Imbalance of programmed cell death patterns mediated by dendritic cell subsets in systemic lupus erythematosus and lupus nephritis. / æ ‘çªçŠ¶ç»†èƒžäºšç¾¤ä»‹å¯¼ç³»ç»Ÿæ€§çº¢æ–‘ç‹¼ç–®å’Œç‹¼ç–®è‚¾ç‚Žçš„ç¨‹åºæ€§ç»†èƒžæ­»äº¡æ¨¡å¼å¤±è¡¡ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns. METHODS: Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 µmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 µmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group. RESULTS: Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group. CONCLUSIONS: This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.

Identification of the shared genes and immune signatures between systemic lupus erythematosus and idiopathic pulmonary fibrosis.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder which could lead to inflammation and fibrosis in various organs. Pulmonary fibrosis is a severe complication in patients with SLE. Nonetheless, SLE-derived pulmonary fibrosis has unknown pathogenesis. Of pulmonary fibrosis, Idiopathic pulmonary fibrosis (IPF) is a typicality and deadly form. Aiming to investigate the gene signatures and possible immune mechanisms in SLE-derived pulmonary fibrosis, we explored common characters between SLE and IPF from Gene Expression Omnibus (GEO) database. RESULTS: We employed the weighted gene co-expression network analysis (WGCNA) to identify the shared genes. Two modules were significantly identified in both SLE and IPF, respectively. The overlapped 40 genes were selected out for further analysis. The GO enrichment analysis of shared genes between SLE and IPF was performed with ClueGO and indicated that p38MAPK cascade, a key inflammation response pathway, may be a common feature in both SLE and IPF. The validation datasets also illustrated this point. The enrichment analysis of common miRNAs was obtained from the Human microRNA Disease Database (HMDD) and the enrichment analysis with the DIANA tools also indicated that MAPK pathways' role in the pathogenesis of SLE and IPF. The target genes of these common miRNAs were identified by the TargetScan7.2 and a common miRNAs-mRNAs network was constructed with the overlapped genes in target and shared genes to show the regulated target of SLE-derived pulmonary fibrosis. The result of CIBERSORT showed decreased regulatory T cells (Tregs), naïve CD4+ T cells and rest mast cells but increased activated NK cells and activated mast cells in both SLE and IPF. The target genes of cyclophosphamide were also obtained from the Drug Repurposing Hub and had an interaction with the common gene PTGS2 predicted with protein-protein interaction (PPI) and molecular docking, indicating its potential treatment effect. CONCLUSIONS: This study originally uncovered the MAPK pathway, and the infiltration of some immune-cell subsets might be pivotal factors for pulmonary fibrosis complication in SLE, which could be used as potentially therapeutic targets. The cyclophosphamide may treat SLE-derived pulmonary fibrosis through interaction with PTGS2, which could be activated by p38MAPK.

Application of clinical indices in pathological acute and chronic index prediction in lupus nephritis. / ä¸´åºŠæŒ‡æ ‡åœ¨ç‹¼ç–®è‚¾ç‚Žç— ç†å­¦æ€¥æ ¢æ€§æŒ‡æ•°é¢„æµ‹ä¸­çš„åº”ç”¨.

OBJECTIVES: To predict renal biopsy with pathological acute and chronic index via comprehensive analysis of clinical indices from lupus nephritis patients. METHODS: We collected 107 inpatients' data from the Third Xiangya Hospital of Central South University from January 2011 to September 2018. These inpatients have already had renal biopsy results. These clinical indices were set as variables. Using multiple linear regression, we built the prediction models for renal pathological acute and chronic index. Simultaneously, we evaluated each vital variable's importance in models by standardized coefficient of regression equation, and model prediction accuracy by 5-fold cross validation. RESULTS: Acute index and chronic index prediction models were built with 19 and 23 clinical variables, respectively. To evaluate the two models' accuracy of prediction, we used 5-fold cross validation and found that the prediction accuracy level were satisfactory with the acute index model (Q2=0.649, R2=0.791) and chronic index model (Q2=0.563, R2=0.744). Standardized coefficient of regression equation showed serum total cholesterol, serum iron, NAG, ß2 microglobulin and BUN were important variables to predict acute index while total cholesterol, ß2 microglobulin, homocystein and serum low density lipoprotein-cholesterol were important for chronic index prediction. CONCLUSIONS: Clinical indices are able to effectively predict renal biopsy conditions, which are valuable to assess lupus nephritis severity.

Identification of ethyl pyruvate as a NLRP3 inflammasome inhibitor that preserves mitochondrial integrity.

