Genotype-guided new approach for dose optimisation of hydroxychloroquine administration in Chinese patients with SLE.

OBJECTIVES: The study aims to investigate the impact of gene polymorphisms on blood hydroxychloroquine (HCQ) concentrations in patients with SLE and provide guidelines for individualised care. METHODS: 489 Chinese patients with SLE taking HCQ for more than 3 months were collected in this study. The blood HCQ, desethylhydroxychloroquine (DHCQ) and desethylchloroquine concentrations were measured. The optimal blood concentration of HCQ was determined by receiver operating characteristic curve analysis. Single nucleotide polymorphisms of metabolic enzymes involved in HCQ metabolism were genotyped and the associations with treatment effects were investigated. RESULTS: The cut-off value of HCQ was 559.67 ng/mL, with sensitivity and specificity values of 0.51 and 0.89, respectively. The TC and CC genotypes of CYP2C8 (rs7910936) were significantly related to the increase in blood HCQ concentrations, and the CYP2C8 (rs10882521) TT genotype was associated with lower blood HCQ concentrations. The DHCQ:HCQ ratio was highest in patients with the GG genotype of the CYP2D6*10 (rs1065852) polymorphism and lowest in those with the AA genotype. Patients with the CYP2C8 (rs7910936) CC genotype were more likely to achieve the optimal blood concentration (p=0.030) in HCQ 200 mg/day group and patients with the CYP2D6*10 (rs1065852) GG genotype were more likely to reach the optimal blood concentration (p=0.049) in 400 mg/day group. CONCLUSIONS: Our results suggest that the optimal blood concentration of HCQ measured approximately 12-18 hours after the last dosage may be between 500 and 600 ng/mL in Chinese patients with SLE. The observed variations in HCQ concentrations between individuals can potentially be attributed to genetic polymorphisms in CYP2D6*10 (rs1065852) and CYP2C8 (rs7910936 and rs10882521). Genotypical testing of patients and regular monitoring of blood levels are recommended for optimising HCQ dosage management in Chinese patients with SLE. TRIAL REGISTRATION NUMBER: ChiCTR2300070628.

Allogenic Umbilical Cord-Derived Mesenchymal Stromal Cells Sustain Long-Term Therapeutic Efficacy Compared With Low-Dose Interleukin-2 in Systemic Lupus Erythematosus.

OBJECTIVES: Mesenchymal stromal cells (MSCs) and low-dose interleukin-2 (IL-2) both have demonstrated efficacy in treating systemic lupus erythematosus (SLE). The aim of this study is to conduct a head-to-head comparison between the 2 treatments and provide insights for clinical applications. METHODS: Lupus-prone mice were treated with umbilical cord-derived MSCs (UC-MSCs), IL-2, or a combination of UC-MSCs and IL-2, respectively. The lupus-like symptoms, renal pathology, and T-cell response were assessed 1 or 4 weeks later. Modulation of IL-2 production by MSCs on immune cells was investigated by the coculture assay. Disease activity and serum IL-2 of SLE patients were determined before and after receiving UC-MSCs. RESULTS: Both UC-MSCs and IL-2 improved lupus symptoms in lupus-prone mice 1 week after treatment, while the effects of UC-MSCs lasted up to 4 weeks. Moreover, the UC-MSC-treated group showed better renal pathology improvement. Importantly, UC-MSCs combined with IL-2 did not provide better efficacy than UC-MSCs alone. Consistent with this, UC-MSCs alone and UC-MSCs + IL-2 resulted in similar levels of serum IL-2 and frequencies of Tregs. Neutralization of IL-2 partly reduced the promotion of Tregs by UC-MSCs, suggesting that IL-2 was involved in the upregulation of Tregs by UC-MSCs. Lastly, an increase in serum IL-2 positively correlated with the reduction of disease activity of SLE patients by UC-MSCs. CONCLUSION: Both the single injection of UC-MSCs and repeated IL-2 administration exerted comparable efficacy in alleviating SLE manifestations, but UC-MSCs provided sustained alleviation and showed better improvement in renal pathology.

Correlation of serum adenosine deaminase activity with disease activity in patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune disease primarily affecting the exocrine glands, which has a variety of clinical manifestations and unclear pathogenic mechanisms. Adenosine deaminase (ADA) is an enzyme involved in the breakdown of purines, and changes in its activity have been associated with a number of autoimmune diseases. This study aims to investigate the relationship between serum ADA activity and disease activity in patients with pSS. METHODS: In this study, 196 patients with pSS and 196 healthy controls were enrolled. Serum ADA activity and clinical laboratory parameters were collected and analyzed in both groups. Pearson correlation analysis was used to examine the correlation between ADA activity and clinical laboratory parameters, as well as the correlation between ADA activity and the disease activity score. RESULTS: Compared with healthy controls, the activity of ADA in the serum of pSS patients was significantly increased (P < 0.0001), and the ADA activity was significantly decreased after immunosuppressive treatment (P < 0.0001). Correlation analysis revealed that the activity of ADA was significantly positively correlated with erythrocyte sedimentation rate (ESR) (r = 0.3, P < 0.0001) and serum immunoglobulin G (IgG) levels (r = 0.5, P < 0.0001), and significantly negatively correlated with high-density lipoprotein (HDL) (r = -0.4, P < 0.0001). Furthermore, there was a significant positive correlation between ADA activity and the disease activity score as measured by the Sjögren's Syndrome Disease Activity Index (SSDAI) (r = 0.4, P < 0.0001). CONCLUSION: This study found that patients with pSS have higher activity of ADA in serum, which is associated with disease activity as measured by SSDAI. These results suggest that ADA activity may be a potential biomarker for evaluating disease activity and treatment efficacy in pSS patients. Additionally, ADA may be a potential target for the treatment of pSS patients.

Mesenchymal stem cells inhibit MRP-8/14 expression and neutrophil migration via TSG-6 in the treatment of lupus nephritis.

Abnormal infiltration and activation of neutrophils play a pathogenic role in the development of lupus nephritis (LN). Myeloid-related proteins (MRPs), MRP-8 and -14, also known as the damage-associated molecular patterns (DAMPs), are mainly secreted by activated neutrophils in systemic lupus erythematosus (SLE). Mesenchymal stem cells (MSCs) regulate a variety of immune cells to treat LN, but it is not clear whether MSCs can regulate neutrophils and the expression of MRP-8/14 in LN. Here, we demonstrated that neutrophil infiltration and MRP-8/14 expression were increased in the kidney of MRL/lpr mice and both decreased after MSCs transplantation. Further, the results showed that tumor necrosis factor- (TNF) stimulated gene-6 (TSG-6) in MSCs is necessary for MSCs to inhibit MRP-8/14 expression in neutrophils and neutrophil migration. In addition, small-molecule immunosuppressant had no significant effect on the expression of MRP-8/14 in neutrophils. Therefore, our results suggest that MSCs inhibited MRP-8/14 expression and neutrophil migration by secreting TSG-6 in the treatment of LN.

The Associations of rs1799724 and rs361525 With the Risk of Ankylosing Spondylitis Are Dependent on HLA-B27 Status in a Chinese Han Population.

Objectives: Human leucocyte antigen B27 (HLA-B27) is an important biomarker for ankylosing spondylitis (AS). However, delay in the diagnosis of AS is still common in clinical practice. Several single nucleotide polymorphisms (SNPs) in the coding gene of tumor necrosis factor alpha (TNFα) have been reported to be AS susceptibility loci. Our aim was to explore whether SNPs in TNFα could be used to improve the performance of HLA-B27 for predicting AS. Methods: Five SNPs (rs1799964, rs1800630, rs1799724, rs1800629, and rs361525) spanning TNFα were genotyped by qPCR-Invader assay in 93 AS patients and 107 healthy controls for association analysis and linkage disequilibrium (LD) analysis. Random forest algorithm was utilized to construct the predictive classifiers for AS. HLA-B was genotyped by PCR-sequence-based typing in a subset of the HLA-B27-positive subjects (38 AS patients and 5 healthy controls). Results: The T allele of rs1799724 was verified to significantly increase the risk of AS (OR = 4.583, p < 0.0001), while the A allele of rs361525 showed an association with the reduced AS risk (OR = 0.168, p = 0.009). In addition, the rs1799964T-rs1800630C-rs1799724T-rs1800629G-rs361525G haplotype was significantly associated with a higher risk of AS (p < 0.0001). The optimal set of variables for classifiers to predict AS only consisted of HLA-B27. Strong associations with HLA-B27 status were found in both rs1799724 (p < 0.0001) and rs361525 (p = 0.001), and all the analyzed HLA-B27-positive subjects carried HLA-B*27:04 or HLA-B*27:05. Conclusion: In the Chinese Han population, the minor allele T of rs1799724 could increase the risk of AS, while the minor allele A of rs361525 protects individuals from AS. However, the contributions of rs1799724 and rs361525 to AS risk were dependent on HLA-B27 status, suggesting the importance of taking the independence and specificity into consideration in AS susceptibility loci studies.

Increased serum adenosine deaminase activity in patients with adult-onset Still's disease.

BACKGROUND: Adult-onset Still's disease (AOSD) is a systemic inflammatory disease of unknown etiology, lacking specific diagnosis and disease activity evaluation indicators. This study will analyze the activity and clinical significance of Adenosine deaminase (ADA) in AOSD patients. METHODS: Totally 53 AOSD patients, 60 patients with other autoimmune diseases including systemic lupus erythematosus (SLE), sjogren syndrome (SS) and rheumatoid arthritis (RA), as well as 60 healthy subjects were included in this study. AOSD activity was determined by Pouchot score. We analyzed the correlation between ADA activity and clinical parameters. In addition, the correlation between ADA activity and disease activity score was also analyzed. RESULTS: This study showed that the activity of ADA in AOSD patients was significantly higher than that of healthy controls, SLE, SS and RA patient groups (p < 0.0001). The ADA activity of AOSD patients decreased significantly after systemic treatment (p < 0.0001). Correlation analysis showed that ADA activity was positively correlated with ALT(r = 0.54, p < 0.0001), AST (r = 0.82, p < 0.0001) and serum ferritin (r = 0.67, p < 0.001). ADA activity was negatively correlated with white blood cell (r = - 0.42, p = 0.002) and platelet counts (r = - 0.44, p = 0.001). We also found a significant positive correlation between the activity of ADA and Pouchot score in AOSD patients (r = 0.51, p = 0.001). Receiver operating characteristic (ROC) curve analysis showed that ADA activity had a sensitivity of 93.3%, and a specificity of 83% for the diagnosis of AOSD, with an area under the curve of 0.93. CONCLUSION: This study showed that serum ADA activity can be used as a potential biomarker for AOSD diagnosis and disease activity assessment.

Human SLE variant NCF1-R90H promotes kidney damage and murine lupus through enhanced Tfh2 responses induced by defective efferocytosis of macrophages.

OBJECTIVES: We previously identified a hypomorphic variant, p.Arg90His (p.R90H) of neutrophil cytosolic factor 1 (NCF1, a regulatory subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 complex), as an putative causal variant for systemic lupus erythematosus (SLE), and established a knock-in (KI) H90 variant in the C57BL/6 background to study how this variant promotes lupus development. METHODS: Wild type (WT) and KI littermates were assessed for immune profiles and lupus-like features. Disease activity and renal damage of patients with SLE were assessed by systemic lupus erythematosus disease activity index (SLEDAI) and renal items of systemic lupus international collaborating clinics (SLICC), respectively. RESULTS: Compared with WT littermates, 5-week-old homozygous KI mice had reduced oxidative burst, splenomegaly, elevated type I interferon (IFN-I) scores, increased ratios of splenic follicular T helper 2 (Tfh2) to either T follicular regulatory (Tfr) or Tfh1 cells, increased ANA+ follicular, germinal centre and plasma cells without spontaneous kidney disease up to 1 year of age. Pristane treatment exacerbated the immune dysregulation and induced IFN-I-dependent kidney disease in 36-week-old H90 KI female mice. Decreased efferocytosis of macrophages derived from KI mice and patients with homozygous H90 SLE promoted elevated ratios of Tfh2/Tfr and Tfh2/Tfh1 as well as dysregulated humoral responses due to reduced voltage-gated proton channel 1 (Hv1)-dependent acidification of phagosome pH to neutralise the decreased electrogenic effect of the H90 variant, resulting in impaired maturation and phagosome proteolysis, and increased autoantibody production and kidney damage in mice and patients with SLE of multiple ancestries. CONCLUSIONS: A lupus causal variant, NCF1-H90, reduces macrophage efferocytosis, enhances Tfh2 responses and promotes autoantibody production and kidney damage in both mice and patients with SLE.

