Peripheral Blood Mononuclear Cells Of Systemic Lupus Erythematosus Patients Research Articles

#2074 Aberrant m6A modification pattern and m6A-related proteins expression in SLE

Abstract Background and Aims Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause. N6-methyladenosine (m6A) is the most common mRNA modification and is involved in various immune processes leading to immune homeostasis disorders. However, the role of m6A in dysregulated immune response of SLE remains unknown. Here, we aim to explore the m6A pattern and expression of m6A-related proteins in the SLE. Method Peripheral blood mononuclear cells (PBMCs) from healthy controls and SLE patients were collected to compare the m6A modification profile by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Expression of 3 m6A-related proteins, including methyltransferase 3 (METTL3), Alkb homologue 5 (ALKBH5), and Fat mass and obesity-associated protein (FTO) and in PBMCs of SLE patients were detected using western blotting (WB). Renal METTL3 expression was further examined in MRL/lpr mice using WB and immunofluorescence. Results SLE patients contained only 28.74% of the same m6A peaks as healthy controls, and 7.881% of the peaks were specific in SLE group (Fig. 1A). The m6A differential peaks were enriched in exon, intron and near 3’ UTR region (Fig. 1B). In addition, compared with healthy controls, m6A modified motifs were obviously aberrant in SLE patients (Fig. 1C). GO and KEGG enrichment analysis showed that some of the m6A peak differential genes were associated with regulation of cellular process, and cytokine-cytokine receptor interaction signaling pathway (Fig. 1D and 1E). Expression of METTL3 and ALKBH5 in SLE patients were significantly increased (METTL3, SLE-AP vs HC, p < 0.001; SLE-SP vs HC, p = 0.0042; ALKBH5, SLE-AP vs HC, p < 0.001; SLE-SP vs HC, p < 0.001; Fig. 1F). There were no significant differences in FTO expressions between the two groups (SLE-AP vs HC, p = 0.167; SLE-SP vs HC, p = 0.477; Fig. 1F). Compared with C57BL/6 mice, renal METTL3 in the MRL/lpr mice was distinctly elevated (p < 0.001; Fig. 1G), which co-localized with CD138 + plasma cells (Fig. 1H). Conclusion These results suggested a distinctly abnormal m6A modification features and m6A-related proteins in SLE patients, indicating the potential significant role of m6A in the pathogenesis of SLE.

Defective Lamtor5 Leads to Autoimmunity by Deregulating v-ATPase and Lysosomal Acidification.

Despite accumulating evidence linking defective lysosome function with autoimmune diseases, how the catabolic machinery is regulated to maintain immune homeostasis remains unknown. Late endosomal/lysosomal adaptor, MAPK and mTOR activator 5 (Lamtor5) is a subunit of the Ragulator mediating mechanistic target of rapamycin complex 1 (mTORC1) activation in response to amino acids, but its action mode and physiological role are still unclear. Here it is demonstrated that Lamtor5 level is markedly decreased in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). In parallel, the mice with myeloid Lamtor5 ablation developed SLE-like manifestation. Impaired lysosomal function and aberrant activation of mTORC1 are evidenced in Lamtor5 deficient macrophages and PBMCs of SLE patients, accompanied by blunted autolysosomal pathway and undesirable inflammatory responses. Mechanistically, it is shown that Lamtor5 is physically associated with ATP6V1A, an essential subunit of vacuolar H+-ATPase(v-ATPase), and promoted the V0/V1 holoenzyme assembly to facilitate lysosome acidification. The binding of Lamtor5 to v-ATPase affected the lysosomal tethering of Rag GTPase and weakened its interaction with mTORC1 for activation. Overall, Lamtor5 is identified as a critical factor for immune homeostasis by intergrading v-ATPase activity, lysosome function, and mTOR pathway. The findings provide a potential therapeutic target for SLE and/or other autoimmune diseases.

Significant heightened methylation levels of RUNX3 gene promoter in patients with systemic lupus erythematosus.

Researchers are actively investigating new diagnostic and prognostic biomarkers that offer improved sensitivity and specificity for systemic lupus erythematosus (SLE). One area of interest is DNA methylation changes. Previous studies have shown a connection between the RUNX3 gene dysfunction and SLE. In this study, the focus was on examining the methylation level of the RUNX3 promoter in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy individuals. A total of 80 individuals diagnosed with SLE from Iran, along with 77 healthy individuals, were included. The methylation levels of the RUNX3 gene in the extracted DNA were evaluated using the MethyQESD method. To determine the diagnostic effectiveness of the RUNX3 promoter methylation level, a receiver operating characteristic (ROC) curve was generated. The methylation of the RUNX3 promoter was found to be significantly higher in patients with SLE compared to healthy individuals (p < .001). This difference in methylation levels was observed between SLE patients and healthy individuals and between SLE patients with renal involvement and those without renal involvement (86.29 ± 10.30 vs 40.28 ± 24.21, p < .001). ROC analyses revealed that the methylation level of the RUNX3 promoter had a diagnostic power of 0.769 [95% CI (0.681-0.814)] for SLE. Additionally, there was a positive correlation between the RUNX3 methylation level and levels of creatinine and C4. The findings of this study emphasize the potential use of RUNX3 methylation levels in PBMCs of SLE patients as biomarkers for diagnosing the disease, predicting renal damage, and assessing disease activity.

Identification of EPSTI1 as a new potential biomarker for SLE based on GEO database.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with highly heterogeneous. The aim of this study is to find the key genes in peripheral blood mononuclear cells (PBMCs) of SLE patients and to provide a new direction for the diagnosis and treatment of lupus. GSE121239, GSE50772, GSE81622, and GSE144390 mRNA expression profiles were obtained from the website of Gene Expression Omnibus (GEO), and differential expressed genes (DEGs) analysis was performed by R. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate signaling pathways for the DEGs. Real-time qPCR (RT-qPCR) was used to verify the key gene EPSTI1 in PBMCs of SLE patients. Finally, the correlation analysis and ROC curve analysis of EPSTI1 for SLE were performed. A total of 12 upregulated DEGs were identified, including MMP8, MX1, IFI44, EPSTI1, OAS1, OAS3, HERC5, IFIT1, RSAD2, USP18, IFI44L, and IFI27. GO and KEGG pathway enrichment analysis showed that those DEGs were mainly concentrated in the response to virus and IFN signaling pathways. Real-time qPCR (RT-qPCR) revealed that EPSTI1 was increased in PBMCs of SLE. EPSTI1 was positively correlated with SLEDAI score in SLE patients. Besides, EPSTI1 was positively correlated with T cell activation- or differentiation-associated genes (BCL6 and RORC). Furthermore, ROC analyses proved EPSTI1 may have diagnostic value for SLE. Together, EPSTI1 was found to be a potential biomarker for SLE, closely related to T cell immune imbalance. Key Points • EPSTI1 expression was significantly increased in PBMCs of SLE patients. • EPSTI1 was positively correlated with disease activity and T cell activation- or differentiation-associated genes in SLE patients. • EPSTI1 might have a good diagnostic value for SLE.

Investigating the causal relationship and potential shared diagnostic genes between primary biliary cholangitis and systemic lupus erythematosus using bidirectional Mendelian randomization and transcriptomic analyses.

The co-occurrence of primary biliary cholangitis (PBC) and systemic lupus erythematosus (SLE) has been consistently reported in observational studies. Nevertheless, the underlying causal correlation between these two conditions still needs to be established. We performed a bidirectional two-sample Mendelian randomization (MR) study to assess their causal association. Five MR analysis methods were utilized for causal inference, with inverse-variance weighted (IVW) selected as the primary method. The Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) and the IVW Radial method were applied to exclude outlying SNPs. To assess the robustness of the MR results, five sensitivity analyses were carried out. Multivariable MR (MVMR) analysis was also employed to evaluate the effect of possible confounders. In addition, we integrated transcriptomic data from PBC and SLE, employing Weighted Gene Co-expression Network Analysis (WGCNA) to explore shared genes between the two diseases. Then, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment methods to perform on the shared genes. The Least Absolute Shrinkage and Selection Operator (LASSO) regression algorithm was utilized to identify potential shared diagnostic genes. Finally, we verified the potential shared diagnostic genes in peripheral blood mononuclear cells (PBMCs)-specific cell populations of SLE patients by single-cell analysis. Our MR study provided evidence that PBC had a causal relationship with SLE (IVW, OR: 1.347, 95% CI: 1.276 - 1.422, P < 0.001) after removing outliers (MR-PRESSO, rs35464393, rs3771317; IVW Radial, rs11065987, rs12924729, rs3745516). Conversely, SLE also had a causal association with PBC (IVW, OR: 1.225, 95% CI: 1.141 - 1.315, P < 0.001) after outlier correction (MR-PRESSO, rs11065987, rs3763295, rs7774434; IVW Radial, rs2297067). Sensitivity analyses confirmed the robustness of the MR findings. MVMR analysis indicated that body mass index (BMI), smoking and drinking were not confounding factors. Moreover, bioinformatic analysis identified PARP9, ABCA1, CEACAM1, and DDX60L as promising diagnostic biomarkers for PBC and SLE. These four genes are highly expressed in CD14+ monocytes in PBMCs of SLE patients and potentially associated with innate immune responses and immune activation. Our study confirmed the bidirectional causal relationship between PBC and SLE and identified PARP9, ABCA1, CEACAM1, and DDX60L genes as the most potentially shared diagnostic genes between the two diseases, providing insights for the exploration of the underlying mechanisms of these disorders.

Proteomic analysis of lysine 2-hydroxyisobutyryl in SLE reveals protein modification alteration in complement and coagulation cascades and platelet activation Pathways

BackgroundPost-translational modifications (PTMs) are considered to be an important factor in the pathogenesis of Systemic lupus erythematosus (SLE). Lysine 2-hydroxyisobutyryl (Khib), as an emerging post-translational modification of proteins, is involved in some important biological metabolic activities. However, there are poor studies on its correlation with diseases, especially SLE.ObjectiveWe performed quantitative, comparative, and bioinformatic analysis of Khib proteins in Peripheral blood mononuclear cells (PBMCs) of SLE patients and PBMCs of healthy controls. Searching for pathways related to SLE disease progression and exploring the role of Khib in SLE.MethodsKhib levels in SLE patients and healthy controls were compared based on liquid chromatography tandem mass spectrometry, then proteomic analysis was conducted.ResultsCompared with healthy controls, Khib in SLE patients was up-regulated at 865 sites of 416 proteins and down-regulated at 630 sites of 349 proteins. The site abundance, distribution and function of Khib protein were investigated further. Bioinformatics analysis showed that Complement and coagulation cascades and Platelet activation in immune-related pathways were significantly enriched, suggesting that differentially modified proteins among them may affect SLE.ConclusionKhib in PBMCs of SLE patients was significantly up- or down-regulated compared with healthy controls. Khib modification of key proteins in the Complement and coagulation cascades and Platelet activation pathways affects platelet activation and aggregation, coagulation functions in SLE patients. This result provides a new direction for the possible significance of Khib in the pathogenesis of SLE patients.

The effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on cytokines and their related molecules: An ex vivo study on patients with systemic lupus erythematosus.

This study aimed to assess the ex vivo impact of Lactobacillus delbrueckii (L. delbrueckii) and Lactobacillus rhamnosus (L. rhamnosus) on inflammatory and anti-inflammatory cytokines as well as their related molecules on the peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients. This study was conducted with 20 newly diagnosed SLE patients (18 females, 2 males; mean age: 33.3±12.4 years; range, 18 to 68 years) between September 2017 and September 2018. Extracted PBMCs from each patient were divided into 4 cell groups in our study. Three cell groups act as treatment groups receiving L. rhamnosus (107 CFU/mL), L. delbrueckii (105 CFU/mL) or a mixture of both, and one group act as our untreated control group in the absence of any probiotic agents. All cell groups were cultured in RPMI 1460 medium for 48 h. Then, total RNA was extracted, and cDNA was synthesized. The gene expression levels of forkhead box P3 (FOXP3), transforming growth factor beta (TGF-β), interleukin (IL)-6, IL-10, and IL-2 were evaluated by a quantitative real-time polymerase chain reaction. The results revealed that expression levels of FOXP3, TGF-β, IL-10, and IL-2 increased and the level of IL-6 decreased in probiotics-receiving groups compared to the control group. Lactobacillus delbrueckii and L. rhamnosus enhanced the expression of regulatory T cell-related molecules such as FOXP3 and IL-2 and also increased the expression of IL-10. These probiotics also reduced the expression of IL-6 as proinflammatory cytokines in the PBMCs of SLE patients. The results of the present study show that these probiotics could be effective in regulating the balance of cytokine gene expression ex vivo , and due to their beneficial effects, they can be an intriguing option in the production of new complement drugs for SLE.

Significant hypomethylation of MMP9 gene promoter in patients with systemic lupus erythematosus.

Scholars are exploring novel diagnostic and prognostic biomarkers with higher sensitivity and specificity for systemic lupus erythematosus (SLE). In this regard, DNA methylation alterations have aroused attention. The association between the dysfunction of MMP9 and TNFAIP3 genes and SLE has been previously demonstrated. Therefore, in this study, we investigated the methylation level of MMP9 and TNFAIP3 promoters in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls. Eighty Iranian SLE patients and 77 healthy individuals were enrolled. The methylation quantification endonuclease-resistant DNA (MethyQESD) method was used to assess methylation levels of MMP9 and TNFAIP3 in extracted DNA of PBMCs. To quantify the diagnostic utility of the promoter methylation level of these genes, the receiver operating characteristic (ROC) curve was constructed. MMP9 promoter was significantly hypomethylated in SLE patients compared with healthy people (p < 0.001), while there was no significant difference in terms of TNFAIP3 promoter methylation levels (p = 0.167). Also, this differential MMP9 methylation was observed in patients with renal involvement and patients without renal involvement (42.07 ± 25.73 vs 56.74 ± 29.71, p = 0.007). ROC analyses indicated that the diagnostic power of the MMP9 promoter methylation level for SLE was 0.839 [95% CI (0.781-0.911)]. Moreover, MMP9 methylation level was negatively correlated with creatinine and anti-dsDNA concentration and positively correlated with C3 and C4 levels. The results of this study highlight the application of MMP9 methylation level in PBMCs of SLE patients as a diagnostic biomarker.

Mesenchymal Stem Cells Suppress Dendritic Cells and Modulate Proinflammatory Milieu Through Interleukin-10 Expression in Peripheral Blood Mononuclear Cells of Human Systemic Lupus Erythematosus.

Immune-mediated inflammatory injury among systemic lupus erythematosus (SLE) individuals may be involved by dendritic cells (DCs) abnormality though the underlying mechanism remains incompletely understood. This study aimed to elaborate MSCs' potential in suppressing abnormal DCs cell function on peripheral blood mononuclear cells (PBMCs) among SLE patients. MSCs were isolated from human umbilical cord blood. On the other side, human PBMCs were isolated from 20 active SLE patients and 5 healthy controls. The PBMCs of SLE patients were divided into 5 groups: sham (Sh) and control (C) groups were treated with standard medium, and the treatment groups (T1, T2 and T3) was co-cultured with hUC-MSC at doses of 1:1, 1:25, and 1:50 (MSCs:PBMCs). The expression of CD11c in DCs was analyzed using flow cytometry, while the level of TNF-α, IFN-γ, IL-6 and IL-10 was analyzed using cytometric bead array (CBA). The MSCs significantly downregulates CD11c of dendritic cells in all treatment groups. MSCs also significantly suppress the level of TNF-α, IFN-γ, IL-6 and the significantly enhance IL-10 level in all treatment groups. Therefore, MSCs could suppress DCs through regulating the proinflammatory milieu in PBMCs of SLE patients.

MiR-31-5p Regulates Type I Interferon by Targeting SLC15A4 in Plasmacytoid Dendritic Cells of Systemic Lupus Erythematosus.

Plasmacytoid dendritic cells (pDCs) are the main producers of type I interferon (IFN-I), and the excessive production of IFN-I is a hallmark of systemic lupus erythematosus (SLE). Both SLC15A4 and miR-31-5p are SLE susceptibility-related genes, and SLC15A4 has been implicated an important role in endolysosomal toll-like receptor (TLR) activation in pDCs. However, whether miR-31-5p exerts a regulating effect on SLC15A4 expression in pDCs is unclear. The expression of SLC15A4 and miR-31-5p in peripheral blood mononuclear cells (PBMCs) of SLE patients was measured by RT-qPCR analyses. The quantitative analysis of IFN-α secretion in the patients' serum was performed by ELISA assay. Luciferase-reporter assay was applied to confirm the interaction between miR-31-5p and SLC15A4. The expression of miR-31-5p, SLC15A4 and IFN-stimulated genes (ISGs, such as MX1, OAS1 and IFIT3) was detected by Western blot and RT-qPCR assays and further IRF5 phosphorylation was evaluated by immunofluorescence after transfected with miR-31-5p mimics or inhibitor in THP-1 and CAL-1 cells. The expression of miR-31-5p was downregulated and negatively correlated with the overexpression of SLC15A4 in PBMCs of SLE patients. In addition to this, the secretion of IFN-α was overexpressed in sera of SLE and positively correlated with SLC15A4 level. We found that miR-31-5p directly targeted SLC15A4 and negatively regulated the expression of SLC15A4 in THP-1 and CAL-1 cells. In vitro inhibition of miR-31-5p increased the phosphorylation of IRF5 and the induction of ISGs stimulated by R848, overexpression of miR-31-5p get the reverse results. miR-31-5p might involve in SLE pathogenesis through regulating IFN-I expression by negatively regulating SLC15A4 to increase the levels of IFN-α and ISGs in pDCs.

Methylation of TET2 Promoter Is Associated with Global Hypomethylation and Hypohydroxymethylation in Peripheral Blood Mononuclear Cells of Systemic Lupus Erythematosus Patients.

