Lysosomal pH Is Regulated in a Sex Dependent Manner in Immune Cells Expressing CXorf21

Background:CXorf21 and SLC15a4 both contain risk alleles for systemic lupus erythematosus (SLE) and Sjögren's syndrome (pSS). The former escapes X inactivation. Our group predicts specific endolysosomal-dependent immune responses are driven by the protein products of these genes, which form a complex at the endolysosomal surface. Our previous studies have shown that knocking out CXorf21 increases lysosomal pH in female monocytes, and the present study assesses whether the lysosomal pH in 46,XX women, who overexpress CXorf21 in monocytes, B cells, and dendritic cells (DCs), differs from 46,XY men.Methods: To determine endolysosome compartment pH we used both LysoSensor™ Yellow/Blue DND-160 and pHrodo® Red AM Intracellular pH Indicator in primary monocyte, B cells, DCs, NK cells, and T cells from healthy men and women volunteers.Results: Compared to male samples, female monocytes, B cells, and DCs had lower endolysosomal pH (female/male pH value: monocytes 4.9/5.6 p < 0.0001; DCs 4.9/5.7 p = 0.044; B cells 5.0/5.6 p < 0.05). Interestingly, T cells and NK cells, which both express low levels of CXorf21, showed no differential pH levels between men and women.Conclusion: We have previously shown that subjects with two or more X-chromosomes have increased CXorf21 expression in specific primary immune cells. Moreover, knockdown of CXorf21 increases lysosomal pH in female monocytes. The present data show that female monocytes, DC, B cells, where CXorf21 is robustly expressed, have lower lysosomal pH compared to the same immune cell populations from males. The lower pH levels observed in specific female immune cells provide a function to these SLE/SS-associated genes and a mechanism for the reported inflated endolysosomal-dependent immune response observed in women compared to men (i.e., TLR7/type I Interferon activity)

Dietary Strawberries Improve Biomarkers of Antioxidant Status and Endothelial Function in Adults With Cardiometabolic Risks in a Randomized Controlled Crossover Trial

Strawberries, a popularly consumed berry fruit, are rich in bioactive compounds with antioxidant effects. In this study, we examined the effects of two dietary achievable doses of strawberries on the antioxidant status and biomarkers of endothelial function in adults with features of metabolic syndrome and a confirmed low baseline of fruit and vegetable intake. In a 14-week randomized controlled crossover study, participants were assigned to one of three groups for four weeks separated by a one-week washout period: control powder, one serving (low dose: 13 g strawberry powder/day), or 2.5 servings (high dose: 32 g strawberry powder/day). Blood samples and health data were collected at baseline and at the end of each four-week phase of intervention. Thirty-three participants completed all three phases of the trial. Significant increases were observed in serum antioxidant capacity and superoxide dismutase activity as well as decreases in lipid peroxidation after both low and high dose strawberry phases when compared with the control phase. Significant decreases were also observed in soluble vascular cell adhesion molecule-1 and tumor necrosis factor-α with the high dose strawberry phase. These data confirm that consuming strawberries for four weeks significantly improves antioxidant status, endothelial function, and inflammation in adults with cardiometabolic risks

Dietary Strawberries Improve Cardiometabolic Risks in Adults With Obesity and Elevated Serum Ldl Cholesterol in a Randomized Controlled Crossover Trial

Background and aims: Dietary berries, such as strawberries, are rich in bioactive compounds and have been shown to lower cardiometabolic risk. We examined the effects of two dietary achievable doses of strawberries on glycemic control and lipid profiles in obese adults with elevated serum LDL cholesterol (LDL-C). Methods: In this 14-week randomized controlled crossover study, participants were assigned to one of the three arms for four weeks separated by a one-week washout period: control powder, one serving (low dose: 13 g strawberry powder/day), or two-and-a-half servings (high dose: 32 g strawberry powder/day). Participants were instructed to follow their usual diet and lifestyle while refraining from consuming other berries and related products throughout the study interval. Blood samples, anthropometric measures, blood pressure, and dietary and physical activity data were collected at baseline and at the end of each four-week phase of intervention. Results: In total, 33 participants completed all three phases of the trial [(mean ± SD): Age: 53 ± 13 y; BMI: 33 ± 3.0 kg/m ). Findings revealed significant reductions in fasting insulin (p = 0.0002) and homeostatic model of assessment of insulin resistance (p = 0.0003) following the high dose strawberry phase when compared to the low dose strawberry and control phases. Glucose and conventional lipid profiles did not differ among the phases. Nuclear magnetic resonance-determined particle concentrations of total VLDL and chylomicrons, small VLDL, and total and small LDL were significantly decreased after the high dose strawberry phase, compared to control and low dose phases (all p \u3c 0.0001). Among the biomarkers of inflammation and adipokines measured, only serum PAI-1 showed a decrease after the high dose strawberry phase (p = 0.002). Conclusions: These data suggest that consuming strawberries at two-and-a-half servings for four weeks significantly improves insulin resistance, lipid particle profiles, and serum PAI-1 in obese adults with elevated serum LDL-C.

