Tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways.

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Multiscale topology classifies cells in subcellular spatial transcriptomics.

Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3-6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7-9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.

The Lupus Susceptibility Locus Sgp3 Encodes the Suppressor of Endogenous Retrovirus Expression SNERV.

Elevated endogenous retrovirus (ERV) transcription and anti-ERV antibody reactivity are implicated in lupus pathogenesis. Overproduction of non-ecotropic ERV (NEERV) envelope glycoprotein gp70 and resultant nephritis occur in lupus-prone mice, but whether NEERV mis-expression contributes to lupus etiology is unclear. Here we identified suppressor of NEERV (Snerv) 1 and 2, Krüppel-associated box zinc-finger proteins (KRAB-ZFPs) that repressed NEERV by binding the NEERV long terminal repeat to recruit the transcriptional regulator KAP1. Germline Snerv1/Snerv2 deletion increased activating chromatin modifications, transcription, and gp70 expression from NEERV loci. F1 crosses of lupus-prone New Zealand Black (NZB) and 129 mice to Snerv1/Snerv2-/- mice failed to restore NEERV repression, demonstrating that loss of SNERV underlies the lupus autoantigen gp70 overproduction that promotes nephritis in susceptible mice and that SNERV encodes for Sgp3 (in NZB mice) and Gv-1 loci (in 129 mice). Increased ERV expression in lupus patients inversely correlated with three putative ERV-suppressing KRAB-ZFPs, suggesting that loss of KRAB-ZFP-mediated ERV control may contribute to human lupus pathogenesis.

C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.

Regulation of macrophage polarization by targeted metabolic reprogramming for the treatment of lupus nephritis.

Lupus nephritis (LN) is a severe and common manifestation of systemic lupus erythematosus (SLE) that is frequently identified with a poor prognosis. Macrophages play an important role in its pathogenesis. Different macrophage subtypes have different effects on lupus-affected kidneys. Based on their origin, macrophages can be divided into monocyte-derived macrophages (MoMacs) and tissue-resident macrophages (TrMacs). During nephritis, TrMacs develop a hybrid pro-inflammatory and anti-inflammatory functional phenotype, as they do not secrete arginase or nitric oxide (NO) when stimulated by cytokines. The infiltration of these mixed-phenotype macrophages is related to the continuous damage caused by immune complexes and exposure to circulating inflammatory mediators, which is an indication of the failure to resolve inflammation. On the other hand, MoMacs differentiate into M1 or M2 cells under cytokine stimulation. M1 macrophages are pro-inflammatory and secrete pro-inflammatory cytokines, while the M2 main phenotype is essentially anti-inflammatory and promotes tissue repair. Conversely, MoMacs undergo differentiation into M1 or M2 cells in response to cytokine stimulation. M1 macrophages are considered pro-inflammatory cells and secrete pro-inflammatory mediators, whereas the M2 main phenotype is primarily anti-inflammatory and promotes tissue repair. Moreover, based on cytokine expression, M2 macrophages can be further divided into M2a, M2b, and M2c phenotypes. M2a and M2c have anti-inflammatory effects and participate in tissue repair, while M2b cells have immunoregulatory and pro-inflammatory properties. Further, memory macrophages also have a role in the advancement of LN. Studies have demonstrated that the polarization of macrophages is controlled by multiple metabolic pathways, such as glycolysis, the pentose phosphate pathway, fatty acid oxidation, sphingolipid metabolism, the tricarboxylic acid cycle, and arginine metabolism. The changes in these metabolic pathways can be regulated by substances such as fish oil, polyenylphosphatidylcholine, taurine, fumaric acid, metformin, and salbutamol, which inhibit M1 polarization of macrophages and promote M2 polarization, thereby alleviating LN.

Moesin deficiency leads to lupus-like nephritis with accumulation of CXCL13-producing patrolling monocytes.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins to the cortical cytoskeleton and thus regulate diverse cellular processes. Mutations in the human moesin gene cause a primary immunodeficiency called X-linked moesin-associated immunodeficiency (X-MAID), which may be complicated by an autoimmune phenotype with kidney involvement. We previously reported that moesin-deficient mice exhibit lymphopenia similar to that of X-MAID and develop a lupus-like autoimmune phenotype with age. However, the mechanism through which moesin defects cause kidney pathology remains obscure. Here, we characterized immune cell infiltration and chemokine expression in the kidney of moesin-deficient mice. We found accumulation of CD4+ T and CD11b+ myeloid cells and high expression of CXCL13, whose upregulation was detected before the onset of overt nephritis. CD4+ T cell population contained IFN-γ-producing effectors and expressed the CXCL13 receptor CXCR5. Among myeloid cells, Ly6Clo patrolling monocytes and MHCIIlo macrophages markedly accumulated in moesin-deficient kidneys and expressed high CXCL13 levels, implicating the CXCL13-CXCR5 axis in nephritis development. Functionally, Ly6Clo monocytes from moesin-deficient mice showed reduced migration toward sphingosine 1-phosphate. These findings suggest that moesin plays a role in regulating patrolling monocyte homeostasis, and that its defects lead to nephritis associated with accumulation of CXCL13-producing monocytes and macrophages.