(1) Background: It is widely accepted that aberrant methylation patterns contribute to the development of systemic lupus erythematosus (SLE). Ten-eleven translocation (TET) methylcytosine dioxygenase is an essential enzyme of which there are three members, TET1, 2, and 3, involved in hydroxymethylation, a newly uncovered mechanism of active DNA methylation. The epigenomes of gene transcription are regulated by 5-hydroxymethylcytocine (5-hmC) and TETs, leading to dysregulation of the immune system in SLE. The purpose of this study was to investigate the global hydroxymethylation status in SLE peripheral blood mononuclear cells (PBMCs) and to explore the role of TETs in changing the patterns of methylation. (2) Methods: We collected PBMCs from 101 SLE patients and 100 healthy donors. TaqMan real-time polymerase chain-reaction assay was performed for the detection of 5-methylcytosine (5-mC), 5-hmC, and TET2 mRNA expression and single-nucleotide polymorphism genotyping. The methylation rates in different CpG sites of TET2 promoters were examined using next-generation sequencing-based deep bisulfite sequencing. Putative transcription factors were investigated using the UCSC Genome Browser on the Human Dec. 2013 (GRCh38/hg38) Assembly. (3) Results: 5-mC and 5-hmC were both decreased in SLE. The mRNA expression level of TET2 was notably high and found to be correlated with the levels of immunologic biomarkers that are indicative of SLE disease activity. The analysis of methylation rates in the TET2 promoter revealed that SLE patients had significantly higher and lower rates of methylation in TET2 105146072-154 and TET2 105146218-331, respectively. (4) Conclusions: TET2 may play an important role in 5-mC/5-hmC dynamics in the PBMCs of SLE patients. The epigenetic modification of TET2 promoters could contribute to the pathogenesis of SLE and the intensity of the immunologic reaction.

Identification of key interferon-stimulated genes for indicating the condition of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with highly heterogeneous clinical symptoms and severity. There is complex pathogenesis of SLE, one of which is IFNs overproduction and downstream IFN-stimulated genes (ISGs) upregulation. Identifying the key ISGs differentially expressed in peripheral blood mononuclear cells (PBMCs) of patients with SLE and healthy people could help to further understand the role of the IFN pathway in SLE and discover potential diagnostic biomarkers.The differentially expressed ISGs (DEISG) in PBMCs of SLE patients and healthy persons were screened from two datasets of the Gene Expression Omnibus (GEO) database. A total of 67 DEISGs, including 6 long noncoding RNAs (lncRNAs) and 61 messenger RNAs (mRNAs) were identified by the “DESeq2” R package. According to Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, those DEISGs were mainly concentrated in the response to virus and immune system processes. Protein-protein interaction (PPI) network showed that most of these DEISGs could interact strongly with each other. Then, IFIT1, RSAD2, IFIT3, USP18, ISG15, OASL, MX1, OAS2, OAS3, and IFI44 were considered to be hub ISGs in SLE by “MCODE” and “Cytohubba” plugins of Cytoscape, Moreover, the results of expression correlation suggested that 3 lncRNAs (NRIR, FAM225A, and LY6E-DT) were closely related to the IFN pathway.The lncRNA NRIR and mRNAs (RSAD2, USP18, IFI44, and ISG15) were selected as candidate ISGs for verification. RT-qPCR results showed that PBMCs from SLE patients had substantially higher expression levels of 5 ISGs compared to healthy controls (HCs). Additionally, statistical analyses revealed that the expression levels of these ISGs were strongly associated to various clinical symptoms, including thrombocytopenia and facial erythema, as well as laboratory indications, including the white blood cell (WBC) count and levels of autoantibodies. The Receiver Operating Characteristic (ROC) curve demonstrated that the IFI44, USP18, RSAD2, and IFN score had good diagnostic capabilities of SLE.According to our study, SLE was associated with ISGs including NRIR, RSAD2, USP18, IFI44, and ISG15, which may contribute to the future diagnosis and new personalized targeted therapies.

UXT attenuates the CGAS-STING1 signaling by targeting STING1 for autophagic degradation

ABSTRACT STING1 (stimulator of interferon response cGAMP interactor 1), the pivotal adaptor protein of CGAS (cyclic GMP-AMP synthase)-STING1 signaling, is critical for type I IFN production of innate immunity. However, excessive or prolonged activation of STING1 is associated with autoinflammatory and autoimmune diseases. Thus, preventing STING1 from over-activation is important to maintain immune homeostasis. Here, we reported that UXT (ubiquitously expressed prefoldin like chaperone), a small chaperone-like protein, was essential to prevent the excessive activation of STING1-mediated type I IFN signaling through autophagic degradation of STING1 via SQSTM1 (sequestosome 1). Upon DNA mimics or cyclic GMP-AMP (cGAMP) stimulation, UXT specifically interacted with STING1 and promoted STING1 degradation through selective macroautophagy/autophagy. Moreover, UXT was required for more efficient autophagic degradation of STING1 by facilitating the interaction of SQSTM1 and STING1. The in vivo role of UXT in attenuating the CGAS-STING1 signaling was further confirmed in the mouse model of DNA-virus infection and the TMPD (2,6,10,14-tetramethylpentadecane)-induced murine lupus model. Intriguingly, the expression of UXT was consistently impaired and exhibited a remarkable inverse correlation with type I IFN signature in the leukocytes and PBMCs (peripheral blood mononuclear cells) of several large SLE (systemic lupus erythematosus) cohorts. Importantly, the replenishment of UXT effectively suppressed the production of IFNs and ISGs in the PBMCs of SLE patients. Taken together, our study reveals a novel regulatory role of UXT in autophagic degradation of STING1 to maintain immune homeostasis. UXT might be a potential therapeutic target for alleviating aberrant type I IFNs in autoimmune diseasesAbbreviations: 3-MA: 3-methyladenine; BMDMs: bone marrow-derived macrophages; cGAMP: cyclic GMP-AMP; CGAS: cyclic gmp-amp synthase; cKO: conditional knockout; CXCL10: C-X-C motif chemokine ligand 10; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HSV-1: herpes simplex virus type 1; HTDNA: herring testes DNA; IFIT1: interferon induced protein with tetratricopeptide repeats 1; IFNA4: interferon alpha 4; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISD: interferon stimulatory DNA; ISGs: IFN-stimulated genes; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; RNA-seq: RNA sequencing; PBMCs: peripheral blood mononuclear cells; RSAD2: radical S-adenosyl methionine domain containing 2; SLE: systemic lupus erythematosus; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TMPD: 2,6,10,14-tetramethylpentadecane; UXT: ubiquitously expressed prefoldin like chaperone.

