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

Abstract

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key regulator of the innate immune system, particularly in the skin where, in response to molecular triggers such as pathogen-associated or damage-associated molecular patterns, the NLRP1 inflammasome promotes caspase-1-dependent processing of bioactive interleukin-1β (IL-1β), resulting in IL-1β secretion and downstream inflammatory responses. NLRP1 is genetically associated with risk of several autoimmune diseases including generalized vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, and others. Here we identify a repertoire of variation in NLRP1 by deep DNA resequencing. Predicted functional variations in NLRP1 reside in several common high-risk haplotypes that differ from the reference by multiple nonsynonymous substitutions. The haplotypes that are high risk for disease share two substitutions, L155H and M1184V, and are inherited largely intact due to extensive linkage disequilibrium across the region. Functionally, we found that peripheral blood monocytes from healthy subjects homozygous for the predominant high-risk haplotype 2A processed significantly greater (P < 0.0001) amounts of the IL-1β precursor to mature bioactive IL-1β under basal (resting) conditions and in response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) compared with monocytes from subjects homozygous for the reference haplotype 1. The increase in basal release was 1.8-fold greater in haplotype 2A monocytes, and these differences between the two haplotypes were consistently observed three times over a 3-mo period; no differences were observed for IL-1α or TNFα. NLRP1 RNA and protein levels were not altered by the predominant high-risk haplotype, indicating that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.

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.

Fig. 2.

IL-1β production from adherent monocytes. (A) Mean ± SEM levels of total IL-1β produced from monocytes cultured from haplotype 1 homozygotes (Hapl 1; n = 6 subjects) and haplotype 2A homozygotes (Hapl 2A; n = 10 subjects). (B) Mean ± SEM percent processed IL-1β from monocytes cultured from haplotype 1 homozygotes (Hapl 1) and haplotype 2A homozygotes (Hapl 2A). Each subject was tested on three occasions, and on each occasion, in triplicate wells. The stimulants are indicated under the horizontal axis: RPMI (no stimulant), LPS (1 ng/mL), MDP (10 μg/mL, LPS + MDP, S. epidermidis (Materials and Methods).

Fig. 3.

Production of IL-1α and TNFα from adherent monocytes. (A) Mean ± SEM levels of intracellular IL-1α produced from monocytes cultured from haplotype 1 homozygotes (same samples as in Fig. 2A) and haplotype 2A homozygotes (same samples as in Fig. 2B). (B) Mean ± SEM level of extracellular TNFα in same samples shown in Fig. 2B. The stimulants are indicated under the horizontal axis as in Fig. 2.

Fig. 4.

NLRP1 RNA and protein levels. Levels of NLRP1 (A) RNA and (B) protein in lymphoblastoid cells from haplotype 1 homozygotes (Hapl 1) and haplotype 2A homozygotes (Hapl 2A). NLRP1 protein was assayed by Western blot (Fig. S2) and quantitated using ImageJ. NLRP1 RNA and protein were normalized to GAPDH RNA or β-actin protein, respectively.