Graham R. Wallace

Characterization of vitamin D production by human ocular barrier cells

PURPOSEnVitamin D3 is a secosteroid mainly synthesized from the conversion of the skin precursor 7-dehydrocholesterol (7DHC) to vitamin D3 by ultraviolet (UV) B sunlight. Extrarenal synthesis of vitamin D3 has been reported in many tissues and cells, including barrier sites. This study characterizes the expression of components of vitamin D3 signaling in human ocular barrier cells.nnnMETHODSnPrimary human scleral fibroblasts (HSF), human corneal endothelial (HCEC-12), nonpigmented ciliary body epithelial (ODM-2), and adult retinal pigment epithelial (ARPE-19) cell lines were analyzed for the expression of vitamin D receptor (VDR), the vitamin D3 activating enzymes 1α-hydroxylase (CYP27B1), 25-hydroxylases (CYP27A1 and CYP2R1), the vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and the endocytic receptors cubilin and megalin using a combination of RT-PCR, immunocytochemistry, and enzyme immunoassay (EIA).nnnRESULTSnThe HSF, HCEC-12, ODM-2, and ARPE-19 express mRNA and protein for all vitamin D3 synthesizing and metabolizing components. The cell types tested, except HSF, are able to convert inactive 25-hydroxyvitamin D3 (25[OH]D3) into active 1,25-hydroxyvitamin D3 (1,25[OH]2D3).nnnCONCLUSIONSnThis novel study demonstrated that ocular barrier epithelial cells express the machinery for vitamin D3 and can produce 1,25(OH)2D3. We suggest that vitamin D3 might have a role in immune regulation and barrier function in ocular barrier epithelial cells.

Genome-wide association study in an admixed case series reveals IL12A as a new candidate in Behçet disease.

Introduction The etiology of Behçet’s disease (BD) is unknown, but widely considered an excessive T-cell mediated inflammatory response in a genetically susceptible host. Recent genome-wide association studies (GWAS) have shown limited number of novel loci-associations. The rarity and unequal distribution of the disease prevalence amongst different ethnic backgrounds have hampered the use of GWAS in cohorts of mixed ethnicity and sufficient sample size. However, novel statistical approaches have now enabled GWAS in admixed cohorts. Methods We ran a GWAS on 336 BD cases and 5,843 controls. The cases consisted of Western Europeans, Middle Eastern and Turkish individuals. Participants from the Generation R study, a multiethnic birth cohort in Rotterdam, The Netherlands were used as controls. All samples were genotyped and data was combined. Linear regression models were corrected for population stratification using Genomic Principal Components and Linear Mixed Modelling. Meta-analysis was performed on selected results previously published. Results We identified SNPs associated at genome-wide significant level mapping to the 6p21.33 (HLA) region. In addition to this known signal two potential novel associations on chromosomes 6 and 18 were identified, yet with low minor allele frequencies. Extended meta-analysis reveal a GWS association with the IL12A variant rs17810546 on chromosome 3. Discussion We demonstrate that new statistical techniques enable GWAS analyses in a limited sized cohort of mixed ethnicity. After implementation, we confirmed the central role of the HLA region in the disease and identified new regions of interest. Moreover, we validated the association of a variant in the IL2A gene by meta-analysis with previous work. These findings enhance our knowledge of genetic associations and BD, and provide further justification for pursuing collective initiatives in genetic studies given the low prevalence of this and other rare diseases.

HLA-B*51 the primary risk in Behçet disease

Behcet disease (BD) is a multisystem autoinflammatory condition characterized by mucosal ulceration and predominantly neutropilic inflammation of immune privileged sites including the eye, brain, and synovial joint (1). In PNAS, Ombrello et al. analyze a combination of directly ascertained and imputated single nucleotide polymorphisms (SNPs) in the HLA-B region in Turkish patients with BD (2). HLA-B*51 was the largest single risk factor for BD, with smaller, independent effects of HLA-B*15 and HLA-B*27. HLA-B*15 was also significantly associated in HLA-B*51−ve patients. Analysis of 32,869 SNPs across the MHC region identified HLA and MHC class I polypeptide-related sequence A (MICA) as the strongest association. Conditioning on HLA-B*51 and rs79556279, the strongest associated SNPs, 4.5 kb upstream of HLA-B loci showed no other significant SNPs. Moreover HLA-B*51 and rs79556279 are in strong linkage disequilibrium. Two other regions, one tetrameric to HLA-C and the second in a region that includes HLA-A, were associated with BD, but the former was lost on conditioning for rs79556279. Associations with HLA-A, particularly HLA-A26, have been previously reported, particularly in Japanese patients with BD.

Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly

It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay. The results show that Sal reduced the percentage of IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA(*009) (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells. Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells.

