Gethin P. Thomas

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10−8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10−6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27–positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Hypothalamic Y2 receptors regulate bone formation

Neuropeptide Y (NPY) is a downstream modulator of leptin action, possibly at the level of the arcuate nucleus where NPY neurons are known to express both leptin receptors and Y2 receptors. In addition to the well-described role of NPY and leptin in energy balance and obesity, intracerebroventricular administration of NPY or leptin also causes bone loss. Here we show that Y2 receptor-deficient mice have a twofold increase in trabecular bone volume as well as greater trabecular number and thickness compared with control mice. We also demonstrate that central Y2 receptors are crucial for this process, since selective deletion of hypothalamic Y2 receptors in mature conditional Y2 knockout mice results in an identical increase in trabecular bone volume within 5 weeks. This hypothalamus-specific Y2 receptor deletion stimulates osteoblast activity and increases the rate of bone mineralization and formation, with no effect on osteoblast or osteoclast surface measurements. The lack of any changes in plasma total calcium, leptinemia, or hypothalamo-pituitary-corticotropic, -thyrotropic, -somatotropic, or -gonadotropic output suggests that Y2 receptors do not modulate bone formation by humoral mechanisms, and that alteration of autonomic function through hypothalamic Y2 receptors may play a key role in a major central regulatory circuit of bone formation.

Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage

The microarchitecture of bone is regulated by complex interactions between the bone‐forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone‐forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.—Gardiner, E. M., Baldock, P. A., Thomas, G. P., Sims, N. A., Henderson, N. K., Hollis, B., White, C. P., Sunn, K. L., Morrison, N. A., Walsh, W. R., Eisman, J. A. Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage. FASEB J. 14, 1908–1916 (2000)

Enrichment of circulating interleukin‐17–secreting interleukin‐23 receptor–positive γ/δ T cells in patients with active ankylosing spondylitis

OBJECTIVE Ankylosing spondylitis (AS) is a common inflammatory arthritis affecting primarily the axial skeleton. IL23R is genetically associated with AS. This study was undertaken to investigate and characterize the role of interleukin-23 (IL-23) signaling in AS pathogenesis. METHODS The study population consisted of patients with active AS (n = 17), patients with psoriatic arthritis (n = 8), patients with rheumatoid arthritis, (n = 9), and healthy subjects (n = 20). IL-23 receptor (IL-23R) expression in T cells was determined in each subject group, and expression levels were compared. RESULTS The proportion of IL-23R-expressing T cells in the periphery was 2-fold higher in AS patients than in healthy controls, specifically driven by a 3-fold increase in IL-23R-positive γ/δ T cells in AS patients. The proportions of CD4+ and CD8+ cells that were positive for IL-17 were unchanged. This increased IL-23R expression on γ/δ T cells was also associated with enhanced IL-17 secretion, with no observable IL-17 production from IL-23R-negative γ/δ T cells in AS patients. Furthermore, γ/δ T cells from AS patients were heavily skewed toward IL-17 production in response to stimulation with IL-23 and/or anti-CD3/CD28. CONCLUSION Recently, mouse models have shown IL-17-secreting γ/δ T cells to be pathogenic in infection and autoimmunity. Our data provide the first description of a potentially pathogenic role of these cells in a human autoimmune disease. Since IL-23 is a maturation and growth factor for IL-17-producing cells, increased IL-23R expression may regulate the function of this putative pathogenic γ/δ T cell population.

Vitamin D action and regulation of bone remodeling : suppression of osteoclastogenesis by the mature osteoblast

Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone. This study indicates that vitamin D–mediated osteoclastogenesis is regulated locally by OPG production in the mature osteoblast.

Genetics and genomics of ankylosing spondylitis

Summary:  Ankylosing spondylitis (AS) is a common, highly heritable arthropathy, the pathogenesis of which is poorly understood. The mechanism by which the main gene for the disease, HLA‐B27, leads to AS is unknown. Genetic and genomic studies have demonstrated involvement of the interleukin‐23 (IL‐23) signaling pathway in AS, a finding which has stimulated much new research into the disease and has led to therapeutic trials. Several other genes and genetic regions, including further major histocompatibility complex (MHC) and non‐MHC loci, have been shown to be involved in the disease, but it is not clear yet how they actually induce the condition. These findings have shown that there is a strong genetic overlap between AS and Crohn’s disease in particular, although there are also major differences in the genes involved in the two conditions, presumably explaining their different presentations. Genomic and proteomic studies are in an early phase but have potential both as diagnostic/prognostic tools and as a further hypothesis‐free tool to investigate AS pathogenesis. Given the slow progress in studying the mechanism of association of HLA‐B27 with AS, these may prove to be more fruitful approaches to investigating the pathogenesis of the disease.

