data thus suggest that the predominant NLRP1 multivariant haplotype associated with common autoimmune diseases up-regulates IL-1β processing via the NLRP1 inflammasome, in a manner analogous to that by which Fig. 2. IL-1β production from adherent monocytes. (A) Mean ± SEM levels dominant activating mutations of NLRP3 result in elevated IL-1β of total IL-1β produced from monocytes cultured from haplotype 1 homo- fl = = processing and severe autoin ammatory disease. zygotes (Hapl 1; n 6 subjects) and haplotype 2A homozygotes (Hapl 2A; n The specific mechanism by which the NLRP1 inflammasome 10 subjects). (B) Mean ± SEM percent processed IL-1β from monocytes cul- manifests greater functional activity due to haplotype 2A tured from haplotype 1 homozygotes (Hapl 1) and haplotype 2A homo- β zygotes (Hapl 2A). Each subject was tested on three occasions, and on each remains unknown. Increase in processing to mature IL-1 was occasion, in triplicate wells. The stimulants are indicated under the hori- not due to increased levels of pro–IL-1β, increased numbers of zontal axis: RPMI (no stimulant), LPS (1 ng/mL), MDP (10 μg/mL, LPS + MDP, monocytes, or greater expression of TLR4 and TLR2. Indeed, S. epidermidis (Materials and Methods). we observed that total IL-1β, total IL-1α, and total TNFα

Levandowski et al. PNAS Early Edition | 3of5 Downloaded by guest on October 1, 2021 individuals, and are associated only with increased risk of autoimmune disease, rather than with frank disease. CAPS and similar diseases associated with increased IL-1β secretion are effectively treated with IL-1β blocking agents (32), supporting the concept that modestly increased IL-1β secretion is causal for disease and suggesting that similar therapeutic approaches may be beneficial in treating or even preventing autoimmune diseases in individuals who are genetically predisposed due to NLRP1. These relatively common autoimmune diseases are inherited as complex traits, in which no single gene variant is sufficient to cause disease. Thus, although common NLRP1 variation is an important predisposing factor to autoimmune disease, the high- risk NLRP1 haplotypes are clearly not sufficient by themselves to result in disease. Rather, increased mature IL-1β may contribute to the pathogenesis of autoimmunity, perhaps by functioning as an “adjuvant,” facilitating presentation of autoantigens that may trigger or provide specificity for the autoimmune response. It will be of considerable interest to extend these studies to systemati- cally compare IL-1β processing in males versus females homozygous for haplotype 2A, as ∼70% of autoimmune diseases occur in females. Materials and Methods Study Populations. Vitiligo family probands comprised 114 unrelated indi- Fig. 3. Production of IL-1α and TNFα from adherent monocytes. (A) Mean ± viduals of self-reported EUR ancestry from North America or the United SEM levels of intracellular IL-1α produced from monocytes cultured from Kingdom. Each was an affected proband of a family with multiple cases of haplotype 1 homozygotes (same samples as in Fig. 2A) and haplotype 2A generalized vitiligo and at least one case of any of the other autoimmune homozygotes (same samples as in Fig. 2B). (B) Mean ± SEM level of extra- diseases epidemiologically associated with vitiligo (33), previously used to α cellular TNF in same samples shown in Fig. 2B. The stimulants are indicated establish linkage and association of NLRP1 with vitiligo (9). All cases met under the horizontal axis as in Fig. 2. strict clinical diagnostic criteria for generalized vitiligo. Normal subjects were 117 unrelated individuals of EUR ancestry from North America with no his- tory of any autoimmune disease, no recent illness, and not consuming production were the same in cells of the two haplotypes, sup- chronic medications, including nonsteroidal anti-inflammatory drugs. This porting the conclusion that increased processing of IL-1β by monocytes from haplotype 2A homozygotes is directly attribut- able to NLRP1 itself. However, haplotype 2A is not associated with significantly altered NLRP1 transcription, or expression level or stability of the NLRP1 protein, suggesting that the predominant effect is due to altered function of the corresponding multivariant NLRP1 polypeptide encoded by this haplotype. Compared with the reference NLRP1 polypeptide encoded by haplotype 1, the NLRP1 polypeptide encoded by haplotype 2A contains three amino acid substitutions, L155H–V1059M– M1184V. Although only L155H is predicted to be deleterious, recently M1184V has been shown to markedly increase the rate of posttranslational autoproteolytic activation of NLRP1 polypeptide (30, 31), indicating that this substitution is also functionally significant. Sharing of both the L155H and M1184V (or the derivative M1184T) substitutions across disease-associated haplotypes suggests the possibility that these two substitutions together may result in greater disease risk than either would alone. Indeed, the haplotype 3 NLRP1 polypeptide contains nine nonsynonymous substitutions, L155H–T246S–T782S–T878M–T995I– M1119V–M1184V–V1241L–R1366C, including L155H and M1184V, and this haplotype is largely inherited intact due to vir- tually complete linkage disequilibrium and appears to confer even greater disease risk than does haplotype 2A. In patients with autosomal dominant CAPS, nonsynonymous substitutions in the NACHT domain of NLRP3 result in even greater processing and secretion of IL-1β, about fivefold that in normal individuals (28). This relatively modest increase in IL-1β secretion causes patients severe local and systemic inflammation and in some cases renal amyloidosis and death (25–27). A similar severe autoinflammatory phenotype has recently been associated Nlrp1 Neut1 Fig. 4. NLRP1 RNA and protein levels. Levels of NLRP1 (A) RNA and (B) with a mutation of in the mouse (29). In contrast, protein in lymphoblastoid cells from haplotype 1 homozygotes (Hapl 1) and nonsynonymous mutations in the common NLRP1 haplotype haplotype 2A homozygotes (Hapl 2A). NLRP1 protein was assayed by 2A are located outside the NACHT domain, result in ∼1.8-fold Western blot (Fig. S2) and quantitated using ImageJ. NLRP1 RNA and protein up-regulation of IL-1β processing in monocytes from healthy were normalized to GAPDH RNA or β-actin protein, respectively.