Cortisol Biosynthesis in the Human Ocular Surface Innate Immune Response

Innate immune responses have a critical role in regulating sight-threatening ocular surface (OcS) inflammation. While glucocorticoids (GCs) are frequently used to limit tissue damage, the role of intracrine GC (cortisol) bioavailability via 11-beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in OcS defense, remains unresolved. We found that primary human corneal epithelial cells (PHCEC), fibroblasts (PHKF) and allogeneic macrophages (M1, GM-CSF; M2, M-CSF) were capable of generating cortisol (M1>PHKF>M2>PHCEC) but in corneal cells, this was independent of Toll-like receptor (TLR) activation. While PolyI∶C induced maximal cytokine and chemokine production from both PHCEC (IFNγ, CCL2, CCL3, and (CCL4), IL6, CXCL10, CCL5, TNFα) and PHKF (CCL2, IL-6, CXCL10, CCL5), only PHKF cytokines were inhibited by GCs. Both Poly I∶C and LPS challenged-corneal cells induced M1 chemotaxis (greatest LPS-PHKF (250%), but down-regulated M1 11β-HSD1 activity (30 and 40% respectively). These data were supported by clinical studies demonstrating reduced human tear film cortisol∶cortisone ratios (a biomarker of local 11β-HSD1 activity) in pseudomonas keratitis (1∶2.9) versus healthy controls (1∶1.3; p<0.05). This contrasted with putative TLR3-mediated OcS disease (Stevens-Johnson Syndrome, Mucous membrane pemphigoid) where an increase in cortisol∶cortisone ratio was observed (113.8∶1; p<0.05). In summary, cortisol biosynthesis in human corneal cells is independent of TLR activation and is likely to afford immunoprotection under physiological conditions. Contribution to ocular mucosal innate responses is dependent on the aetiology of immunological challenge.

Gene-Based Genome-Wide Association Analysis in European and Asian Populations Identified Novel Genes for Rheumatoid Arthritis.

Objective Rheumatoid arthritis (RA) is a complex autoimmune disease. Using a gene-based association research strategy, the present study aims to detect unknown susceptibility to RA and to address the ethnic differences in genetic susceptibility to RA between European and Asian populations. Methods Gene-based association analyses were performed with KGG 2.5 by using publicly available large RA datasets (14,361 RA cases and 43,923 controls of European subjects, 4,873 RA cases and 17,642 controls of Asian Subjects). For the newly identified RA-associated genes, gene set enrichment analyses and protein-protein interactions analyses were carried out with DAVID and STRING version 10.0, respectively. Differential expression verification was conducted using 4 GEO datasets. The expression levels of three selected ‘highly verified’ genes were measured by ELISA among our in-house RA cases and controls. Results A total of 221 RA-associated genes were newly identified by gene-based association study, including 71‘overlapped’, 76 ‘European-specific’ and 74 ‘Asian-specific’ genes. Among them, 105 genes had significant differential expressions between RA patients and health controls at least in one dataset, especially for 20 genes including 11 ‘overlapped’ (ABCF1, FLOT1, HLA-F, IER3, TUBB, ZKSCAN4, BTN3A3, HSP90AB1, CUTA, BRD2, HLA-DMA), 5 ‘European-specific’ (PHTF1, RPS18, BAK1, TNFRSF14, SUOX) and 4 ‘Asian-specific’ (RNASET2, HFE, BTN2A2, MAPK13) genes whose differential expressions were significant at least in three datasets. The protein expressions of two selected genes FLOT1 (P value = 1.70E-02) and HLA-DMA (P value = 4.70E-02) in plasma were significantly different in our in-house samples. Conclusion Our study identified 221 novel RA-associated genes and especially highlighted the importance of 20 candidate genes on RA. The results addressed ethnic genetic background differences for RA susceptibility between European and Asian populations and detected a long list of overlapped or ethnic specific RA genes. The study not only greatly increases our understanding of genetic susceptibility to RA, but also provides important insights into the ethno-genetic homogeneity and heterogeneity of RA in both ethnicities.

Progenitor cells are mobilized by acute psychological stress but not beta-adrenergic receptor agonist infusion

