Peripheral Blood Of Systemic Lupus Erythematosus Patients Research Articles

Shared molecular signatures between systemic lupus erythematosus and osteoporosis.

This study explores the molecular interplay between systemic lupus erythematosus (SLE) and osteoporosis (OP), aiming to uncover shared gene signatures and pathways for better treatment approaches. Leveraging microarray data from the Gene Expression Omnibus (GEO) database, we employed weighted gene coexpression network analysis to identify coexpression modules in SLE and OP, with subsequent protein-protein interaction analysis clarifying the connections among shared genes. Key genes were pinpointed using CytoHubba and random forest algorithms, validated across independent GEO datasets, and further analyzed through gene set enrichment analysis (GSEA) and immune infiltration studies. We discovered two highly correlated modules in SLE and OP, isolating 30 shared genes and identifying GBP1, SOCS1, IFI16, and XAF1 as central to both conditions. Notably, XAF1 and GBP1 mRNA levels were significantly elevated in the peripheral blood of SLE patients compared with healthy and RA counterparts, underscoring their potential as biomarkers. GSEA and immune infiltration analyses indicated pronounced immune and inflammatory responses, especially in interferon signaling pathways, implicating these core-shared gene networks in the diseases' pathogenesis. The findings highlight the involvement of GBP1, SOCS1, IFI16, and XAF1 in SLE with concurrent OP and suggest that targeting immune and inflammatory responses, particularly through interferon pathways, may offer therapeutic promise for these intertwined conditions.

Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

Circulating TIGIT±PD1+TPH, TIGIT ± PD1+TFH cells are elevated and their predicting role in systemic lupus erythematosus

It is well established that increased peripheral helper T cells (TPH) and follicular helper T cells (TFH) was found in systemic lupus erythematosus (SLE) patients. However, the expression patterns and immunomodulatory roles of TIGIT and PD1 on TPH/TFH in SLE are poorly understood. The expression patterns of TIGIT and PD1 on TPH and TFH cells were examined using flow cytometry and their expression patterns in SLE patients were then further evaluated for their correlation with auto-antibodies, disease activity and severity, B cell differentiation. Logistic regression was used to analyze the risk factors. And the receiver operating characteristic curves and logistic regression model were created to evaluate the predicting role in SLE. TIGIT±PD1+TPH, TIGIT±PD1+TFH cells in the peripheral blood of SLE patients were upregulated, whereas TIGIT+PD1−TFH was downregulated. TIGIT ± PD1+TPH, TIGIT ± PD1+TFH cells positively correlated with auto-antibodies production, disease activity and severity, whereas TIGIT+PD1−TFH cells negatively correlated. TIGIT ± PD1+TPH, TIGIT−PD1+TFH were positively correlated with the frequency of plasmablasts. Furthermore, higher TIGIT+PD1+TPH and TIGIT+PD1+TFH were shown to be risk factors for SLE, whereas TIGIT+PD1−TFH was found to be a protective factor, according to logistic regression analysis. A further logistic regression model showed that combination of TPH/TFH and routine blood indicators may has potential predicting value for SLE, with AUC of 0.957. The increased TIGIT ± PD1+TPH, increased TIGIT ± PD1+TFH, decreased TIGIT+PD1−TFH correlates with disease severity and activity, may boost our comprehending of the role of TIGIT and PD1 on TPH/TFH in SLE, and a logistic regression model based on combination of TPH/TFH and routine blood indicators shows prominent value for predicting SLE.