BACKGROUND: The NLRP3 inflammasome, a cytosolic complex that mediates the maturation of IL-1ß and IL-18 as well as the release of high mobility group box 1 (HMGB1), contributes to the lethality of endotoxic shock. Ethyl pyruvate (EP) was previously shown to inhibit HMGB1 release and promote survival during endotoxemia and experimental sepsis. However, the underlying protective mechanism remains elusive. RESULT: EP dose-dependently inhibited the ATP-, nigericin-, alum-, and silica-induced caspase-1 activation and HMGB1 release in mouse macrophages. EP failed to inhibit DNA transfection- or Salmonella Typhimurium-induced caspase-1 activation and HMGB1 release. Mechanistically, EP significantly attenuated mitochondrial damage and cytoplasmic translocation of mitochondrial DNA, a known NLRP3 ligand, without influencing the potassium efflux, the lysosomal rupture or the production of mitochondrial reactive oxygen species (mtROS). CONCLUSION: Ethyl pyruvate acts as a novel NLRP3 inflammasome inhibitor that preserves the integrity of mitochondria during inflammation.

Granzyme B-mediated damage of CD8+ T cells impairs graft-versus-tumor effect.

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and other malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the treatment. Using multiple tumor models and diverse donor-host combinations, we have studied the role of granzyme B (GzmB) in GVT effect. We first confirmed previous findings that GzmB deficiency diminished the ability of a high dose of CD8(+) T cells to cause lethal graft-versus-host disease. However, when GVT studies were performed using a moderate cell dose that the hosts could tolerate, GzmB(-/-) CD8(+) T cells demonstrated a significantly enhanced GVT effect. GzmB-mediated, activation-induced cell death in wild-type CD8(+) T cells was found responsible for their reduced GVT activity. Conversely, GzmB(-/-) CD8(+) T cells exhibited enhanced expansion, skewed toward an effector or effector memory phenotype, and produced higher amounts of IFN-γ and Fas ligand that might contribute to GzmB-independent tumor control. These findings demonstrate for the first time, to our knowledge, that GzmB-mediated damage of CD8(+) T cells impairs the desired GVT effect. This study suggests that inhibiting donor-derived GzmB function may represent a promising strategy to improve GVT effect without exacerbating graft-versus-host disease.

[Advances in post-translational modifications of cGAS in innate immunity].

As a member of the nucleotidyltransferase family, cyclic guanosine monophosphate-adenosine monophosphate synthase (cyclic GMP-AMP synthase, or cGAS) is primarily involved in innate immunity as a nucleic acids sensor that activates its downstream pathway and regulates type I interferon synthesis. The regulation of cGAS function is correlated with the bacterial and viral infections, autoimmune diseases, tumors, and other diseases. Besides, post-translational modification is one of the most in-depth and extensive ways of cGAS function adjustment. There are mainly six types of post-translational modifications (PTMs) of cGAS, including phosphorylation, acetylation, ubiquitination, sumoylation, peptide chain cleavage, and glutamylation. This article not only systematically summarizes how PTMs of cGAS regulate the functions of cGAS under different physiological and pathological conditions, but also probes deep into the potential of PTMs as therapeutic targets.

Effect of tonsillar mononuclear cell supernatants in patients with IgA nephropathy on renal tubular epithelial cells.

OBJECTIVE: This study aimed to investigate the immunological relationship and the mechanism between tonsillar inflammation and immunoglobulin A nephropathy (IgAN). SUBJECTS: Tonsillar mononuclear cells (TMCs) were prepared from 13 patients with IgAN and 13 patients with chronic tonsillitis but without renal disease. The human renal tubular epithelial cell (TEC) line, HK-2, was used to test the effects of secretions from the TMCs. METHODS: Phytohemagglutinin (PHA) was used to induce the inflammatory responses in TMCs. The secretions from TMCs, stimulated with or without PHA, were collected and applied to HK-2 cells. The proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of HK-2 cells were evaluated. The expression of key components during apoptosis and EMT was measured. RESULTS: The secretions from PHA-stimulated IgAN TMCs significantly inhibited proliferation, promoted apoptosis, and down-regulated Bcl-2 in HK-2 cells (P < 0.05) in time- and concentration-dependent manners. They also modulated the expression of key components during EMT, E-cadherin and α-SMA (P < 0.05). CONCLUSIONS: The secretions from PHA-stimulated IgAN TMCs can cause the inhibition of proliferation, promotion of apoptosis, down-regulation of Bcl-2, and EMT effects in HK-2 TECs, which may reflect the in-vivo remote modulation of functions of renal TECs by tonsillar inflammation.

Immune landscape and the key role of APOE+ monocytes of lupus nephritis under the single-cell and spatial transcriptional vista.

