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

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-1ß (IL-1ß), resulting in IL-1ß 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-1ß precursor to mature bioactive IL-1ß 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.

Variant of TYR and autoimmunity susceptibility loci in generalized vitiligo.

BACKGROUND: Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair, and is associated with an elevated risk of other autoimmune diseases. METHODS: To identify generalized vitiligo susceptibility loci, we conducted a genomewide association study. We genotyped 579,146 single-nucleotide polymorphisms (SNPs) in 1514 patients with generalized vitiligo who were of European-derived white (CEU) ancestry and compared the genotypes with publicly available control genotypes from 2813 CEU persons. We then tested 50 SNPs in two replication sets, one comprising 677 independent CEU patients and 1106 CEU controls and the other comprising 183 CEU simplex trios with generalized vitiligo and 332 CEU multiplex families. RESULTS: We detected significant associations between generalized vitiligo and SNPs at several loci previously associated with other autoimmune diseases. These included genes encoding major-histocompatibility-complex class I molecules (P=9.05x10(-23)) and class II molecules (P=4.50x10(-34)), PTPN22 (P=1.31x10(-7)), LPP (P=1.01x10(-11)), IL2RA (P=2.78x10(-9)), UBASH3A (P=1.26x10(-9)), and C1QTNF6 (P=2.21x10(-16)). We also detected associations between generalized vitiligo and SNPs in two additional immune-related loci, RERE (P=7.07x10(-15)) and GZMB (P=3.44x10(-8)), and in a locus containing TYR (P=1.60x10(-18)), encoding tyrosinase. CONCLUSIONS: We observed associations between generalized vitiligo and markers implicating multiple genes, some associated with other autoimmune diseases and one (TYR) that may mediate target-cell specificity and indicate a mutually exclusive relationship between susceptibility to vitiligo and susceptibility to melanoma.

Development of murine lupus involves the combined genetic contribution of the SLAM and FcgammaR intervals within the Nba2 autoimmune susceptibility locus.

Autoantibodies are of central importance in the pathogenesis of Ab-mediated autoimmune disorders. The murine lupus susceptibility locus Nba2 on chromosome 1 and the syntenic human locus are associated with a loss of immune tolerance that leads to antinuclear Ab production. To identify gene intervals within Nba2 that control the development of autoantibody-producing B cells and to determine the cellular components through which Nba2 genes accomplish this, we generated congenic mice expressing various Nba2 intervals where genes for the FcgammaR, SLAM, and IFN-inducible families are encoded. Analysis of congenic strains demonstrated that the FcgammaR and SLAM intervals independently controlled the severity of autoantibody production and renal disease, yet are both required for lupus susceptibility. Deregulated homeostasis of terminally differentiated B cells was found to be controlled by the FcgammaR interval where FcgammaRIIb-mediated apoptosis of germinal center B cells and plasma cells was impaired. Increased numbers of activated plasmacytoid dendritic cells that were distinctly CD19+ and promoted plasma cell differentiation via the proinflammatory cytokines IL-10 and IFNalpha were linked to the SLAM interval. These findings suggest that SLAM and FcgammaR intervals act cooperatively to influence the clinical course of disease through supporting the differentiation and survival of autoantibody-producing cells.

Expression profiling identifies Klf15 as a glucocorticoid target that regulates airway hyperresponsiveness.

Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15(-/-) mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15(-/-) mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15(-/-) murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function.

A role of the LIM-homeobox gene Lhx2 in the regulation of pituitary development.

The mammalian pituitary gland originates from two separate germinal tissues during embryonic development. The anterior and intermediate lobes of the pituitary are derived from Rathke's pouch, a pocket formed by an invagination of the oral ectoderm. The posterior lobe is derived from the infundibulum, which is formed by evagination of the neuroectoderm in the ventral diencephalon. Previous studies have shown that development of Rathke's pouch and the generation of distinct populations of hormone-producing endocrine cell lineages in the anterior/intermediate pituitary lobes is regulated by a number of transcription factors expressed in the pouch and by inductive signals from the ventral diencephalon/infundibulum. However, little is known about factors that regulate the development of the posterior pituitary lobe. In this study, we show that the LIM-homeobox gene Lhx2 is extensively expressed in the developing ventral diencephalon, including the infundibulum and the posterior lobe of the pituitary. Deletion of Lhx2 gene results in persistent cell proliferation, a complete failure of evagination of the neuroectoderm in the ventral diencephalon, and defects in the formation of the distinct morphological features of the infundibulum and the posterior pituitary lobe. Rathke's pouch is formed and endocrine cell lineages are generated in the anterior/intermediate pituitary lobes of the Lhx2 mutant. However, the shape and organization of the pouch and the anterior/intermediate pituitary lobes are severely altered due to the defects in development of the infundibulum and the posterior lobe. Our study thus reveals an essential role for Lhx2 in the regulation of posterior pituitary development and suggests a mechanism whereby development of the posterior lobe may affect the development of the anterior and intermediate lobes of the pituitary gland.