OBJECTIVESnStimuli that activate the sympathetic nervous system, such as acute psychological stress, rapidly invoke a robust mobilization of lymphocytes into the circulation. Experimental animal studies suggest that bone marrow-derived progenitor cells (PCs) also mobilize in response to sympathetic stimulation. Here we tested the effects of acute psychological stress and brief pharmacological β-adrenergic (βAR) stimulation on peripheral PC numbers in humans.nnnMETHODSnIn two studies, we investigated PC mobilization in response to an acute speech task (n=26) and βAR-agonist (isoproterenol) infusion (n=20). A subset of 8 participants also underwent the infusion protocol with concomitant administration of the βAR-antagonist propranolol. Flow cytometry was used to enumerate lymphocyte subsets, total progenitor cells, total haematopoietic stem cells (HSC), early HSC (multi-lineage potential), late HSC (lineage committed), and endothelial PCs (EPCs).nnnRESULTSnBoth psychological stress and βAR-agonist infusion caused the expected mobilization of total monocytes and lymphocytes and CD8(+) T lymphocytes. Psychological stress also induced a modest, but significant, increase in total PCs, HSCs, and EPC numbers in peripheral blood. However, infusion of a βAR-agonist did not result in a significant change in circulating PCs.nnnCONCLUSIONnPCs are rapidly mobilized by psychological stress via mechanisms independent of βAR-stimulation, although the findings do not exclude βAR-stimulation as a possible cofactor. Considering the clinical and physiological relevance, further research into the mechanisms involved in stress-induced PC mobilization seems warranted.

Behçet's disease: do natural killer cells play a significant role?

Behçet’s disease (BD) is a complex inflammatory disease, of unknown etiology. While disease pathogenesis remains unclear, a strong relationship between BD and HLA-B*51 has been established over the last 30u2009years. A number of theories exist regarding the cause of BD; however, few are able to account for the increased rates of HLA-B*51 positive individuals, particularly around the Mediterranean basin and Middle-East where the prevalence is highest. This review outlines current immunogenetic data on BD and the immunoregulatory role natural killer cells may play. It also describes the interaction of the killer immunoglobulin-like receptor – KIR3DL1 with its ligand Bw4, which is found on HLA-B51. Finally, CD94/NKG2D, MICA, and ERAP are outlined with regard to their potential roles in BD.

Association analysis of TGFBR3 gene with Behçet's disease and idiopathic intermediate uveitis in a Caucasian population

Background Transforming growth factor β (TGFβ) is an important immunoregulatory cytokine in regulatory T cell (Treg) and Th17-mediated pathology, including uveitis due to Behçets disease (BD). Of the three isoforms, TGFβ2 is found at highest levels in the aqueous humour of uninflamed eyes. TGFβ signals through a cell-surface receptor comprising three subunits (TGFBR1, 2 and 3). TGFBR3 is considered necessary for TGFβ2 signal transduction, but not for other isoforms. A polymorphism in TGFBR3 (rs1805110) has previously been identified in Han Chinese patients with BD. We investigated the frequency of this polymorphism in a Caucasian population with BD and idiopathic intermediate uveitis (IIU). Methods The single-nucleotide polymorphism (SNP) rs1805110 in TGFBR3 was genotyped in 75 BD patients, 92 IIU disease controls and 85 disease-free controls. The association with both diseases was analysed using Fishers exact test. Results No significant difference in rs1805110 allele or genotype frequency was observed. A low frequency of the T allele was observed (5.88% control, 9.33% BD, 10.33% IIU) with the TT genotype absent in patients with BD and IIU (1.18% control, 0% BD and 0% IIU). Stratification analysis according to clinical features of BD did not associate with the tested SNP. Conclusions RS1805110 is not associated with BD or IIU in Caucasian patients. The T allele frequency is consistent with that presented for Caucasian populations in the HapMap database (p>0.05). Our results differ from the previous analysis in Han Chinese patients (p<0.0001), however, the possibility of having a much smaller effect due to the low minority frequency cannot be excluded.

Glucocorticoid sensitivity in Behçet's disease

Objective Glucocorticoid (GC) sensitivity is highly variable among individuals and has been associated with susceptibility to develop (auto-)inflammatory disorders. The purpose of the study was to assess GC sensitivity in Behçets disease (BD) by studying the distribution of four GC receptor (GR) gene polymorphisms and by measuring in vitro cellular GC sensitivity. Methods Healthy controls and patients with BD in three independent cohorts were genotyped for four functional GR gene polymorphisms. To gain insight into functional differences in in vitro GC sensitivity, 19 patients with BD were studied using two bioassays and a whole-cell dexamethasone-binding assay. Finally, mRNA expression levels of GR splice variants (GR-α and GR-β) were measured. Results Healthy controls and BD patients in the three separate cohorts had similar distributions of the four GR polymorphisms. The Bcll and 9β minor alleles frequency differed significantly between Caucasians and Mideast and Turkish individuals. At the functional level, a decreased in vitro cellular GC sensitivity was observed. GR number in peripheral blood mononuclear cells was higher in BD compared with controls. The ratio of GR-α/GR-β mRNA expression levels was significantly lower in BD. Conclusions Polymorphisms in the GR gene are not associated with susceptibility to BD. However, in vitro cellular GC sensitivity is decreased in BD, possibly mediated by a relative higher expression of the dominant negative GR-β splice variant. This decreased in vitro GC sensitivity might play an as yet unidentified role in the pathophysiology of BD.