Resveratrol Does Not Benefit Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Nonalcoholic fatty liver disease (NAFLD), characterized by accumulation of hepatic triglycerides (steatosis), is associated with abdominal obesity, insulin resistance, and inflammation. Although weight loss via calorie restriction reduces features of NAFLD, there is no pharmacologic therapy. Resveratrol is a polyphenol that prevents high-energy diet-induced steatosis and insulin resistance in animals by up-regulating pathways that regulate energy metabolism. We performed a placebo-controlled trial to assess the effects of resveratrol in patients with NAFLD. METHODS Overweight or obese men diagnosed with NAFLD were recruited from hepatology outpatient clinics in Brisbane, Australia from 2011 through 2012. They were randomly assigned to groups given 3000 mg resveratrol (n = 10) or placebo (n = 10) daily for 8 weeks. Outcomes included insulin resistance (assessed by the euglycemic-hyperinsulinemic clamp), hepatic steatosis, and abdominal fat distribution (assessed by magnetic resonance spectroscopy and imaging). Plasma markers of inflammation, as well as metabolic, hepatic, and antioxidant function, were measured; transcription of target genes was measured in peripheral blood mononuclear cells. Resveratrol pharmacokinetics and safety were assessed. RESULTS Eight-week administration of resveratrol did not reduce insulin resistance, steatosis, or abdominal fat distribution when compared with baseline. No change was observed in plasma lipids or antioxidant activity. Levels of alanine and aspartate aminotransferases increased significantly among patients in the resveratrol group until week 6 when compared with the placebo group. Resveratrol did not significantly alter transcription of NQO1, PTP1B, IL6, or HO1 in peripheral blood mononuclear cells. Resveratrol was well-tolerated. CONCLUSIONS Eight weeks administration of resveratrol did not significantly improve any features of NAFLD, compared with placebo, but it increased hepatic stress, based on observed increases in levels of liver enzymes. Further studies are needed to determine whether agents that are purported to mimic calorie restriction, such as resveratrol, are safe and effective for complications of obesity. Clinical trials registration no: ACTRN12612001135808.

Association of ERAP1, but not IL23R, with ankylosing spondylitis in a Han Chinese population

OBJECTIVE The results of a recent genome-wide association study have shown that ERAP1 and IL23R are associated with ankylosing spondylitis (AS) in Caucasian populations from North America and the UK. Based on these findings, we undertook the current study to investigate whether single-nucleotide polymorphisms (SNPs) covering the genes ERAP1 and IL23R are associated with AS in a Han Chinese population. METHODS A case-control study was performed in Han Chinese patients with AS (n = 527) and controls (n = 945) from Shanghai and Nanjing. All patients met the modified New York criteria for AS. The Sequenom iPlex platform was used to genotype cases and controls for 21 tag SNPs covering IL23R and 38 tag SNPs covering ERAP1. Statistical analysis was performed using the Cochran-Armitage test for trend. RESULTS Multiple SNPs in ERAP1 were significantly associated with AS (for rs27980, P = 0.0048; for rs7711564, P = 0.0081). However, no association was observed between IL23R and AS (for all SNPs, P > 0.1). The nonsynonymous SNP in IL23R, rs11209026, widely thought to be the primary AS-associated SNP in IL23R in Europeans, was found not to be polymorphic in Chinese. CONCLUSION Our results demonstrate that genetic polymorphisms in ERAP1 are associated with AS in Han Chinese, suggesting a common pathogenic mechanism for the disease in Chinese and Caucasian populations, and that IL23R is not associated with AS in Chinese, indicating a difference in the mechanism of disease pathogenesis between Chinese and Caucasian populations. This may result from the fact that rs11209026, the nonsynonymous SNP in IL23R, is not polymorphic in Chinese patients, providing further evidence that rs11209026 is the key polymorphism associated with AS (and likely inflammatory bowel disease and psoriasis) in this gene.

Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone “secretome” using a signal‐trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm‐like protein (Bril). Bril encodes a 14.8‐kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone‐specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus‐mediated Bril overexpression and lentivirus‐mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose‐dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

β-Glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice

OBJECTIVE The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohns disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohns disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.