47XXY and 47XXX in Scleroderma and Myositis.

Objective: We undertook this study to examine the X chromosome complement in participants with systemic sclerosis (SSc) as well as idiopathic inflammatory myopathies. Methods: The participants met classification criteria for the diseases. All participants underwent single-nucleotide polymorphism typing. We examined X and Y single-nucleotide polymorphism heterogeneity to determine the number of X chromosomes. For statistical comparisons, we used χ2 analyses with calculation of 95% confidence intervals. Results: Three of seventy men with SSc had 47,XXY (P = 0.0001 compared with control men). Among the 435 women with SSc, none had 47,XXX. Among 709 men with polymyositis or dermatomyositis (PM/DM), seven had 47,XXY (P = 0.0016), whereas among the 1783 women with PM/DM, two had 47,XXX. Of 147 men with inclusion body myositis (IBM), six had 47,XXY, and 1 of the 114 women with IBM had 47,XXX. For each of these myositis disease groups, the excess 47,XXY and/or 47,XXX was significantly higher compared with in controls as well as the known birth rate of Klinefelter syndrome or 47,XXX. Conclusion: Klinefelter syndrome (47,XXY) is associated with SSc and idiopathic inflammatory myopathies, similar to other autoimmune diseases with type 1 interferon pathogenesis, namely, systemic lupus erythematosus and Sjögren syndrome.We thank Drs. Elaine Remmers and Robert Colbert for their insightful comments on the article

Common Variants within MECP2 Confer Risk of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a predominantly female autoimmune disease that affects multiple organ systems. Herein, we report on an X-chromosome gene association with SLE. Methyl-CpG-binding protein 2 (MECP2) is located on chromosome Xq28 and encodes for a protein that plays a critical role in epigenetic transcriptional regulation of methylation-sensitive genes. Utilizing a candidate gene association approach, we genotyped 21 SNPs within and around MECP2 in SLE patients and controls. We identify and replicate association between SLE and the genomic element containing MECP2 in two independent SLE cohorts from two ethnically divergent populations. These findings are potentially related to the overexpression of methylation-sensitive genes in SLE

A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)

Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767-777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells.Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells.In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines

Lupus risk variants in the PXK locus alter B-cell receptor internalization

Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus), rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR) in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb) common haplotype marking a single causal variant that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3' UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 × 10-10, OR 0.81 (0.75 - 0.86). Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 202 kb. Functionally, we found that PXK operates on the B-cell antigen receptor (BCR); we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity

Preferential binding to elk-1 by sle-associated il10 risk allele upregulates il10 expression

Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10−8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans

ABIN1 dysfunction as a genetic basis for lupus nephritis

The genetic factors underlying the pathogenesis of lupus nephritis associated with systemic lupus erythematosus are largely unknown, although animal studies indicate that nuclear factor (NF)-?B is involved. We reported previously that aknockin mouse expressinganin active form of ABIN1 (ABIN1[D485N]) develops lupus-like autoimmune disease and demonstrates enhanced activation of NF-?B and mitogen-activated protein kinases in immune cells after toll-like receptor stimulation. In the current study, we show that ABIN1[D485N] mice develop progressive GN similar to class III and IV lupus nephritis in humans. To investigate the clinical relevance of ABIN1 dysfunction, we genotyped five single-nucleotide polymorphisms in the gene encoding ABIN1, TNIP1, in samples from European-American, African American, Asian, Gullah, and Hispanic participants in the Large Lupus Association Study 2. Comparing cases of systemic lupus erythematosus with nephritis and cases ofsystemic lupus erythematosus without nephritis revealed strong associations with lupus nephritis at rs7708392 in European Americans and rs4958881 in African Americans. Comparing cases of systemic lupus erythematosus with nephritis and healthy controls revealed a stronger association at rs7708392 in European Americans but not at rs4958881 in African Americans. Our data suggest that variants in the TNIP1 gene are associated with the risk for lupus nephritis and could be mechanistically involved in disease development via aberrant regulation of NF-?B and mitogen-activated protein kinase activity. Copyright © 2013 by the American Society of Nephrology

Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10-8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10-7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ~146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10-7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10-4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE