T cell expressions of aberrant gene signatures and Co-inhibitory receptors (Co-IRs) as predictors of renal damage and lupus disease activity.

BACKGROUND: Systemic lupus erythematosus (SLE) is distinguished by an extensive range of clinical heterogeneity with unpredictable disease flares and organ damage. This research investigates the potential of aberrant signatures on T cell genes, soluble Co-IRs/ligands, and Co-IRs expression on T cells as biomarkers for lupus disease parameters. METHODS: Comparative transcriptome profiling analysis of non-renal and end-stage renal disease (ESRD) phenotypes of SLE was performed using CD4 + and CD8 + cDNA microarrays of sorted T cells. Comparing the expression of Co-IRs on T cells and serum soluble mediators among healthy and SLE phenotypes. RESULTS: SLE patients with ESRD were downregulated CD38, PLEK, interferon-γ, CX3CR1, FGFBP2, and SLCO4C1 transcripts on CD4 + and CD8 + T cells simultaneously and NKG7, FCRL6, GZMB/H, FcγRIII, ITGAM, Fas ligand, TBX21, LYN, granulysin, CCL4L1, CMKLR1, HLA-DRß, KIR2DL3, and KLRD1 in CD8 T cells. Pathway enrichment and PPI network analyses revealed that the overwhelming majority of Differentially Expressed Genes (DEGs) have been affiliated with novel cytotoxic, antigen presentation, and chemokine-cell migration signature pathways. CD8 + GZMK + T cells that are varied in nature, including CD161 + Mucosal-associated invariant T (MAIT) cells and CD161- aged-associated T (Taa) cells and CD161-GZMK + GZMB + T cells might account for a higher level of GZMK in CD8 + T cells associated with ESRD. SLE patients have higher TIGIT + , PD1 + , and lower CD127 + cell percentages on CD4 + T cells, higher TIM3 + , TIGIT + , HLA-DR + cell frequency, and lower MFI expression of CD127, CD160 in CD8 T cells. Co-IRs expression in T cells was correlated with soluble PD-1, PDL-2, and TIM3 levels, as well as SLE disease activity, clinical phenotypes, and immune-therapy responses. CONCLUSION: The signature of dysfunctional pathways defines a distinct immunity pattern in LN ESRD patients. Expression levels of Co-IRs in peripheral blood T cells and serum levels of soluble PD1/PDL-2/TIM3 can serve as biomarkers for evaluating clinical parameters and therapeutic responses.

Comprehensive Co-Inhibitory Receptor (Co-IR) Expression on T Cells and Soluble Proteins in Rheumatoid Arthritis.

Co-inhibitory receptors (Co-IRs) are essential in controlling the progression of immunopathology in rheumatoid arthritis (RA) by limiting T cell activation. The objective of this investigation was to determine the phenotypic expression of Co-IR T cells and to assess the levels of serum soluble PD-1, PDL-2, and TIM3 in Taiwanese RA patients. METHODS: Co-IRs T cells were immunophenotyped employing multicolor flow cytometry, and ELISA was utilized for measuring soluble PD-1, PDL-2, and TIM3. Correlations have been detected across the percentage of T cells expressing Co-IRs (MFI) and different indicators in the blood, including ESR, high-sensitivity CRP (hsCRP), 28 joint disease activity scores (DAS28), and soluble PD-1/PDL-2/TIM3. RESULTS: In RA patients, we recognized elevated levels of PD-1 (CD279), CTLA-4, and TIGIT in CD4+ T cells; TIGIT, HLA-DR, TIM3, and LAG3 in CD8+ T cells; and CD8+CD279+TIM3+, CD8+HLA-DR+CD38+ T cells. The following tests were revealed to be correlated with hsCRP: CD4/CD279 MFI, CD4/CD279%, CD4/TIM3%, CD8/TIM3%, CD8/TIM3 MFI, CD8/LAG3%, and CD8+HLA-DR+CD38+%. CD8/LAG3 and CD8/TIM3 MFIs are linked to ESR. DAS28-ESR and DAS28-CRP exhibited relationships with CD4/CD127 MFI, CD8/CD279%, and CD8/CD127 MFI, respectively. CD4+CD279+TIM3+% was correlated with DAS28-ESR (p = 0.0084, N = 46), DAS28-CRP (p = 0.007, N = 47), and hsCRP (p = 0.002, N = 56), respectively. In the serum of patients with RA, levels of soluble PD-1, PDL-2, and Tim3 were extremely elevated. CD4+ TIM3+% (p = 0.0089, N = 46) and CD8+ TIM3+% (p = 0.0305, N = 46) were correlated with sTIM3 levels; sPD1 levels were correlated with CD4+CD279+% (p < 0.0001, N = 31) and CD3+CD279+% (p = 0.0084, N = 30). CONCLUSIONS: Co-IR expressions on CD4+ and CD8+ T cells, as well as soluble PD-1, PDL-2, and TIM3 levels, could function as indicators of disease activity and potentially play crucial roles in the pathogenesis of RA.

Functional MICA Variants Are Differentially Associated with Immune-Mediated Inflammatory Diseases.

As the principal ligand for NKG2D, MICA elicits the recruitment of subsets of T cells and NK cells in innate immunity. MICA gene variants greatly impact the functionality and expression of MICA in humans. The current study evaluated whether MICA polymorphisms distinctively influence the pathogenesis of psoriasis (PSO), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) in Taiwanese subjects. The distributions of MICA alleles and levels of serum soluble NKG2D were compared between healthy controls and patients with PSO, RA, and SLE, respectively. The binding capacities and cell surface densities of MICA alleles were assessed by utilizing stable cell lines expressing four prominent Taiwanese MICA alleles. Our data revealed that MICA*010 was significantly associated with risks for PSO and RA (PFDR = 1.93 × 10-15 and 0.00112, respectively), while MICA*045 was significantly associated with predisposition to SLE (PFDR = 0.0002). On the other hand, MICA*002 was associated with protection against RA development (PFDR = 4.16 × 10-6), while MICA*009 was associated with a low risk for PSO (PFDR = 0.0058). MICA*002 exhibited the highest binding affinity for NKG2D compared to the other MICA alleles. Serum concentrations of soluble MICA were significantly elevated in SLE patients compared to healthy controls (p = 0.01). The lack of cell surface expression of the MICA*010 was caused by its entrapment in the endoplasmic reticulum. As a prevalent risk factor for PSO and RA, MICA*010 is deficient in cell surface expression and is unable to interact with NKG2D. Our study suggests that MICA alleles distinctively contribute to the pathogenesis of PSO, RA, and SLE in Taiwanese people.

Long-Term Survival of Patients with Connective Tissue Disease-Associated Pulmonary Arterial Hypertension: A Single-Center Cohort.

Pulmonary arterial hypertension (PAH) is a rare but severe complication of connective tissue disease (CTD). CTD-associated PAH (CTD-PAH) is the most common subgroup of PAH in East Asia. We prospectively collected 41 patients with CTD-PAH and followed them for a mean period of 43 ± 36 months. The long-term survival rates of the CTD-PAH patients at 1, 2, 3 and 5 years were 90%, 80%, 77%, and 60%, respectively. The non-survivors had more dilated main pulmonary arteries, higher pulmonary artery pressure and pulmonary vascular resistance (PVR). PAH-specific therapy resulted in improvements in functional class, 6-minute walk distance, serum uric acid, right ventricular function and PVR. Increased C-reactive protein during follow-up, indicating inflammatory processes, was also crucial for the management of CTD-PAH. Therefore targeting both PAH and inflammation is important in this specific subgroup of PAH. The results of this study may help develop treatment strategies for CTD-PAH patients.

Functional ERAP1 Variants Distinctively Associate with Ankylosing Spondylitis Susceptibility under the Influence of HLA-B27 in Taiwanese.

Epistasis of ERAP1 single nucleotide variations (SNVs) and HLA-B27 has been linked to ankylosing spondylitis susceptibility (AS). The current study examined how prevalent ERAP1 allelic variants (SNV haplotypes) in Taiwan affect ERAP1 functions and AS susceptibility in the presence or absence of HLA-B27. Sanger sequencing was used to discover all ERAP1 coding SNVs and common allelic variants in Taiwanese full-length cDNAs from 45 human patients. For the genetic association investigation, TaqMan genotyping assays were utilized to establish the genotypes of ERAP1 SNVs in 863 AS patients and 1438 healthy controls. Ex vivo biological analysis of peripheral blood mononuclear cells from homozygous donors of two common-risk ERAP1 allelic variants was performed. Two common-risk ERAP1 allelic variants were also cloned and functionally studied. In Taiwanese, eleven frequent ERAP1 SNVs and six major ERAP1 allelic variants were discovered. We discovered that in Taiwanese, the most prevalent ERAP1-001 variant with 56E, 127R, 276I, 349M, 528K, 575D, 725R, and 730Q interacting with HLA-B27 significantly contributed to the development of AS. In HLA-B27 negative group, however, the second most prevalent ERAP1-002 variant with 56E, 127P, 276M, 349M, 528R, 575D, 725R, and 730E was substantially related with an increased risk of AS. Ex vivo and in vitro research demonstrated that ERAP1 allelic variants have a significant impact on ERAP1 functions, suggesting that ERAP1 plays a role in the development of AS. In an HLA-B27-dependent manner, common ERAP1 allelic variants are related with AS susceptibility.

Genetic effects of B3GNT2 on ankylosing spondylitis susceptibility and clinical manifestations in Taiwanese.

BACKGROUND/PURPOSE: The intergenic SNP rs10865331 at 2p15 was identified as a major risk factor for ankylosing spondylitis (AS) susceptibility in genome-wide association studies (GWAS). B3GNT2 gene regulates polylactosamine synthesis is potentially functionally relevant to AS disease development. We investigated whether SNP rs10865331 and two B3GNT2 SNPs (rs11900673 and rs1136151) are associated with AS susceptibility and disease severity in Taiwanese. METHODS: Distributions of genotypes, alleles, and haplotypes of three SNPs were compared between 1,472 AS patients and 2,117 healthy blood donors and among AS patients stratified by clinical characteristics. RESULTS: The intergenic SNP rs10865331 was significantly associated with AS (PFDR = 1.02E-05) in Taiwanese. In AS patients stratified by positivity of HLA-B27 and syndesmophyte formation, all three B3GNT2 locus SNPs (rs11900673, rs1136151, and rs10865331) were significantly associated with syndesmophyte formation among HLA-B27 positive AS patients. Haplotype analyses revealed that the "CTA" (rs11900673C/rs1136151T/rs10865331A) haplotype was significantly associated with AS susceptibility (Padj = 0.0177) and syndesmophyte formation (Padj = 0.016) in HLA-B27 positive patients. In contrast, "TCG" (rs11900673T/rs1136151C/rs10865331G) haplotype showed protection against AS development (Padj = 0.0005 for HLA-B27 positive and Padj = 0.004 for HLA-B27 negative, respectively) and syndesmophyte formation (Padj = 0.0017) in HLA-B27 positive patients. Furthermore, B3GNT2 mRNA expressions were negatively associated with erythrocyte sedimentation rate (ESR, P = 0.0103), C-reactive protein (CRP, P = 0.0353), Bath ankylosing spondylitis functional index (BASFI, P = 0.0171), and syndesmophyte formation (P = 0.0148). CONCLUSION: Our data suggest that B3GNT2 gene may contribute to AS development and affect AS severity by interacting with HLA-B27 in Taiwanese.

MICA*019 Allele and Soluble MICA as Biomarkers for Ankylosing Spondylitis in Taiwanese.

MICA (major histocompatibility complex class I chain-related gene A) interacts with NKG2D on immune cells to regulate host immune responses. We aimed to determine whether MICA alleles are associated with AS susceptibility in Taiwanese. MICA alleles were determined through haplotype analyses of major MICA coding SNP (cSNP) data from 895 AS patients and 896 normal healthy controls in Taiwan. The distributions of MICA alleles were compared between AS patients and normal healthy controls and among AS patients, stratified by clinical characteristics. ELISA was used to determine soluble MICA (sMICA) levels in serum of AS patients and healthy controls. Stable cell lines expressing four major MICA alleles (MICA*002, MICA*008, MICA*010 and MICA*019) in Taiwanese were used for biological analyses. We found that MICA*019 is the only major MICA allele significantly associated with AS susceptibility (PFDR = 2.25 × 10-115; OR, 14.90; 95% CI, 11.83-18.77) in Taiwanese. In addition, the MICA*019 allele is associated with syndesmophyte formation (PFDR = 0.0017; OR, 1.69; 95% CI, 1.29-2.22) and HLA-B27 positivity (PFDR = 1.45 × 10-33; OR, 28.79; 95% CI, 16.83-49.26) in AS patients. Serum sMICA levels were significantly increased in AS patients as compared to healthy controls. Additionally, MICA*019 homozygous subjects produced the highest levels of sMICA, compared to donors with other genotypes. Furthermore, in vitro experiments revealed that cells expressing MICA*019 produced the highest level of sMICA, as compared to other major MICA alleles. In summary, the MICA*019 allele, producing the highest levels of sMICA, is a significant risk factor for AS and syndesmophyte formation in Taiwanese. Our data indicate that a high level of sMICA is a biomarker for AS.

Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and joint destruction. Previous studies have shown that natural killer (NK) cells may play an important role in the pathogenesis of RA. Interleukin (IL)-15, a pro-inflammatory cytokine which induces proliferation and differentiation of NK cells, is overexpressed in RA. In this present study, we examine various NKRs and adhesion molecule expression on NK cells from RA patients and their response to IL-15 stimulation. We also sought to study cytokine-induced memory-like (CIML) NK cells in RA patients. We established that 1. RA patients had higher NK cell percentages in peripheral blood and their serum IL-15 levels were higher compared to healthy volunteers; 2. NK cells from RA patients showed lower NKp46 expression and an impaired CD69 response to IL-15; 3. NK cells from RA patients showed higher CD158b and CD158e expression but lower CD62L expression; 4. exogenous IL-15 up-regulated CD69, CD158b, CD158e but down-regulated NKp46 and CD62L expression in RA; 5. As to CIML NK cells, restimulation - induced NK cytotoxicity and IFN-γ production was impaired in RA patients, 6. Reduced NKp46, perforin, and granzyme B expression on NK cells was found in RA patients with bone deformity and erosion, 7. RA disease activity (DAS28) showed inverse correlation with the percentages of CD56+CD3- NK cells, and NKp46 and perforin expression on NK cells, respectively. Taken together, our study demonstrated differential expression of various NK receptors in RA patients. NKp46, CD158e, and perforin expression on NK cells may serve as markers of RA severity.

Signaling Pathways of Type I and Type III Interferons and Targeted Therapies in Systemic Lupus Erythematosus.

Type I and type III interferons (IFNs) share several properties in common, including the induction of signaling pathways, the activation of gene transcripts, and immune responses, against viral infection. Recent advances in the understanding of the molecular basis of innate and adaptive immunity have led to the re-examination of the role of these IFNs in autoimmune diseases. To date, a variety of IFN-regulated genes, termed IFN signature genes, have been identified. The expressions of these genes significantly increase in systemic lupus erythematosus (SLE), highlighting the role of type I and type III IFNs in the pathogenesis of SLE. In this review, we first discussed the signaling pathways and the immunoregulatory roles of type I and type III IFNs. Next, we discussed the roles of these IFNs in the pathogenesis of autoimmune diseases, including SLE. In SLE, IFN-stimulated genes induced by IFN signaling contribute to a positive feedback loop of autoimmunity, resulting in perpetual autoimmune inflammation. Based on this, we discussed the use of several specific IFN blocking strategies using anti-IFN-α antibodies, anti-IFN-α receptor antibodies, and IFN-α-kinoid or downstream small molecules, which intervene in Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways, in clinical trials for SLE patients. Hopefully, the development of novel regimens targeting IFN signaling pathways will shed light on promising future therapeutic applications for SLE patients.

Features of trunk muscle weakness in patients with ankylosing spondylitis: A cross-sectional study.

BACKGROUND: Ankylosing spondylitis (AS) is an inflammatory autoimmune disorder that manifested with sacroiliitis at its early stage and developed extensive inflammation with syndesmophytes of the lumbar, thoracic and cervical spines at its later stage. In the present study, we characterized the trunk isometric strength in patients with AS with different disease severity, defined by the radiological images. METHODS: In a cross-sectional study conducted in a university-affiliated hospital, thirty-eight male AS patients (23 in the early AS group whose radiological findings showed no syndesmophyte, Modified Stoke Ankylosing Spinal Score (m-SASSS <3); and 15 in the syndesmophyte group, m-SASSS ≥24), and 22 healthy controls were recruited. All subjects received assessments of maximum isometric strength of trunk flexor and extensor muscles at a variety of trunk postures measured by an isokinetic device. RESULTS: Under all examined trunk postures, the syndesmophyte AS patient group had the lowest isometric trunk muscle strength among the three groups. The flexion/extension ratio, defined by the ratio between isometric trunk flexor and extensor strengths, was highest among the three groups. CONCLUSIONS: Trunk muscle strength significantly decreases in patients with syndesmophyte AS. The decrease of trunk muscle is inhomogeneous, which is more profound in extensor than in flexor muscles.

Cytotoxic Function and Cytokine Production of Natural Killer Cells and Natural Killer T-Like Cells in Systemic Lupus Erythematosis Regulation with Interleukin-15.

Natural killer cells and NKT-like cells are the first line immune defense against tumor and virus infection. Deficient NK and NKT-like cell effector function may contribute to increased susceptibility to infection in SLE patients. We sought to examine the perforin and granzyme B expression, interferon-gamma (IFN-γ), and tumor-necrosis factor-alpha (TNF-α) production and CD107a degranulation of NK and NKT-like cells from SLE patients and their regulation by IL-15. We established that (1) perforin expression on SLE NK cells was decreased but unrelated to disease activity; (2) the MFI of granzyme B was increased in NK cells from SLE patients with active disease, associated with increased percentages of granzyme B+ CD56bright NK cells; (3) NK cells from active SLE patients, both CD56dim and CD56bright NK subsets, produced higher IFN-γ compared to controls; (4) CD56dim, but not CD56bright NK cells from active SLE patients, produced lower TNF-α, compared to inactive SLE patients and controls; (5) CD107a degranulation of SLE NK cells was comparable to controls; (6) IL-15 enhanced perforin/granzyme B expression, IFN-γ/TNF-α production, and CD107a degranulation of NK cells from SLE patients; and (7) similar observations were found for CD56+CD3+ NKT-like cells. Taken together, we demonstrated the differential expression of the heightened granzyme B and decreased TNF-α in NK and NKT-like cells in SLE patients. Higher granzyme B expression of NK and NKT-like cells in active SLE patients, further enhanced by circulating IL-15, may contribute to the maintenance of inflammation in SLE.

Association of Genetic Variants of RANK, RANKL, and OPG with Ankylosing Spondylitis Clinical Features in Taiwanese.

Ankylosing spondylitis (AS) is a chronic inflammatory disease that leads to spinal ankylosis. The receptor activator of the nuclear factor-kappa (RANK), RANK ligand, and osteoprotegerin (OPG) (RANK/RANKL/OPG) pathway plays critical roles in bone metabolism and the immune system. The current study was aimed at investigating whether six single-nucleotide polymorphisms (SNPs) within the RANK, RANKL, and OPG genes essential for bone homeostasis are associated with AS. Genotype distributions, allele and haplotype frequencies, were compared between 1120 AS patients and 1435 healthy controls and among AS patients with stratification by syndesmophyte formation, onset age, and HLA-B27 positivity. We found that RANKL SNPs were associated with AS syndesmophyte formation. Notably, the RANKL SNP haplotype rs7984870C/rs9533155G/rs9525641C was negatively associated with AS susceptibility and appeared to protect against syndesmophyte formation in AS. Functionally, RANKL promoter SNPs (rs9525641 C/T and rs9533155 G/C) affected DNA-protein complex formation and promoter activity in promoter reporter analyses. The OPG SNP haplotype rs2073618G/rs3102735T was significantly associated with HLA-B27 negativity in AS patients. Furthermore, AS patients with syndesmophyte formation had significantly lower levels of soluble RANKL levels than those without syndesmophyte formation. Our data suggested a role for RANKL in AS susceptibility and severity.

Interferon-λ3/4 genetic variants and interferon-λ3 serum levels are biomarkers of lupus nephritis and disease activity in Taiwanese.

BACKGROUND: Type III interferons (IFNs) or IFN-λs are the newly discovered cytokines that primarily target the cells of epithelial and myeloid lineages, which are major components of kidneys. The current study aimed to investigate whether IFN-λs are involved in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis. METHODS: TaqMan allele discrimination assays were used to determine IFNL3/4 SNP genotypes of 1620 healthy controls and 1013 SLE patients (two independent cohorts consisting of 831 and 182 subjects, respectively) from Taiwan. The distributions of IFNL3/4 SNP genotypes and allele frequencies were compared between SLE patients and healthy controls and among SLE patients stratified by clinical phenotypes. ELISA was used to determine the serum IFN-λ3 concentrations of SLE patients. RESULTS: All major IFN3/4 SNP alleles were significantly associated with the risk for lupus nephritis (rs8099917T, PFDR = 0.0021, OR 1.75, 95% CI 1.24-2.47; rs12979860C, PFDR = 0.0034, OR 1.65, 95% CI 1.18-2.30; rs4803217C, PFDR = 0.0021, OR 1.76, 95% CI 1.25-2.48; and ss469415590TT, PFDR = 0.0021, OR 1.73, 95% CI 1.23-2.42) among SLE patients. Similarly, the major IFNL3/4 SNP haplotype rs8099917T-ss469415590TT-rs12979860C-rs4803217C (or T-TT-C-C) was a significant risk factor for lupus nephritis (P = 0.0015, OR 1.68, 95% CI 1.22-2.32). Additionally, all minor IFN3/4 SNP alleles were significantly associated with SLE susceptibility in nephritis-negative SLE patients as compared to normal healthy controls (rs8099917G, PFDR = 0.00177, OR 1.68, 95% CI 1.24-2.28; rs12979860T, PFDR = 0.00299, OR 1.58, 95% CI 1.18-2.32; rs4803217A, PFDR = 0.00176, OR 1.65, 95% CI 1.22-2.23; and ss469415590ΔG, PFDR = 0.00176, OR 1.70, 95% CI 1.26-2.29). Furthermore, the elevated serum levels of IFN-λ3 were significantly correlated with the complement depression and the high SLE disease activities in SLE patients. CONCLUSIONS: IFN-λ3/4 genetic variants play a unique role in the development of lupus nephritis and SLE.

Role of Interleukin- (IL-) 17 in the Pathogenesis and Targeted Therapies in Spondyloarthropathies.

Spondyloarthropathy (SpA) is a unique type of joint inflammation characterized by coexisting erosive bone damage and pathological new bone formation. Previous genetic association studies have demonstrated that several cytokine pathways play a critical role in the pathogenesis of ankylosing spondylitis (AS), psoriatic arthritis (PsA), and other types of SpA. In addition to several well-known proinflammatory cytokines, recent studies suggest that IL-17 plays a pivotal role in the pathogenesis of SpA. Further evidence from human and animal studies have defined that IL-17 and IL-17-producing cells contribute to tissue inflammation, autoimmunity, and host defense, leading to the following pathologic events associated with SpA. Recently, several clinical trials targeting IL-17 pathways demonstrated the positive response of IL-17 blockade in treating AS, indicating a great potential of IL-17-targeting therapy in SpA. In this review article, we have discussed the contributing role of IL-17 and different IL-17-producing cells in the pathogenesis of SpA and provided an outline of therapeutic application of the IL-17 blockade in the treatment of SpA. Other targeted cytokines associated with IL-17 axis in SpA will also be included.

Activating and inhibitory receptors on natural killer cells in patients with systemic lupus erythematosis-regulation with interleukin-15.

Natural killer (NK) cells may play an important role in the pathogenesis of SLE. Interleukin(IL)-15, an NK-enhancing cytokine, is over-expressed in SLE patients. In the present study, we examined the effect of IL-15 on NK cytotoxicity of SLE patients, and the expression of various activating and inhibitory NK receptors on NK cells from SLE patients in relation to disease activity. We also sought to determine how IL-15 would affect the NK receptor expression on NK cells from SLE patients. PBMCs were collected from 88 SLE patients with inactive disease activity (SLEDAI score<6) and active disease activity (SLEDAI score≥6), 26 age-matched healthy adults were used as controls. PBMC were incubated in the presence or absence of IL-15 (10ng/ml) for eighteen hours. CD3-CD56+ lymphoctes were gated using flow cytometry and further divided into CD56dim and CD56bright subsets according to the MFI of CD56. We observed that 1. Serum IL-15 was elevated in SLE patients, and higher in active disease than in inactive disease; 2. NK cytotoxicity of SLE patients was deficient compared to controls and showed an impaired response to IL-15 compared to controls; 3.CD69, CD94, NKG2A, NKp30, and CD158b on NK cells from SLE patients were higher than controls, and could be further enhanced by IL-15; 4. NKp46 expression from SLE patients was higher than controls, but down-regulated by IL-15; 5.Deficient NKG2D and NKAT-2 expression were found on NK cells from SLE patients, which were enhanced by IL-15; 6. A unique NKp46- subset and CD158b+ subsets were observed in NK cells from SLE patients but not controls. 7. Unlike controls, CD158k on NK cells from SLE patients failed to respond to IL-15. Taken together, we demonstrated the aberrant NCR and iNKR expression on NK cells and their distinct response to IL-15 in SLE patients. As IL-15 predominantly aggravates the aberrant NKR expression found in SLE, IL-15 antagonist may have therapeutic benefits in SLE patients.

Human Leukocyte Antigen C*12:02:02 and Killer Immunoglobulin-Like Receptor 2DL5 are Distinctly Associated with Ankylosing Spondylitis in the Taiwanese.

Human leukocyte antigen (HLA) class I ligands and Killer immunoglobulin-like receptors (KIRs) regulate the cytolytic activity of natural killer (NK) cells and certain T cells. We examined their genetic predisposition to disease susceptibility and clinical phenotypes in Taiwanese ankylosing spondylitis (AS) patients. KIR genotyping and Human Leucocyte Antigen C (HLA-C) sequencing were performed in 653 Taiwanese AS patients and 952 healthy controls. KIR genotype distributions and HLA-C allele frequencies were compared in patients and controls and among patients with and without HLA-B27 positivity, early age onset and spinal syndesmophytes. HLA-C alleles were functionally characterized using 3D structural modelling with peptide simulation. This study discovered that the HLA-C*12:02:02 allele (43.42% vs. 3.31%; p < 0.00001 odds ratio (OR), 16.88; 95% confidence intervals (CI): 11.27-25.28) confers a strong risk for Taiwanese AS development. The 3D modelling results identified four unique amino acid polymorphisms, Ala73, Trp156, Arg219 and Met304, that may affect the function of the HLA-C*12:02:02 allele. KIR2DL5 (p = 0.0047; pFDR = 0.0423) and the KIR Bx haplotype (p = 0.0000275) were protective against Taiwanese AS, while KIR 2DS4/1D (22 base pair truncated deletion; p = 0.0044; pFDR = 0.1998) appeared to be a risk factor for it. KIR2DL5 combined with the HLA-C1/C2 heterozygous genotype showed a protective effect (AS 5.97% vs. normal 11.66%; p = 0.002; pFDR = 0.0127, OR, 0.48 95% CI: 0.33-0.70); in contrast, KIR 2DS4/1D combined with the HLA-C1C1 homozygous genotype (AS 45.33% vs. normal 35.92%; p = 0.002; pFDR = 0.0127, OR, 1.48 95% CI: 1.21-1.81) represented a risk factor for AS development. Our data suggested that interactions between KIRs and their cognate HLA-C ligands may contribute to the pathogenesis of AS.

B cell epitope of human cytomegalovirus phosphoprotein 65 (HCMV pp65) induced anti-dsDNA antibody in BALB/c mice.

BACKGROUND: HCMV phosphoprotein 65 (HCMVpp65) is a putative immunogen that acts as an accelerator, inducing autoantibody and exacerbating autoimmune response in susceptible animals. The immunity to pp65336-439 instigates autoimmunity, suggesting that pp65336-439 contains crucial B cell epitope(s) for the development of nephritis. This study narrowed down the target epitope to pp65422-439 for immunization of BALB/c mice and mapping of B cell epitope. METHODS: The target epitope pp65422-439 reactivity and B cell epitope mapping was examined in serum from pp65422-439-immunized mice and patients with systemic lupus erythematosus (SLE). Kidney tissue from immunized mice was examined for signs of immune complex nephritis. RESULTS: Anti-pp65422-439 antibody in serum either from patients with SLE or from pp65422-439-immunized mice exhibited cross-reactivity to several nuclear components such as double-stranded DNA (dsDNA). Moreover, the pp65422-439-immunized mice developed initial signs of glomerulonephritis such as deposition of immunoglobulin G/M (IgG/IgM) and third complement component (C3). With B cell epitope mapping by pp65422-439-derived decapeptides, one dominant epitope, pp65428-437, was identified in serum from pp65422-439-immunized mice and patients with SLE with anti-pp65422-439 antibody. Epitope spreading from pp65428-437 to pp65430-439 was found in pp65422-439-immunized mice in which we generated monoclonal antibodies to pp65425-434 and pp65430-439. However, dsDNA positive reactivity was exclusively observed in Crithidia luciliae stains with pp65430-439-reactive monoclonal antibody. Additionally, we observed the amelioration of autoimmunity following the elevation of IgM targeting pp65428-437. CONCLUSIONS: Our data suggest that pp65428-437 may be an autoimmune or lupus-prone B cell epitope and may catalyze further epitope spreading for inducing autoantibodies in lupus-susceptible individuals.

The role of endothelial microparticles in autoimmune disease patients with Raynaud's phenomenon.

BACKGROUND AND AIM: Raynaud's phenomenon (RP) is a microvascular disorder characterized by episodic peripheral vasospasm and ischemia and is commonly found in patients with autoimmune diseases (AID). The vasomotor homoeostasis and endothelial cells damage are involved in RP. Endothelial microparticles (EMPs) may act as a biomarker for endothelial damage. The aim of this study is to investigate the correlation between the levels of microparticles (MPs) and microvasculopathy in AID with RP. METHODS: Thirty-seven patients with AID and RP (RP group) and 27 patients with AID but without RP (non-RP group) were enrolled. The microvasculopathy score of RP was graded by nailfold capillary microscopy. The plasma levels of MPs were measured by flow cytometry utilizing specific labels for endothelial MPs (CD105 and CD144) and annexin V staining for phosphatidylserine bearing-MPs (annexin V+MPs). RESULTS: The levels of circulating EMPs (CD105+ p = 0.005, CD144+ p = 0.004), and the annexin V+ MPs (p < 0.001) were significantly elevated in the RP group compared with the non-RP group. Moreover, the high microvasculopathy scores were closely related with annexinV+ MPs levels in the RP group (p = 0.041). CONCLUSIONS: Levels of circulating EMPs and annexin V+ MPs are elevated in AID patients with RP indicate the endothelial damage and endothelial dysfunctions. In addition, levels of annexin V+ MPs can predict the severity of microvasculopathy in AID with RP.

Effect of Interleukin-15 on CD11b, CD54, and CD62L Expression on Natural Killer Cell and Natural Killer T-Like Cells in Systemic Lupus Erythematosus.

Adhesion molecules may play an important role in systemic lupus erythematosus (SLE) pathogenesis. We investigated the effect of interleukin- (IL-) 15 on CD11b, CD54, and CD62L expression on natural killer (NK) cells, T cells, and CD56+CD3+ NKT-like cells from SLE subjects and healthy controls. SLE patients had decreased circulating NK cells and NKT-like cells compared to controls. NK cells from SLE patients showed higher CD11b and CD62L expression compared to controls. IL-15 enhanced CD11b and CD54 but downregulated CD62L expression on NK cells from SLE patients. Similar observations were found for T cells and NKT-like cells. NK cells from SLE patients expressed higher CD56 than controls; both could be further enhanced by IL-15. IL-15 also enhanced CD56 expression of NKT-like cells from SLE patients. A greater degree of IL-15 induced downregulation of CD62L on NKT-like cells noted in SLE patients compared to controls. The percentage of CD11b expressing NK cells and the % inhibition of CD62L expression on NKT-like cells by IL-15 correlated with serum anti-dsDNA levels in SLE patients, respectively. Taken together, we demonstrated the dysfunctional NK and NKT-like cells in SLE patients with regard to CD11b and CD62L expression and their response to IL-15.

Effects of Complement C4 Gene Copy Number Variations, Size Dichotomy, and C4A Deficiency on Genetic Risk and Clinical Presentation of Systemic Lupus Erythematosus in East Asian Populations.

OBJECTIVE: Human complement C4 is complex, with multiple layers of diversity. The aims of this study were to elucidate the copy number variations (CNVs) of C4A and C4B in relation to disease risk in systemic lupus erythematosus (SLE), and to compare the basis of race-specific C4A deficiency between East Asians and individuals of European descent. METHODS: The East Asian study population included 999 SLE patients and 1,347 healthy subjects. Variations in gene copy numbers (GCNs) of total C4, C4A, and C4B, as well as C4-Long and C4-Short genes, were determined and validated using independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein occurring concurrently with C4A deficiency. RESULTS: In East Asians, high GCNs of total C4 and C4A were strongly protective against SLE, whereas low and medium GCNs of total C4 and C4A, and the absence of C4-Short genes, were risk factors for SLE. Homozygous C4A deficiency was infrequent in East Asian subjects, but had an odds ratio (OR) of 12.4 (P = 0.0015) for SLE disease susceptibility. Low serum complement levels were strongly associated with low GCNs of total C4 (OR 3.19, P = 7.3 × 10(-7) ) and C4B (OR 2.53, P = 2.5 × 10(-5) ). Patients with low serum complement levels had high frequencies of anti-double-stranded DNA antibodies (OR 4.96, P = 9.7 × 10(-17) ), hemolytic anemia (OR 3.89, P = 3.6 × 10(-10) ), and renal disease (OR 2.18, P = 8.5 × 10(-6) ). The monomodular-Short haplotype found to be prevalent in European Americans with C4A deficiency, which was in linkage disequilibrium with HLA-DRB1*0301, was scarce in East Asians. Instead, most East Asian subjects with C4A deficiency were found to have a recombinant haplotype with bimodular C4-Long and C4-Short genes, encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA sequencing revealed an E920K polymorphism in C4B96. CONCLUSION: C4 CNVs and deficiency of C4A both play an important role in the risk and manifestations of SLE in East Asian and European populations.