PGLYRP2 as a novel biomarker for the activity and lipid metabolism of systemic lupus erythematosus

BackgroundTo assess the value of peptidoglycan recognition protein 2 (PGLYRP2) in assessing the disease activity and lipid metabolism in patients with systemic lupus erythematosus (SLE).MethodsSLE patients with stable disease (n = 15), active lupus nephritis (LN) (n = 15) and neuropsychiatric systemic lupus erythematosus (NP-SLE) (n = 15) admitted to Northern Jiangsu People’s Hospital (Jiangsu, China) in 2019–2020 were recruited. In addition, volunteers with matched age and sex (n = 15) were enrolled as controls. The level of PGLYRP2 in the serum and its expression in peripheral blood mononuclear cells (PBMCs) were measured. The link between PGLYRP2 level and clinical parameters (including lipid profile) was described.ResultsSerum PGLYRP2 level in SLE cases exceeded that in healthy volunteers (3938.56 ± 576.07 pg/mL), and significantly higher in active LN (5152.93 ± 446.13 pg/mL) and NP-SLE patients (5141.52 ± 579.61 pg/mL). As shown by quantitative real-time PCR results, the expression of PGLYRP2 in PBMCs of SLE patients with active LN and NP-SLE surpassed that in healthy volunteers (P < 0.01). Receiver operating characteristic (ROC) curves demonstrated that PGLYRP2 was capable of distinguishing stable SLE from active LN (AUC = 0.841, 95%CI = 0.722–0.960, P = 0.000). PGLYRP2 level positively correlated with SLEDAI of SLE patients (r = 0.5783, P < 0.01). Moreover, its level varied with serological and renal function parameters (complement 3, complement 4, estimated glomerular filtration rate and 24-h urine protein) and immunoglobulin A (IgA) of SLE. A potential correlation between PGLYRP2 level and lipid profile (HLD-c, Apo-A1 and Apo B/A1) was determined in SLE patients. The linear regression analysis indicated SLEDAI as an independent factor of PGLYRP2 level, with a positive correlation in between (P < 0.05).ConclusionsSerum PGLYRP2 level significantly increases in SLE patients, and is positively correlated to SLEDAI. Moreover, serum PGLYRP2 level is correlated with renal damage parameters and the abnormal lipid profile of SLE. PGLYRP2 could be used to predict SLE activity, dyslipidemia and cardiovascular disease risks in SLE patients.

Increased MLKL mRNA level in the PBMCs is correlated with autoantibody production, renal involvement, and SLE disease activity

BackgroundNecroptosis is a form of regulated necrosis that is involved in various autoimmune diseases. Mixed lineage kinase domain-like pseudokinase (MLKL) has been identified as a key executor of necroptosis; however, the significance of MLKL in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) has not been investigated. In this study, we aimed to determine the mRNA level of MLKL in PBMCs and examine its relationship with clinical features and serological parameters in SLE.MethodsReal-time transcription-polymerase chain reaction (RT-PCR) analysis was used to determine the expression of MLKL mRNA in PBMCs from 59 patients with SLE, 25 patients with rheumatoid arthritis (RA), and 30 age- and sex-matched healthy controls (HC). Spearman’s correlation test was performed to assess the correlation of MLKL mRNA with clinical variables. The receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value.ResultsOur results showed MLKL mRNA in PBMCs was upregulated in SLE patients compared to that in RA and HC individuals. SLE patients positive for antinuclear antibodies had significantly higher MLKL mRNA than antibody-negative patients. In SLE patients, MLKL mRNA was found to be upregulated in patients with lupus nephritis (LN) as compared with patients without LN, and also higher in active patients than in stable patients. MLKL mRNA level was significantly and positively correlated with c-reaction protein (CRP) (r = 0.3577, p = 0.0237), erythrocyte sedimentation rate (ESR) (r = 0.4091, p = 0.0043), serum immunoglobulin G (IgG) concentration (r = 0.3546, p = 0.0289), and the numbers of positive antinuclear antibodies (ANAs) (r = 0.3945, p = 0.0432). ROC analysis showed that MLKL mRNA in PBMCs had an area under the curve of 0.9277 (95% CI 0.8779–0.9775, p < 0.001) to discriminate SLE from controls.ConclusionsThese results suggest that increased MLKL mRNA level in the PBMCs of SLE patients is correlated with renal involvement and disease activity, identifying a subgroup of patients with SLE or LN who may benefit from early diagnosis and therapies targeting MLKL.