The presumable effects of hydroxychloroquine and its metabolites in the treatment of systemic lupus erythematosus

ObjectiveHydroxychloroquine (HCQ) is an essential drug in the treatment of systemic lupus erythematosus (SLE). This study aimed to detect the concentrations of HCQ and its metabolites from peripheral blood of SLE patients and to investigate the relationship between those concentrations and SLE disease activity. Methods176 SLE patients treated with HCQ were enrolled in this study. The concentrations of HCQ and its metabolites in their peripheral blood were measured by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Patients’ disease activity was evaluated with the systemic lupus erythematosus disease activity index (SLEDAI). The variables between different concentrations or treatments were statistically analyzed. Linear regression was employed to explore relationships between the concentrations of HCQ and its metabolites with the disease activity. ResultsThe SLEDAI was lower in patients with higher concentrations of HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ) (P = 0.024, P = 0.018, and P = 0.003, respectively). There were no significant differences in SLEDAI and the concentrations of HCQ and its metabolites among groups with different treatments (P > 0.05). After adjusting age, gender, disease duration, HCQ dose adjusted to actual body weight, and glucocorticoid (GC) dose, the SLEDAI was negatively correlated with the concentrations of HCQ, DHCQ, DCQ and bisdesethylchloroquine (BDCQ) (P = 0.007, P = 0.011, P = 0.029, and P = 0.008, respectively). After grouping analysis, in patients treated with HCQ and GC, the SLEDAI was negatively correlated with concentrations of HCQ, DHCQ and BDCQ (P = 0.011, P = 0.035, and P = 0.036, respectively). ConclusionsThe concentrations of HCQ and metabolites were correlated with the SLE disease activity after adjusting possible confounding factors, indicating that HCQ and its metabolites might play certain immunoregulatory roles in SLE treatment. Moreover, GC might have a synergistic effect with HCQ. It is helpful in clinical management and follow-up to monitor the concentrations of HCQ and its metabolites in SLE patients.

The effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B cells and CD4+T cells in peripheral blood of patients with systemic lupus erythematosus

BackgroundThe defect of B cell self-tolerance and the continuous antigen presentation by T cells (TCs) mediated by autoreactive B cells (BCs) play a key role in the occurrence and development of systemic lupus erythematosus (SLE). PD-1/PD-L1 signaling axis negatively regulates the immune response of TCs after activation and maintains immune tolerance. However, the effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B/CD4+TCs in the peripheral blood of patients with SLE has not been studied in detail.MethodsPD-1/PD-L1 and Ki-67 levels in peripheral blood (PB) of 50 SLE patients and 41 healthy controls (HCs) were detected through flow cytometry, and then the expression of PD-1+/−cells and PD-L1+/−cells Ki-67 was further analyzed. CD19+B/CD4+TCs were separated for cell culture and the supernatant was collected to determine proliferation and differentiation of TCs. IL-10 and IFN-γ secretion in the supernatant was also determined using ELISA.ResultsThe PD-1, PD-L1, and Ki-67 levels on CD19+B/CD4+TCs in patients with SLE were higher than HCs. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ cells was higher than that of PD-L1− cells, and the proliferative activity of PD-1+ cells was higher than that of PD-1− cells. In the system co-culturing CD19+B/CD4+TCs from HCs/SLE patients, activated BCs promoted TCs proliferation and PD-L1 expression among TCs. Addition of anti-PD-L1 to co-culture system restored the proliferation of TCs, and inhibited IL-10/IFN-γ level. The addition of anti-PD-L1 to co-culture system also restored Tfh and downregulated Treg in HCs.ConclusionsAxis of PD-1/PD-L1 on CD19+B/CD4+TCs in PB of SLE patients is abnormal, and cell proliferation is abnormal. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ and PD-1+ cells compared with PD-L1− and PD-1− cells in SLE patients, respectively. CD19+B/CD4+TCs in SLE patients can interact through PD-1/PD-L1.

Blood platelets in chronic graft-versus-host disease: association with Th1/Th2 ratio