BACKGROUND: Lupus nephritis (LN) is among the most common complication of systemic lupus erythematosus (SLE) with high mortality and morbidity. The analysis of LN kidney's local immune response through single-cell and spatial transcriptome enables the study of potential therapeutic targets. METHODS: By single cell sequencing and spatial transcriptome, we profile cells from LN kidney and normal kidney tissues to characterize cellular composition and elucidate the potential upstream monocyte/macrophage (Mono/MΦ) initiating the auto-immune response. After the high-throughput synergy screening, we performed the immunofluorescence to identify the specific cells in LN patients. The function experiments were finished by flow cytometry and Elisa. RESULTS: By immunofluorescence and spatial transcriptome, we identified differential subsets of Mono/MΦ and demonstrated that they exhibit temporal expression of TIMP1, IL1B, SPP1 and APOE. With the function experiments, we found that the APOE+ Mono may be compensatorily increased in LN, and the capacity of antigen presenting was decreased with the overexpression of APOE. Furthermore, how do the LN-specific Mono/MΦ transport in and out the glomerulus to active the local immune response remains unclear. Our results showed that lymphangiogenesis occurred in LN kidneys but not in normal kidneys, suggesting the presence of a new lymphatic vessel may serve as a 'green channel' for LN-specific Mono/MΦ. CONCLUSIONS: In LN, APOE+ Mono are compensatorily elevated, with decreased antigen presenting ability and reduced secretion of interferons. The lymphangiogenesis in LN prompts the trafficking of Mono/MΦ in LN kidney.

Achieving physical examination competence through optimizing hands-on practice cycles: a prospective cohort comparative study of medical students.

BACKGROUND: Deliberate practice (DP) was proposed for effective clinical skill training, which highlights focused, repetitive practice and feedback as the key points for practice. Although previous studies have investigated the effect of feedback in DP, little is known about the proper repetitive cycles of clinical skills training especially in physical examination (PE) training. METHODS: We drew learning curves and designed a comparative study to find out the optimal number of hands-on practice cycles, an important aspect of DP, in abdominal PE training for medical students. A comparative study was conducted to validate the optimal number of hands-on practice by dividing students into two cohorts including Cohort A (high-frequency hand-on training) and B (low-frequency hand-on training). RESULTS: The learning curve study of 16 students exhibited a threshold of four repetitive practices when 81.25% students reached the competence score. A total of 74 students' final exam scores were collected for analysis. Students in Cohort A (4-5 PEs) scored significantly higher than those in Cohort B (≤3 PEs) (84.41 ± 11.78 vs 76.83 ± 17.51] in the final exam (P = 0.030)). CONCLUSION: High-frequency practice can improve students' competence of abdominal PE skill. We recommend four cycles of hands-on practice for each student in a training course like PE training.

A preliminary study of KAT2A on cGAS-related immunity in inflammation amplification of systemic lupus erythematosus.

Previous studies demonstrated that cGAS pathway is related to the inflammation amplification in a variety of autoimmune diseases. Lysine acetyltransferase family (KATs) can regulate the nuclear transcription or cytoplasmic activation of cGAS through different mechanisms. However, its role and related immunity patterns in systemic lupus erythematosus (SLE) have not been explored. In this study, RNA-seq and scRNA-seq profiling were performed for peripheral blood mononuclear cells (PBMCs) from patients with SLE. R packages were used for bioinformatic analysis. Cell culture, RT-PCR, western blotting, immunofluorescence, immunohistochemistry, and ELISA were used to explore gene expression in vitro or clinical specimens. Plasmid transfection and mass spectrometry were used to detect protein modifications. Eight acetyltransferase and deacetylase family members with significantly differential expression in SLE were found. Among them, KAT2A was abnormally upregulated and positively correlated with disease activity index. Further, KAT2A-cGAS pathway was aberrantly expressed in specific immune cell subsets in SLE. In vitro studies showed KAT2A modulated cGAS through increasing expression and post-translational modification. Our research provides novel insights for accurately positioning specific immune-cell subgroups in which KAT2A-cGAS reaction mainly works and KAT2A regulation patterns.

In lupus nephritis, how do extracellular DNAs trigger type I interferon secretion: Under the assistance of HMGB1-cGAS?

Systemic lupus erythematosus (SLE) is an autoimmune disease with multiple organs involved. Kidney damage is common among SLE patients. In lupus nephritis, extracellular DNA accumulation from necrosis cells and activated cells is perceived as initial step of inflammation. The up-regulated type I IFN is one pivotal cytokine causing downstream inflammation enlargement. Currently, intracellular DNA sensor cGAS signaling has been found to be related to lupus nephritis and the aberrant up-regulation of type I IFN. However, how extracellular accumulated DNA activates intracellular cGAS is still unknown. It was reported that nuclear protein HMGB1 takes part in multiple autoimmune diseases and inflammation induction. When HMGB1 is secreted to extracellular environment under certain conditions, it combines with DNA and triggers IFN-I secretion. It has been reported that HMGB1 level in renal tissue and cGAS level in peripheral blood mononuclear cells were both significantly up-regulated in SLE patients. Hence, we present a hypothesis that in lupus nephritis, the released HMGB1 helps extracellular accumulated DNA endocytosis and cGAS signaling pathway activation, followed by IFN-I secretion. We infer this is one pivotal pro-inflammation pathway in lupus nephritis progression.