Downregulation of microRNA-101-3p participates in systemic lupus erythematosus progression via negatively regulating HDAC9.

This study aimed to investigate the mechanism of microRNA-101-3p (miR-101-3p) on the progression of systemic lupus erythematosus (SLE). The human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples of SLE patients and healthy individuals, followed by cell culture and transfection. Moreover, the flow cytometry assay, quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunoassay were used to assess the effect of miR-101-3p on PBMCs. Bioinformatics analysis was conducted to predict the putative target gene of miR-101-3p, luciferase reporter gene assay, and RNA pull-down assay were applied to verify the interaction between them. Compared with healthy individuals, the expression level of miR-101-3p in PBMCs of SLE patients was significantly decreased, whereas interleukin (IL)-17A, IL-6, and interferon (IFN)-γ were remarkably increased (all P < .001). Correlation analyses showed that there were negative correlations between miR-101-3p and IL-17A, IL-6 and IFN-γ. The expression level of miR-101-3p in PBMCs of SLE patients was positively correlated with C3 expression (rs = .4075; P = .0229), while negatively associated with erythrocyte sedimentation rate (ESR) (rs = -.4238; P = .0175) and IgG expression (rs = -.4949; P = .0047). Overexpression of miR-101-3p could inhibit the differentiation of CD4 + T cells into Th17 lineage. Histone deacetylase 9 (HDAC9) was identified as a potential target gene of miR-101-3p. Furthermore, HDAC9 abolished the effect of miR-101-3p on Th17 cell differentiation and IL-17A expression in SLE. In conclusion, downregulated miR-101-3p in PBMCs of SLE patients inhibited Th17 cell differentiation by directly targeting HDAC9, which could be used as a novel therapeutic therapy for SLE treatment.

MiR-98 Modulates Cytokine Production from Human PBMCs in Systemic Lupus Erythematosus by Targeting IL-6 mRNA.

Objective There is evidence that interleukin-6 (IL-6) upregulation plays a critical role in immunopathology of systemic lupus erythematosus (SLE). MicroRNA- (miRNA-) 98 was predicted to bind with the 3′-untranslated region (3′-UTR) of IL-6 gene. We hypothesized miR-98 through its regulation of IL-6 gene expression to influence cytokine production from peripheral blood mononuclear cells (PBMCs) in SLE. Methods The expression of miR-98 and IL-6 mRNA in the PBMCs of 41 SLE patients and 20 healthy controls (HC) was detected by quantitative reverse transcription PCR (qRT-PCR). The correlations between miR-98 expression and clinical features were evaluated. Luciferase reporter assay was performed to identify miR-98 targets. miR-98 mimics, miR-98 inhibitor, and IL-6 overexpression vector were generated. Cell viability of PBMCs was assessed using MTT assay. Gene expression and protein level were determined by qRT-PCR and Western blotting. TNF-α, IL-8, IL-1β, and IL-10 levels in cultured supernatants were quantified using ELISA. Results The expression of miR-98 was downregulated in PBMCs of SLE patients, and its expression is negatively associated with IL-6 levels. miR-98 expression was correlated with disease activity, lupus nephritis, and anti-dsDNA antibody. IL-6 mRNA was a target gene of miR-98. IL-6 overexpression promoted the proliferation of PBMCs and increased the levels of TNF-α, IL-8, IL-1β, and IL-10. Those effects were further enhanced by miR-98 inhibitor, while were suppressed by miR-98 mimics. miR-98 regulated the levels of STAT3 phosphorylation via its target gene IL-6. Conclusion The current study revealed that miR-98 could ameliorate STAT3-mediated cell proliferation and inflammatory cytokine production via its target gene IL-6 in patients with SLE. These results suggest that miR-98 might serve as a potential target for SLE treatment and other IL-6-mediated diseases.