As found in clinical and laboratory studies, platelets not only play a key role in the processes of coagulation and thrombosis, but are also able to actively participate in other pathophysiological processes, including the development of immune reactions. It has been shown that changes in the immune system leading to systemic lupus erythematosus (SLE) are often accompanied by changes in the number of platelets and their activity in the peripheral blood of SLE patients, which correlate with the severity of the clinical manifestations of the disease. Earlier we have studied the standard experimental model of SLE in detail, based on the induction of chronic graft-versus-host disease (cGVHD) in the semi-allogeneic system DBA/2 → (C57Bl/6 x DBA/2)F1. However, the participation of platelets in this immunopathological process has not been studied. There are no data in the literature on the behavior of platelets in cGVHD or on their relationship with the state of Th1/Th2 balance. It can been expected that the platelet count changes according to the development of cGVHD in the used experimental model by analogy with the development of SLE in humans.In the experiments, we used female mice of the DBA/2 strain and (C57Bl/6 × DBA/2)F1 hybrids. Chronic GVHD in a semi-allogeneic system was induced by injecting DBA/2 mouse splenocytes into B6D2F1 hybrid mice: 60-70 × 106 cells intravenously twice with an interval of 6 days. The studied parameters were evaluated three months after the start of the experiment and the formation of lupus-like glomerulonephritis in animals with Th2-dependent cGVHD variant.A decrease in the number of erythrocytes and hemoglobin, a decrease in hematocrit and a parallel increase in the number of reticulocytes in the blood of mice with cGVHD are in good agreement with our earlier conclusion that these animals have autoimmune hemolytic anemia. It was found that, platelets increase significantly with the development of cGVHD unlike other blood cells. Secondary thrombocytosis is observed in the case of the Th2-dependent variant of сGVHD in this model of SLE, while in the group with the Th1-dependent variant of сGVHD, the average number of platelets in the blood does not differ from the control group.

Interferon-α regulates abnormally increased expression of RSAD2 in Th17 and Tfh cells in systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that often involves abnormal activation of regulatory IFN genes and regulation of B cells by CD4+ T cells. Radical S-adenosyl methionine domain containing 2 (RSAD2) is a viral suppressor protein regulated by type I IFN, and it has been proven to play an important regulatory role in SLE. However, the mechanism by which RSAD2 participates in the pathogenesis of SLE is unclear. In this study, we observed higher expression levels of RSAD2 in CD4+ T-cell subsets from the peripheral blood of SLE patients than in those from healthy controls by bioinformatics analysis and validation experiments. We analyzed the expression of RSAD2 in CD4+ T cells of patients with SLE and other autoimmune diseases. In addition, we found that the expression of RSAD2 in CD4+ T cells might be regulated by IFN-α, and RSAD2 significantly affected the differentiation of Th17 cells and T follicular helper (Tfh) cells. Our findings underlined that RSAD2 may promote B-cell activation by promoting the differentiation of Th17 and Tfh cells in SLE patients, a process that is regulated by IFN-α.

Aberrant expression of JMJD3 in SLE promotes B-cell differentiation

Systemic lupus erythematosus (SLE) is a typical autoimmune disease distinguished by multiple organ dysfunction, which is related to a variety of causative factors. B-cell overactivation is a key factor in SLE. However, the pathogenesis underlying anomalous B cells has not been well elucidated. B-cell fate is regulated in diverse epigenetic ways apart from traditional ways. As one of the mechanisms of epigenetics, histone modification mainly affects transcription and translation by changing the chemical groups on histones by histone modification enzymes. JMJD3, a histone demethylase, can promote T-cell proliferation in SLE patients, which exacerbates SLE. However, the mechanism of JMJD3 in B cells in SLE has not been studied. Here, we found that the mean fluorescence intensity (MFI) of JMJD3 in classical memory B cells (CMBs) was higher than that in naïve B cells (NBs) from human tonsil tissue; JMJD3 was overexpressed in B cells from the peripheral blood of SLE patients compared with healthy controls (HCs). In vitro, our experiment showed that JMJD3 could regulate B-cell differentiation by promoting naïve B-cell differentiation into CD27+ B cells, and Blimp-1 and Bcl-6 also decreased after inhibitor treatment. These findings provide a new direction for the pathogenesis of SLE and may supply a new idea for subsequent drug development.