NALP1 in vitiligo-associated multiple autoimmune disease.

BACKGROUND: Autoimmune and autoinflammatory diseases involve interactions between genetic risk factors and environmental triggers. We searched for a gene on chromosome 17p13 that contributes to a group of epidemiologically associated autoimmune and autoinflammatory diseases. The group includes various combinations of generalized vitiligo, autoimmune thyroid disease, latent autoimmune diabetes in adults, rheumatoid arthritis, psoriasis, pernicious anemia, systemic lupus erythematosus, and Addison's disease. METHODS: We tested 177 single-nucleotide polymorphisms (SNPs) spanning the 17p13 linkage peak for association with disease and identified a strong candidate gene. We then sequenced DNA in and around the gene to identify additional SNPs. We carried out a second round of tests of association using some of these additional SNPs, thus elucidating the association with disease in the gene and its extended promoter region in fine detail. RESULTS: Association analyses resulted in our identifying as a candidate gene NALP1, which encodes NACHT leucine-rich-repeat protein 1, a regulator of the innate immune system. Fine-scale association mapping with the use of DNA from affected families and additional SNPs in and around NALP1 showed an association of specific variants with vitiligo alone, with an extended autoimmune and autoinflammatory disease phenotype, or with both. Conditional logistic-regression analysis of NALP1 SNPs indicated that at least two variants contribute independently to the risk of disease. CONCLUSIONS: DNA sequence variants in the NALP1 region are associated with the risk of several epidemiologically associated autoimmune and autoinflammatory diseases, implicating the innate immune system in the pathogenesis of these disorders.

The glucocorticoid receptor and KLF15 regulate gene expression dynamics and integrate signals through feed-forward circuitry.

The glucocorticoid receptor (GR) regulates adaptive transcriptional programs that alter metabolism in response to stress. Network properties that allow GR to tune gene expression to match specific physiologic demands are poorly understood. We analyzed the transcriptional consequences of GR activation in murine lungs deficient for KLF15, a transcriptional regulator of amino acid metabolism that is induced by glucocorticoids and fasting. Approximately 7% of glucocorticoid-regulated genes had altered expression in Klf15-knockdown (Klf15(-/-)) mice. KLF15 formed coherent and incoherent feed-forward circuits with GR that correlated with the expression dynamics of the glucocorticoid response. Coherent feed-forward gene regulation by GR and KLF15 was characterized by combinatorial activation of linked GR-KLF15 regulatory elements by both factors and increased GR occupancy, while expression of KLF15 reduced GR occupancy at the incoherent target, MT2A. Serum deprivation, which increased KLF15 expression in a GR-independent manner in vitro, enhanced glucocorticoid-mediated induction of feed-forward targets of GR and KLF15, such as the loci for the amino acid-metabolizing enzymes proline dehydrogenase and alpha-aminoadipic semialdehyde synthase. Our results establish feed-forward architecture as an organizational principle for the GR network and provide a novel mechanism through which GR integrates signals and regulates expression dynamics.

Common variants in FOXP1 are associated with generalized vitiligo.

In a recent genome-wide association study of generalized vitiligo, we identified ten confirmed susceptibility loci. By testing additional loci that showed suggestive association in the genome-wide study, using two replication cohorts of European descent, we observed replicated association of generalized vitiligo with variants at 3p13 encompassing FOXP1 (rs17008723, combined P=1.04x10(-8)) and with variants at 6q27 encompassing CCR6 (rs6902119, combined P=3.94x10(-7)).

LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum.

Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.

Early disease onset and increased risk of other autoimmune diseases in familial generalized vitiligo.

Generalized vitiligo is an autoimmune disorder in which acquired white patches of skin and overlying hair result from autoimmune loss of melanocytes from involved areas. Although usually sporadic, family clustering of vitiligo may occur, in a non-Mendelian pattern typical of multifactorial, polygenic inheritance. Sporadic vitiligo is associated with autoimmune thyroid disease, pernicious anemia, Addison's disease, and lupus; these same disorders occur at increased frequency in patients' first-degree relatives. Here, we studied 133 'multiplex' generalized vitiligo families, with multiple affected family members. The age of onset of vitiligo is earlier in these 'multiplex' families than in patients with sporadic vitiligo. Affected members of the multiplex vitiligo families have elevated frequencies of autoimmune thyroid disease, rheumatoid arthritis, psoriasis, adult-onset insulin-dependent diabetes mellitus, pernicious anemia, and Addison's disease. Probands' unaffected siblings have elevated frequencies of most of these same autoimmune diseases, particularly if the proband had non-vitiligo autoimmune disease. Familial generalized vitiligo is thus characterized by earlier disease onset and a broader repertoire of associated autoimmune diseases than sporadic vitiligo. This mostly likely reflects a greater inherited genetic component of autoimmune susceptibility in these families. These findings have important implications for autoimmune disease surveillance in families in which multiple members are affected with vitiligo.