Reduced Let-7f in Bone Marrow-Derived Mesenchymal Stem Cells Triggers Treg/Th17 Imbalance in Patients With Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) patients exist an imbalance between regulatory T (Treg) and T helper 17 cells (Th17), which might be contributed by defective immune regulation of bone marrow derived mesenchymal stem cells (BM-MSCs) from SLE patients. Our microRNA array analysis showed markedly down-regulated expression levels of microRNA let-7f in BM-MSCs from SLE patients compared to those from normal controls (NOR). To explore the role of let-7f in the disease pathogenesis, we showed that expression levels of let-7f in SLE BM-MSCs were negatively associated with SLE disease activity, and the predicted let-7 family targeted gene expression of interlukin-6 (IL-6) was significantly higher in BM-MSCs from SLE patients compared to normal controls (NOR). Transient transfection of BM-MSCs with let-7f mimics or inhibitors showed reduced levels of let-7f impaired the proliferation rate of BM-MSCs, BM-MSC-mediated downregulation of Th17 cells and upregulation of Treg cells, increased the apoptosis rate of BM-MSCs through targeting IL-6 and activating signal transducers and activators of transcription-3 (STAT3) pathway, but had no significant effect on the differentiation of Th1 and Th2. Our findings showed a key role of let-7f in the imbalance of Treg/Th17 mediated by SLE BM-MSCs, suggesting the potential of manipulating let-7f expression in BM-MSCs for treating SLE patients.

MicroRNA-663 induces immune dysregulation by inhibiting TGF-ß1 production in bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus.

Mesenchymal stem cells (MSCs) are critical for immune regulation. Although several microRNAs (miRNAs) have been shown to participate in autoimmune pathogenesis by affecting lymphocyte development and function, the roles of miRNAs in MSC dysfunction in autoimmune diseases remain unclear. Here, we show that patients with systemic lupus erythematosus (SLE) display a unique miRNA signature in bone marrow-derived MSCs (BMSCs) compared with normal controls, among which miR-663 is closely associated with SLE disease activity. MiR-663 inhibits the proliferation and migration of BMSCs and impairs BMSC-mediated downregulation of follicular T helper (Tfh) cells and upregulation of regulatory T (Treg) cells by targeting transforming growth factor ß1 (TGF-ß1). MiR-663 overexpression weakens the therapeutic effect of BMSCs, while miR-663 inhibition improves the remission of lupus disease in MRL/lpr mice. Thus, miR-663 is a key mediator of SLE BMSC regulation and may serve as a new therapeutic target for the treatment of lupus.

Endothelial cell injury is involved in atherosclerosis and lupus symptoms in gld.apoE- / - mice.