Regulatory effect of IL-38 on NF-κB pathway in systemic lupus erythematosus

BackgroundIL-38 is a newly identified cytokine that exhibits immunosuppression effects. However, there are few studies focusing on the effects and mechanisms of IL-38 in the systemic lupus erythematosus (SLE). AimWe investigated the effects and mechanisms of IL-38 on NF-κB signaling pathway in SLE. MethodsLevels of IL-38, IL-36R, IL-1RAcP, IKKα/β, NF-κB, TNF-α and anti-dsDNA antibody levels in peripheral blood of SLE patients, and in peripheral blood and kidney tissues of MRL/lpr mice, were examined with real-time PCR, ELISA, Western blot and immunohistochemistry. Pathological changes of kidney were detected with PAS staining. Recombinant human IL-38 protein and IL-38 siRNA were used to intervene the PBMCs of SLE patients and MRL/lpr mice. ResultsThe mRNA and protein levels of IL-38 in peripheral blood of SLE patients decreased and were positively correlated. The mRNA and protein levels of IKKα/β, NF-κB, and TNF-α increased, especially in patients with active SLE. There was a negative correlation between IL-38 and the levels of IKKα/β, NF-κB and TNF-α in SLE patients. In vitro experiments showed that the levels of IKKα/β, NF-κB and TNF-α, and anti-dsDNA antibodies decreased in PBMCs of SLE patients after treatment with human recombinant IL-38 protein. These effects were reversed after IL-38 siRNA intervention. Consistent results were obtained on IL-38, IKKα/β, NF-κB, and TNF-α in MRL/lpr lupus mice after treatment with IL-38 protein or IL-38 shRNA. Additionally, kidney function (reflected by creatinine and blood urea nitrogen), anti-dsDNA antibody, complement C3, and urinary protein levels decreased after treatment with IL-38 protein but increased after IL-38 shRNA treatment. PAS staining showed IL-38 protein treatment induced mild hyperplasia of glomerular mesangial cells and a small amount of lymphocyte infiltration. However, these were aggravated after IL-38 shRNA treatment. ConclusionIL-38 may be involved in the occurrence and development of SLE by regulating the NF-κB signaling pathway. This study only discussed the relationship between IL-38 and NF-κB, and more biological functions of IL-38 need to be further studied.

The Changes of Peripheral Blood FⅫa-AT, TSP-1, LA Ratio in Patients with Systemic Lupus Erythematosus and the Clinical Value of Combined Diagnosis of Thrombotic Events

To explore the changes of Ⅻ antithrombin (FⅫa-AT), thrombospondin-1 (TSP-1), and lupus anticoagulant (LA) ratio in the peripheral blood factor of patients with systemic lupus erythematosus (SLE) and the clinical value of combined diagnosis of thrombotic events. A total of 133 SLE patients treated in Xingtai People's Hospital were selected and divided into simple SLE group (105 cases) and SLE complicated with thrombosis group (28 cases) according to whether thrombotic events occurred, and 102 cases of healthy people in the same period were selected as control. The clinical data of the 3 groups, the level of peripheral blood FⅫa-AT, TSP-1, and LA ratio were compared, the relationship between each peripheral blood index and SLE disease activity index (SLEDAI) score were analyzed. The influencing factors of thrombotic events in SLE patients were analyzed, and the value of each peripheral blood index in the diagnosis of SLE complicated with thrombotic events were evaluated. The proportion of the patients with age ≥60 year, hypertension, and smoking history in SLE complicated with thrombosis group was higher than those in simple SLE group and control group (P＜0.05). The SLEDAI score, peripheral blood FⅫa-AT, TSP-1, LA ratio levels of the patients in SLE complicated with thrombosis group were significantly higher than those in simple SLE group and control group, and the simple SLE group was significantly higher than the control group (P＜0.05). FⅫa-AT, TSP-1, LA ratio in peripheral blood of SLE patients were positively correlated with SLEDAI score (r=0.663, 0.578 and 0.625). Age, blood pressure, smoking history, peripheral blood FⅫa-AT, TSP-1, LA ratio were the important influencing factors of thrombotic events in SLE patients (P＜0.05). The AUC diagnosed by the FⅫa-AT, TSP-1, and LA ratio in peripheral blood was 0.881, the 95% CI was 0.813-0.931, the sensitivity was 82.14%, and the specificity was 91.43%, which was superior to each index alone (P＜0.05). Peripheral blood FⅫa-AT, TSP-1, LA ratio level changes in SLE patients are significantly related to disease activity, and the combined diagnosis of thrombotic events is more reliable.

Activation status of CD56dim natural killer cells is associated with disease activity of patients with systemic lupus erythematosus.

Decreased natural killer (NK) cells have been reported in systemic lupus erythematosus (SLE) patients. However, the role of NK cells in the pathogenesis of SLE is not well understood. In this study, we aimed to characterize NK cell subsets, phenotypes, and cytokine-secreting functions and investigate the clinical relevance of NK cells in SLE patients. Peripheral blood samples from 81 SLE patients and 59 healthy donors (HDs) were collected. The frequency and phenotype of NK cells were measured by flow cytometry. Intracellular interferon-γ (IFN-γ) production by NK cells was evaluated by flow cytometry after stimulation with interleukin-12 (IL-12) and IL-18. The percentages of NK cells in the peripheral blood of SLE patients were significantly lower than those in HDs, and the percentages of CD56dim NK cells among total NK cells showed a trend toward decrease. The CD56dim NK cells in SLE patients showed increased production of IFN-γ and displayed relatively activated phenotypic characteristics, including significant increases in NKp44, NKp46, and CD69 and decreased expression of CD16 and CD158a/h/g. Furthermore, CD56dim NK cells in active SLE patients had higher percentages of NKp44+ cells and lower percentages of CD158a/h/g+ cells than those in inactive SLE patients. The percentages of CD158a/h/g+ cells among CD56dim NK cells were negatively correlated with the systemic lupus erythematosus disease activity index (SLEDAI) and positively correlated with C3 and C4 levels. CD56dim NK cells in SLE patients show a reduced proportion tendency among total NK cells and are activated, which partially reflects the disease activity. CD158a/h/g expression on CD56dim NK cells may be considered an index of disease activity. Key Points • In patients with SLE, the proportion of CD56dim NK cells showed a decreased trend and CD56dim NK cells were phenotypically activated which partially reflects the disease activity. • CD158a/h/g expression on CD56dim NK cells were decreased which may be used as an indicator for evaluating disease activity in SLE patients.

Exploring the Role of Non-Coding RNAs in the Pathophysiology of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic immune-related disorder designated by a lack of tolerance to self-antigens and the over-secretion of autoantibodies against several cellular compartments. Although the exact pathophysiology of SLE has not been clarified yet, this disorder has a strong genetic component based on the results of familial aggregation and twin studies. Variation in the expression of non-coding RNAs has been shown to influence both susceptibility to SLE and the clinical course of this disorder. Several long non-coding RNAs (lncRNAs) such as GAS5, MALAT1 and NEAT1 are dysregulated in SLE patients. Moreover, genetic variants within lncRNAs such as SLEAR and linc00513 have been associated with risk of this disorder. The dysregulation of a number of lncRNAs in the peripheral blood of SLE patients has potentiated them as biomarkers for diagnosis, disease activity and therapeutic response. MicroRNAs (miRNAs) have also been shown to affect apoptosis and the function of immune cells. Taken together, there is a compelling rationale for the better understanding of the involvement of these two classes of non-coding RNAs in the pathogenesis of SLE. Clarification of the function of these transcripts has the potential to elucidate the molecular pathophysiology of SLE and provide new opportunities for the development of targeted therapies for this disorder.

Association of the Polymorphism rs13259960 in SLEAR With Predisposition to Systemic Lupus Erythematosus.

Genome-wide association studies have identified many susceptibility loci for systemic lupus erythematosus (SLE). However, most of these loci are located in noncoding regions of the genome. Long noncoding RNAs (lncRNAs) are pervasively expressed and have been reported to be involved in various diseases. This study aimed to explore the genetic significance of lncRNAs in SLE. A genome-wide survey of SLE risk variants in lncRNA gene loci was performed in Han Chinese subjects (4,556 with SLE and 9,451 healthy controls). The functional relevance of an SLE risk variant in one of the lncRNA genes was explored using biochemical and molecular cell biology analyses. In vitro loss-of-function and gain-of-function strategies were used to clarify the functional and phenotypic relevance of this SLE susceptibility lncRNA. Moreover, correlation of this lncRNA with the degree of apoptosis in the peripheral blood of SLE patients was evaluated. A novel SLE susceptibility locus in a lncRNA gene, designated SLEAR (for SLE-associated RNA), was identified at the single-nucleotide polymorphism rs13259960 (odds ratio 1.35, Pcombined = 1.03 × 10-11 ). The A>G variation at rs13259960, located in an intronic enhancer, was found to impair STAT1 recruitment to the enhancer that loops to the SLEAR promoter, resulting in decreased SLEAR production in peripheral blood mononuclear cells from patients with SLE (3 with the G/G genotype, 22 with A/G, and 103 with A/A at rs13259960; P = 0.0241). Moreover, SLEAR interacted with the RNA binding proteins interleukin enhancer binding factor 2, heterogeneous nuclear RNP F, and TATA-binding protein-associated factor 15, to form a complex for transcriptional activation of the downstream antiapoptotic genes. In addition, SLEAR regulated apoptosis of Jurkat cells in vitro, and its expression level was correlated with the degree of cell death in the peripheral blood of patients with SLE (r = 0.824, P = 2.15 × 10-8 ; n = 30). These findings suggest a mechanism by which the risk variant at rs13259960 modulates SLEAR expression and confers a predisposition to SLE. Taken together, these results may give insights into the etiology of SLE.

Expanded circulating peripheral helper T cells in systemic lupus erythematosus: association with disease activity and B cell differentiation.

Peripheral helper T (TPH) cells are a recently identified Th cell subset that promotes B cell differentiation and antibody production in inflamed tissues. This study investigated circulating TPH cells to determine their involvement in systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells collected from SLE patients and healthy individuals were analysed. TPH cells were identified as CD3+CD4+CD45RA-CXCR5- cells with a high expression of PD-1. The frequency, activation status and subsets of TPH cells were evaluated by flow cytometry. The production of IL-21 was assessed by intracellular staining and the association of TPH cells with disease activity and B cell populations was determined. Circulating TPH cells, identified as CD3+CD4+CD45RA-PD-1highCXCR5- cells were increased in the peripheral blood of SLE patients compared with controls. Circulating TPH cells produced similar amounts of IL-21 compared with follicular Th cells. The expansion and activation of TPH cells were correlated with SLE disease activity. Activated TPH cells, particularly Th1-type TPH cells, were associated with the promotion of B cell differentiation in SLE patients. The association of TPH cells with disease activity suggests the involvement of extrafollicular T-B cell interactions in the pathogenesis of SLE. TPH cells promote autoantibody production in aberrant lymphoid organs and therefore might be a novel therapeutic target in autoantibody-producing disorders.

Circulating Pentraxin3-Specific B Cells Are Decreased in Lupus Nephritis.

Background: Pentraxin3 (PTX3) is overexpressed in kidneys of patients developing lupus nephritis (LN). Active LN is associated with reduced anti-PTX3 antibodies. However, abnormalities of B cell differentiation against PTX3 have not been characterized in systemic lupus erythematosus (SLE).Objective: Characterization of PTX3-specific (PTX3+) B cells in peripheral blood of SLE patients with or without LN and healthy donors (HD).Patients and Methods: SLE patients without LN, biopsy-proven LN and matched HD were analyzed. Active LN was defined as proteinuria>0.5 g/day or CrCl<60 ml/min/1.73 m2 with active urinary sediment. Peripheral B cells were analyzed for direct PTX3 binding by flow cytometry using PTX3 labeled with cyanine 5 (Cy5) and phycoerythrin (PE).Results: Initially, a flow cytometry based assay to identify PTX3+ B cells was developed by demonstrating simultaneous binding of PTX3-Cy5 and PTX3-PE. Specificity of B cells was validated by blocking experiments using unlabeled PTX3. We could identify circulating PTX3+ B-cells in HD and patients. Notably, LN patients showed a significantly diminished number of PTX3+ B cells (SLE vs. LN p = 0.033; HD vs. LN p = 0.008). This decrease was identified in naïve and memory B cell compartments (naïve: SLE vs. LN p = 0.028; HD vs. LN p = 0.0001; memory: SLE vs. LN p = 0.038, HD vs. LN p = 0.011).Conclusions: Decreased PTX3+ B cells in LN within the naïve and memory compartment suggest their negative selection at early stages of B cell development potentially related to a decreased regulatory function. PTX3+ B cells could candidate for autoantigen-defined regulatory B cells as a striking abnormality of LN patients.