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.1222808110 Levandowski et al. Downloaded by guest on October 1, 2021 study was approved by the University of Colorado Denver Combined In- differential centrifugation of blood over Ficoll-Paque (Sigma-Aldrich). To stitutional Review Board, and all donors gave written, informed consent. isolate monocytes, PBMC were suspended at 5 × 106 cells/mL in RPMI 1640 containing penicillin-streptomycin (Cellgro), and 100 μl of PBMC suspension NLRP1 DNA Sequencing. A total of 161.5 kb spanning the NLRP1 region of was added to 96-well flat-bottom polystyrene plates and incubated at 37 °C chromosome 17p13.2 (chr17:5386564–5548062) was sequenced in the 114 for 1 h. Nonadherent cells were then removed, and the adherent monocytes unrelated EUR vitiligo probands. Primers were designed using Primer3 were incubated in 200 μL RPMI 1640 (unstimulated), or in RPMI 1640 con- (http://primer3.sourceforge.net/webif.php), amplifying 517 PCR products taining 1 ng/mL peptidoglycan-free LPS (Sigma), 10 μg/mL MDP, a combina- – ′ sized 200 600 bp (Table S2), spanning all 17 exons and introns, 60.2 kb of 5 tion of LPS plus MDP, or 1 × 106 microorganisms/mL heat-killed S. flanking DNA, and 30.9 kb of 3′ flanking DNA. For each patient, 5 μg ge- epidermidis in triplicate wells. The release of LDH was determined using the nomic DNA was used to prepare DNA sequencing libraries and was se- LDH-Cytotoxicity Assay Kit II (BioVision). After 24 h, cell supernatants were quenced as described previously (34). Functional consequences of all exonic collected and cells were lysed by adding 100 μL of 0.5% triton X-100. sequence variants were assessed using FuncPred (http://snpinfo.niehs.nih. – gov/snpinfo/snpfunc.htm), SeattleSeq Annotation 131 (http://snp.gs.wash- Supernatants and cell lysates were stored at 80 °C. ington.edu/SeattleSeqAnnotation131/), and SKIPPY (http://research.nhgri.nih. Additional detailed materials and methods are presented in SI Materials gov/skippy/index.shtml). and Methods.

Isolation of Monocytes and Cell Stimulation. Healthy individuals homozygous ACKNOWLEDGMENTS. We thank the study participants, whose contribu- for either NLRP1 haplotype 1 (n = 6) or haplotype 2A (n = 10) were identi- tions made this work possible. Also, we thank Tania Azam for her assistance ﬁed, and peripheral venous blood was collected into heparinized tubes in in assays of IL-1α. This work was funded in part by Grants R01AR045584, the morning from each subject on three separate occasions over a period of R01AR056292, R01AI15614, P30AR057212, and UL1RR025780 from the Na- at least 3 mo. Peripheral blood mononuclear cells (PBMC) were prepared by tional Institutes of Health.

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