AIM: Cardiovascular complications related to atherosclerosis are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the underlying mechanisms are not fully understood. Endothelial dysfunction has been identified as having involvement in pathogenesis of cardiovascular diseases and SLE. This study aims to evaluate endothelial cell injury in mice with the combination of lupus and atherosclerosis. METHODS: The mouse model of accelerated atherosclerosis in lupus (gld.apoE- / - mouse) was generated from apolipoprotein E-deficient (apoE- / - ) and Faslgld C57BL/6 mice. The lupus-like autoimmunity and atherosclerotic lesions were evaluated. The endothelial cell injury was determined. RESULTS: The results showed that the double-mutant gld.apoE- / - mice were generated. Spleens from 5-month-old gld.apoE- / - mice were significantly enlarged compared with wild-type mice (WT mice). The gld.apoE- / - mice produced high levels of total immunoglobulin G (IgG) and IgM and showed marked increase of IgG and C3 deposits in the glomeruli. The gld.apoE- / - mice displayed a pattern of glomerulonephritis typically found in SLE. The gld.apoE- / - mice have high levels of serum creatinine. The total cholesterol, low-density lipoprotein cholesterol and triglycerides were significantly increased, while high-density lipoprotein cholesterol decreased in the double-mutant mice. The circulating endothelial progenitor cells were significantly decreased. The serum levels of thrombomodulin and vascular cell adhesion molecule-1 were significantly elevated in gld.apoE- / - mice. The gld.apoE- / - mice simultaneously exhibited SLE and atherosclerosis characteristics. CONCLUSION: Our findings indicated that endothelial cell injury might be a biomarker for evaluating risks of cardiovascular disease in SLE and targeting endothelial cell dysfunction might prevent and treat atherosclerosis in SLE.

Association between Type I interferon and depletion and dysfunction of endothelial progenitor cells in C57BL/6 mice deficient in both apolipoprotein E and Fas ligand.

Patients with systemic lupus erythematosus (SLE) have a tremendously increased risk for cardiovascular disease (CVD), which could not be accounted in entirety by traditional Framingham risk factors. To study whether the accelerated atherosclerosis in SLE patients is mediated by type I interferon (IFN-I) through the regulation of endothelial progenitor cells (EPCs), we created a line of C57BL/6 mice with deficiency in both apolipoprotein E (ApoE-/-) and fas ligand (FasL-/-, gld.). As expected, the resultant gld. ApoE-/- mice exhibited both aggravated lupus-like disease and atherosclerosis under normal diet. Increased expression of IFN-I-stimulated genes (ISGs) was closely associated with depletion and dysfunction of EPCs, as well as with accelerated atherosclerotic lesion in gld. ApoE-/- mice. While only IFN-α instead of other interventions, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IRS423 and IRS661, impaired EPC function in vitro. Mechanistically, activation or inhibition of the TLR7/9 signaling could modulate EPC number and function in vivo. Decreased proliferation rate and increased apoptotic rate of EPCs induced by IFN-α might contribute to the results to a certain extent. Thus, our data suggest that excessive expression of IFN-I through the activation of TLR7/9 signaling may induce accelerated atherosclerosis in lupus through the depletion or dysfunction of EPCs, suggesting that targeting IFN-I might have potential therapeutic effects on both lupus disease and premature atherosclerosis in SLE patients.

Leptin and Neutrophil-Activating Peptide 2 Promote Mesenchymal Stem Cell Senescence Through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway in Patients With Systemic Lupus Erythematosus.

OBJECTIVE: Mesenchymal stem cells (MSCs) derived from patients with systemic lupus erythematosus (SLE) exhibit enhanced senescence. Cellular senescence has been reported to be induced by several inflammatory cytokines, including interferon-α (IFNα) and IFNγ, that are involved in the pathogenesis of SLE. We undertook this study to investigate whether the inflammatory environment in SLE could affect MSC senescence. METHODS: Cellular senescence was measured by staining of senescence-associated ß-galactosidase and by expression of the cell cycle inhibitors p53 and p21. Eighty cytokines and chemokines in serum from healthy controls and patients with SLE were identified by cytokine antibody array. RESULTS: SLE serum promoted senescence of MSCs, which was reversed by the phosphatidylinositol 3-kinase (PI3K)/Akt signaling inhibitor LY294002 but not by the JAK/STAT inhibitor AG490 and not by the MEK/ERK inhibitor PD98059. Cytokine antibody array analysis revealed that leptin and neutrophil-activating peptide 2 (NAP-2) were the 2 factors most significantly elevated in SLE serum compared with normal serum. Blockade of leptin or NAP-2 in MSC cultures abolished SLE serum-induced senescence, while direct addition of these 2 factors could promote senescence in cultures of normal MSCs. Inhibition of PI3K/Akt signaling with LY294002 reduced leptin- and NAP-2-induced senescence in MSCs. CONCLUSION: Taken together, our data show that leptin and NAP-2 act synergistically to promote MSC senescence through enhancement of the PI3K/Akt signaling pathway in SLE patients.