MiR-125b is downregulated in systemic lupus erythematosus patients and inhibits autophagy by targeting UVRAG

Systemic lupus erythematosus (SLE) is a severe autoimmune disease and the pathogenesis remains incompletely understood. This study aimed to investigate the role of miR-125b in the pathogenesis of SLE and explore the underlying mechanism. Compared to healthy controls, the expression of miR-125b decreased in peripheral blood mononuclear cells (PBMCs) of SLE patients. In addition, PBMCs exposed to ultraviolet B had lower miR-125b level compared to those unexposed to radiation. We identified UV radiation resistance associated gene (UVRAG) as a target of miR-125b. Jurkat cells treated with miR-125b-5p agomir showed reduced levels of ATG7, Beclin-1 and LC3 II and decreased autophagy. In contrast, Jurkat cells treated with miR-125b-5p antagomir showed increased levels of ATG7, Beclin-1 and LC3 II and increased autophagy. Furthermore, Jurkat cells transfected with UVRAG expression vector showed higher expression of ATG7, Beclin-1 and LC3 II and increased autophagy. Conversely, cells transfected with UVRAG siRNA had lower expression of ATG7, Beclin-1 and LC3 II and decreased autophagy. Taken together, our data demonstrate that Ultraviolet B radiation can downregulate miR-125b-5p and increase UVRAG expression and autophagy activity in PBMCs of SLE patients. These findings help explain how ultraviolet B exacerbates SLE and suggest that UVRAG is a potential therapeutic target for SLE.

Vitamin D3 Alters the Expression of Toll-like Receptors in Peripheral Blood Mononuclear Cells of Patients With Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by production of inflammatory cytokines and autoreactive antibodies due to the loss of immune tolerance. Recognition of self-nucleic acids by intracellular Toll-like receptors (TLRs) can overactivate immune responses and this abnormal activation of TLRs contributes to the pathogenesis of the disease. In recent years, anti-inflammatory and immunomodulatory effects of 1,25-dihydroxyvitamin D3 (VitD3) on the immune system has received particular attention. The present study investigated the effects of vitamin D3 on the expression of TLR3, TLR7, and TLR9 in SLE patients. Study participants included 20 SLE patients and 20 age- and sex-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured in the presence or absence of vitamin D3 (50 nM). Then RNA was extracted, cDNA was synthesized and gene expression levels of TLR3, TLR7, and TLR9 were assessed using real-time PCR. Up-regulated expression levels of TLR7 and TLR9 were observed in the PBMCs of SLE patients in comparison with controls. Culturing PBMCs with vitamin D3 significantly down-regulated the expression of TLR3 (8.86 ± 4.2 for SLE patients vs. 45.34 ± 18.6 for control; P = 0.03), TLR7 (17.91 ± 7.7 for SLE patients vs. 242.37 ± 89.6 for controls; P = 0.0001) and TLR9 (4.67 ± 1.9 for SLE patients vs. 8.9 ± 1.5 for controls; P = 0.007) in SLE patients in comparison with healthy controls. The results of the current study suggest that vitamin D3 could exert some of its immunomodulatory effects in SLE patients via affecting the expression levels of some TLRs. J. Cell. Biochem. 118: 4831-4835, 2017. © 2017 Wiley Periodicals, Inc.

Unraveling the expression of microRNA-27a* & NKG2D in peripheral blood mononuclear cells and natural killer cells of pediatric systemic lupus erythematosus patients.

The activity of natural killer (NK) cells is known to be decreased in systemic lupus erythematosus (SLE) patients. Nevertheless, the exact contribution of NK cells in the pathogenesis of SLE is still inconclusive. MicroRNAs (miRNAs), are small noncoding RNA molecules that play a fundamental role in regulating NK cell function. The objective of this study was to investigate the expression of miRNAs that might potentially target an essential activating receptor, NKG2D in peripheral blood mononuclear cells (PBMCs) and NK cells of SLE patients. In silico analysis revealed miR-27a* to potentially target NKG2D messenger RNA (mRNA), hence PBMCs and NK cells were isolated from blood samples of SLE patients and healthy controls. Next, the cells were transfected using mimics and antagomirs, after which miRNA/mRNA were quantified using real time quantitative reverse transcription polymerase chain reaction. The results of this study showed that miR-27a* is overexpressed in the PBMCs and NK cells of SLE patients. In contrast, NKG2D was found to be downregulated in PBMCs and NK cells of SLE patients. Forcing the expression of miR-27a* in PBMCs and NK cells enhances the expression of NKG2D in SLE patients. Furthermore, the ligand of NKG2D, ULBP2, was found to be downregulated in the PBMCs of SLE patients. The altered expression of the triad, miR-27a* as well as NKG2D and ULBP2, is thought to be characteristic for NK cells in SLE patients. Hence, the ability of miR-27a* to alter the expression of NKG2D may provide a new groundwork for understanding the role of miRNAs in NK cells of SLE patients.