The role and clinical significance of programmed cell death- ligand 1 expressed on CD19+B-cells and subsets in systemic lupus erythematosus

BackgroundProgrammed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1)-targeted therapies have enhanced T-cell response and demonstrated efficacy in the treatment of multiple cancers. However, the role and clinical significance of PD-L1 expression on CD19+ B-cells and their subsets, with particular reference to systemic lupus erythematosus (SLE), have not yet been studied in detail. ObjectiveThe present study aimed to investigate PD-L1 expression on CD19+ B-cells and their subsets, in addition to exploring its possible role in Tfh-cell activation and B-cell differentiation in SLE. MethodsFrequencies of CD19+ B-cells, their subsets, PD-L1 and Tfh cells in the peripheral blood of SLE patients and healthy controls (HCs) were determined using cytometry. The clinical data of SLE patients were recorded in detail, and the correlation between their laboratory parameters, clinical parameters and disease activity indices was statistically analyzed. CD19+PD-L1+B-cells and CD19+PD-L1− B-cells were sorted and cultured with a stimulant, following which the supernatants were collected for immunoglobulin G and anti-double stranded DNA detection via enzyme-linked immunosorbent assay. ResultsIn SLE patients, CD19+B-cells and partial subgroups were enriched in peripheral blood. Also, the observed increase in the frequency of CD19+PD-L1+B-cells was significantly associated with a higher disease activity index. An in vitro culture test demonstrated that the amounts of anti-dsDNA and immunoglobulin G secreted by the CD19+PD-L1+B-cells of SLE patients and HCs were vastly different. In addition, a strong correlation existed between the frequencies of CD19+PD-L1+B-cells and defined Tfh cells of SLE patients. ConclusionThis study demonstrated that the expression of CD19+PD-L1+B-cells in the peripheral blood of SLE patients was abnormal, and that disease-related laboratory parameters and clinical indicators were correlated. CD19+PD-L1+B-cells were enriched and played a critical role in activating the pathogenic T-cell and B-cell responses in patients with SLE.

Clinical significance of abnormally expressed PD-1 on CD4+ CD28- and CD4+ CD28+ T cells in peripheral blood of SLE patients

Objective To investigate the clinical significance of abnormally expressed PD-1 on CD4+ CD28+ /-T cells in peripheral blood of patients with systemic lupus erythematosus (SLE). Methods Peripheral blood samples were collected form 50 patients with primary SLE and 40 healthy subjects and used to isolated mononuclear cells. Expression of CD4+ CD28-, CD4+ CD28+ , CD4+ CD28+ PD-1+ and CD4+ CD28-PD-1+ T cells in peripheral blood samples of the two groups were detected by flow cytometry. Clinical data of SLE patients were collected. Based on SLE disease activity index (SLEDAI), SLE patients were classified into two groups: stable group (SLEDAI<10) and active group (SLEDAI≥10). Based on the condition of renal damage, they were also divided into two groups: lupus nephritis group and non-lupus nephritis group. Differences in T cell expression were compared among these groups. Statistical analysis was performed to analyze the relationships of different T cell subsets with laboratory and clinical parameters relating to SLE and SLEDAI. Results The percentages of peripheral CD4+ CD28-, CD4+ CD28+ PD-1+ and CD4+ CD28-PD-1+ T cells of active group were higher than those of stable and healthy control groups (P<0.05). Moreover, patients with lupus nephritis had higher percentages of these T cell subsets than those without (P<0.01). SLE patients who were positive for anti-dsDNA or anti-SmRNP antibody, or had decreased complement C3, thrombocytopenia or decreased lymphocytes had higher percentages peripheral CD4+ CD28-T cells than those in the corresponding negative group. SLE patients who were positive for anti-dsDNA or anti-SmRNP antibody, or had decreased complement C3, complement C4 or lymphocytes showed enhanced expression of peripheral CD4+ CD28+ PD-1+ T cells as compared with those in the corresponding negative group. SLE patients positive for anti-dsDNA antibody, or with decreased complement C3 or lymphocytes or suffering from alopecia had higher percentages of peripheral CD4+ CD28-PD-1+ T cell than those in the corresponding negative group. Differences between different groups were statistically significant (P<0.05). Conclusion Abnormal expression of CD4+ CD28-T cells and PD-1 on CD4+ CD28- and CD4+ CD28+ T cells in peripheral blood of patients with SLE has certain correlation with laboratory parameters and clinical indicators. Key words: Systemic lupus erythematosus; PD-1; CD4+ CD28+ /-T cell; Clinical research