Mesenchymal stem cells from patients with rheumatoid arthritis display impaired function in inhibiting Th17 cells.

Mesenchymal stem cells (MSCs) possess multipotent and immunomodulatory properties and are suggested to be involved in the pathogenesis of immune-related diseases. This study explored the function of bone marrow MSCs from rheumatoid arthritis (RA) patients, focusing on immunomodulatory effects. RA MSCs showed decreased proliferative activity and aberrant migration capacity. No significant differences were observed in cytokine profiles between RA and control MSCs. The effects of RA MSCs on proliferation of peripheral blood mononuclear cells (PBMCs) and distribution of specific CD4(+) T cell subtypes (Th17, Treg, and Tfh cells) were investigated. RA MSCs appeared to be indistinguishable from controls in suppressing PBMC proliferation, decreasing the proportion of Tfh cells, and inducing the polarization of Treg cells. However, the capacity to inhibit Th17 cell polarization was impaired in RA MSCs, which was related to the low expression of CCL2 in RA MSCs after coculture with CD4(+) T cells. These findings indicated that RA MSCs display defects in several important biological activities, especially the capacity to inhibit Th17 cell polarization. These functionally impaired MSCs may contribute to the development of RA disease.

Association of TNF-α with impaired migration capacity of mesenchymal stem cells in patients with systemic lupus erythematosus.

Previous studies indicated that bone marrow mesenchymal stem cells (BMSCs) from patients with systemic lupus erythematosus (SLE) exhibited impaired capacities of proliferation, differentiation, and immune modulation. Considering that migration capacity is important for the exertion of BMSCs functions, the defects in migration might contribute to BMSCs dysfunction in SLE patients. In this study, we showed that the migration capacity of SLE BMSCs was remarkably impaired in comparison with those of healthy controls. Increased tumor necrosis factor α (TNF-α) in SLE serum significantly inhibited the migration capacity and in vivo homing capacity of SLE BMSCs via a specific TNF receptor I (TNFRI) manner, in which decreased HGF mRNA production caused by the activation of I kappa B kinase beta (IKK-ß) pathway is partially involved. To our knowledge, this is the first report to discuss the possible mechanisms for impaired migration of BMSCs in SLE patients. Our results suggest that inhibition of TNF-α pathway might be helpful for accelerating BMSCs migration to the inflammatory microenvironment in SLE patients, thereby having a potential role in SLE treatment.

Activated NF-κB in bone marrow mesenchymal stem cells from systemic lupus erythematosus patients inhibits osteogenic differentiation through downregulating Smad signaling.

Osteoporosis in patients with systemic lupus erythematosus (SLE) is thought to be the result of accelerated osteoclastogenesis induced by pro-inflammatory cytokines such as tumor necrosis factor (TNF). However, the molecular mechanisms involved in the osteoblastogenesis in SLE patients are not fully understood. We investigated the bone morphogenetic protein-2 (BMP-2)-induced osteoblastic capacity of bone marrow-derived mesenchymal stem cells (BMMSCs) from SLE patients and the TNF signaling system in determining BMP-2-induced regulatory pathways. It showed that the capacity of osteogenic differentiation of BMMSCs from SLE patients was reduced compared with that from healthy controls. The nuclear factor κB (NF-κB) signaling was activated while the BMP/Smad pathway was repressed in BMMSCs from SLE patients. TNF activated NF-κB pathway and inhibited the phosphorylation of Smad 1/5/8 and BMP-2-induced osteoblastic differentiation in BMMSCs from normal controls, while addition of pyrollidine dithiocarbamate (PDTC), an NF-κB inhibitor, to SLE-BMMSCs could partially reverse these effects. Thus, our findings have shown that the activated NF-κB pathway in SLE-BMMSCs inhibits the BMP-2-induced osteoblastic differentiation through BMP/Smad signaling pathway, suggesting that the impaired osteoblastic differentiation may participate in the pathology of osteoporosis in SLE patients.