Lupus nephritis pathology prediction with clinical indices.

Effective treatment of lupus nephritis and assessment of patient prognosis depend on accurate pathological classification and careful use of acute and chronic pathological indices. Renal biopsy can provide most reliable predicting power. However, clinicians still need auxiliary tools under certain circumstances. Comprehensive statistical analysis of clinical indices may be an effective support and supplementation for biopsy. In this study, 173 patients with lupus nephritis were classified based on histology and scored on acute and chronic indices. These results were compared against machine learning predictions involving multilinear regression and random forest analysis. For three class random forest analysis, total classification accuracy was 51.3% (class II 53.7%, class III&IV 56.2%, class V 40.1%). For two class random forest analysis, class II accuracy reached 56.2%; class III&IV 63.7%; class V 61%. Additionally, machine learning selected out corresponding important variables for each class prediction. Multiple linear regression predicted the index of chronic pathology (CI) (Q2 = 0.746, R2 = 0.771) and the acute index (AI) (Q2 = 0.516, R2 = 0.576), and each variable's importance was calculated in AI and CI models. Evaluation of lupus nephritis by machine learning showed potential for assessment of lupus nephritis.

Association of mtDNA M/N haplogroups with systemic lupus erythematosus: a case-control study of Han Chinese women.

To investigate whether mitochondrial DNA haplogroups M or N are related to occurrence or manifestations of systemic lupus erythematosus (SLE), we collected M/N haplogrouping and clinical characteristics from 868 Han Chinese women with SLE, as well as for 870 age-matched healthy Han Chinese control women. M/N haplogroups were determined in all subjects using allele-specific amplification. The frequency of M haplogroup in all patients was 429 (49.4%) and the frequency of N haplogroup, 439 (50.6%). The corresponding frequencies in controls were 456 (52.4%) and 414 (47.6%) (P = 0.213). Among women older than 50 years at onset age, the N haplogroup was significantly higher in patients than in healthy controls (59.6% vs 41.7%, P = 0.042). The N haplogroup was associated with significantly higher risk for certain SLE characteristics: hematological system damage (OR 2.128, 95%CI 1.610 to 2.813), skin impairment (OR 1.873, 95%CI 1.428 to 2.457), neurological disturbance (OR 3.956, 95%CI 1.874 to 8.352) and alopecia (OR 1.322, 95%CI 1.007 to 1.737). Our results suggest that in Han Chinese women, the mtDNA N haplogroup is associated with higher risk of late-onset SLE, skin impairment, neurological disturbance, hematological system damage and alopecia.

TGF-ß1 induces the dissolution of tight junctions in human renal proximal tubular cells: role of the RhoA/ROCK signaling pathway.

The RhoA/ROCK signaling pathway plays a significant role in transforming growth factor (TGF)-ß1-mediated epithelial-mesenchymal transition (EMT). It remains unclear, however, whether the RhoA/ROCK signaling pathway mediates TGF-ß1-induced EMT by promoting the dissolution of tight junctions (TJs) in renal proximal tubular epithelial cells. In this study, we aimed to investigate the association between TGF-ß1-mediated Rho/ROCK signaling and TJs in a cell line derived from human renal proximal tubular cells (HK-2 cells). HK-2 cells were treated with 5 ng/ml TGF-ß1 for 0, 12, 24 and 48 h. Zona occludens protein 1 (also known as tight junction protein 1; ZO-1) and occludin mRNA and protein levels were determined by real-time PCR and western blot analysis, respectively. The HK-2 cells were then divided into three groups: a control group (serum-free culture medium for 24 h); a TGF-ß1 group (treated with 5 ng/ml TGF-ß1 for 24 h); and a TGF-ß1 + Y-27632 (a specific ROCK inhibitor) group (pre-treated with 10 µM Y-27632 for 2 h and subsequently treated with 5 ng/ml TGF-ß1 for 24 h). The levels of ZO-1 and occludin were detected by real-time PCR, western blot analysis and immunofluorescence. As shown by our results, the mRNA and protein levels of ZO-1 and occludin were decreased in the HK-2 cells following treatment with TGF-ß1 in a time-dependent manner; in addition, ZO-1 and occludin levels in the TGF-ß1 + Y-27632 group were significantly increased compared with those of the TGF-ß1 group (P<0.05), with no significant changes compared with the control group. Our results indicate that the Rho/ROCK signaling pathway mediated by TGF-ß1 plays a role in the dissolution of TJs during EMT.