Circulating UCA1 is highly expressed in patients with systemic lupus erythematosus and promotes the progression through the AKT pathway.

To investigate the role of Urothelial Carcinoma Associated 1 (UCA1) during the progression of systemic lupus erythematosus (SLE) and the underlying mechanism. UCA1 expression in peripheral blood of SLE patients, as well as the expression of protein kinase B (AKT) in the peripheral blood mononuclear cell (PBMC), was detected by qRT-PCR. Expression differences in UCA1 and AKT between different groups were compared by t-test or univariate analysis. Through correlation analysis, the correlation between UCA1, AKT and clinical indicators of patients was analyzed. After overexpression and knockout of UCA1, the effect on phenotypes of BaF3 cell was examined. Finally, we analyzed the correlation between AKT and UCA1, and the effect on AKT pathway after overexpression and knockout of UCA1. We found that plasma level of UCA1 and AKT was significantly enhanced in SLE patients. By analyzing the clinical data, a higher UCA1 level was observed in female patients than in males. In addition, UCA1 level in SLE patients with active stage and pathological lesions was higher than those in a stable stage without organ involvement. Correlation analysis showed that there was a positive correlation between UCA1 and C3, anti-ds-DNA, ESR and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Similarly, there was a positive correlation between AKT and C3, anti-ds-DNA, erythrocyte sedimentation rate (ESR) and SLEDAI, respectively. After overexpression and knockdown of UCA1, it was found that overexpression of UCA1 significantly enhanced cell proliferation, while the interference with UCA1 significantly inhibited cell proliferation. Western blot revealed increased expressions of PI3K and AKT after overexpressing UCA1, whereas knockdown of UCA1 led significantly decreased expressions of PI3K and AKT. UCA1 expression was significantly increased in SLE, which promoted the progression of SLE by activating AKT pathway.

Increased circulating CD4+CXCR5+FoxP3+ follicular regulatory T cells correlated with severity of systemic lupus erythematosus patients

As one specialized subset of regulatory T cells (Tregs), follicular regulatory T cells (TFR) could suppress follicular helper T cells (TFH) and B cells in germinal centers to maintain immune homeostasis. The unbalance of TFR and TFH cells could result in abnormal germinal center responses and contribute to pathogenesis of autoimmune diseases. However, the role of TFR cells in systemic lupus erythematosus (SLE) remains unclear. This study revealed a significant increase of CD4+CXCR5+FOXP3+ TFR cells in peripheral blood of SLE patients compared with healthy controls. Meanwhile, the suppression ability of circulating TFR cells was not altered. The ratios of TFR/TFH were increased in SLE patients and the frequency of TFR was positively correlated with auto-antibodies and SLEDAI scores of SLE patients. Our results demonstrated that circulating TFR cells were increased during SLE, which suggested that elevated TFR might be a response to the pathogenesis of SLE to suppress TFH function and may provide novel insight for the pathogenesis of SLE.

Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients.

Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.