Gasdermin D deficiency aborts myeloid calcium influx to drive granulopoiesis in lupus nephritis.

Gasdermin D (GSDMD) is emerging as an important player in autoimmune diseases, but its exact role in lupus nephritis (LN) remains controversial. Here, we identified markedly elevated GSDMD in human and mouse LN kidneys, predominantly in CD11b+ myeloid cells. Global or myeloid-conditional deletion of GSDMD was shown to exacerbate systemic autoimmunity and renal injury in lupus mice with both chronic graft-versus-host (cGVH) disease and nephrotoxic serum (NTS) nephritis. Interestingly, RNA sequencing and flow cytometry revealed that myeloid GSDMD deficiency enhanced granulopoiesis at the hematopoietic sites in LN mice, exhibiting remarkable enrichment of neutrophil-related genes, significant increases in total and immature neutrophils as well as granulocyte/macrophage progenitors (GMPs). GSDMD-deficient GMPs and all-trans-retinoic acid (ATRA)-stimulated human promyelocytes NB4 were further demonstrated to possess enhanced clonogenic and differentiation abilities compared with controls. Mechanistically, GSDMD knockdown promoted self-renewal and granulocyte differentiation by restricting calcium influx, contributing to granulopoiesis. Functionally, GSDMD deficiency led to increased pathogenic neutrophil extracellular traps (NETs) in lupus peripheral blood and bone marrow-derived neutrophils. Taken together, our data establish that GSDMD deletion accelerates LN development by promoting granulopoiesis in a calcium influx-regulated manner, unraveling its unrecognized critical role in LN pathogenesis.

Upregulation of PD-1 and its ligands and expansion of T peripheral helper cells in the nephritic kidneys of lupus-prone BXSB-Yaa mice.

OBJECTIVE: This study aimed to investigate the role of the programmed cell death protein 1 (PD-1) pathway and T peripheral helper (Tph) cells in the pathogenesis of lupus nephritis using lupus-prone BXSB-Yaa mice. METHODS: Male BXSB-Yaa mice and age-matched male C57BL/6 mice were used. The expression of PD-1 and its ligands (programmed cell death 1 ligand-1, PD-L1 and programmed cell death 1 ligand-2, PD-L2) and the phenotypes of kidney-derived cells and splenocytes expressing these molecules were analyzed by immunofluorescence and flow cytometry. RESULTS: Nephritis spontaneously developed in 16-week-old but not in 8-week-old BXSB-Yaa or C57BL/6 mice. PD-1 was expressed on CD4+ mononuclear cells (MNCs) that infiltrated the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of CD4+PD-1+CXCR5-ICOS+ kidney-derived Tph cells was higher in 16-week-old than in 8-week-old BXSB-Yaa and C57BL/6 mice, whereas the frequency of CD4+PD-1+CXCR5+ICOS+ kidney-derived T follicular helper (Tfh) cells was not significantly different between the mice. PD-L1 was constitutively expressed in the renal tubules. PD-L2 was expressed in the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of PD-L1highCD11c+CD3-CD19- and PD-L2+CD11c+CD3-CD19- kidney-derived MNCs in 16-week-old BXSB-Yaa mice was significantly higher than that of the control mice. The percentage of kidney-derived Tph cells but not Tfh cells was correlated with the urinary protein levels in the nephritic mice. CONCLUSION: The results of this study suggest that kidney-infiltrating PD-1+ Tph cells expanded concomitantly with the upregulation of PD-L1 and PD-L2 in the kidneys and the progression of lupus nephritis.

Restoration of Follicular T Regulatory/Helper Cell Balance by OX40L-JAG1 Cotreatment Suppresses Lupus Nephritis in NZBWF1/j Mice.

Class-switched antinuclear autoantibodies produced by T follicular helper (TFH) cell-dependent germinal center (GC) B cell response play an essential pathogenic role in lupus nephritis (LN). The role of T follicular regulatory (TFR) cells, an effector subset of CD4+Foxp3+ T regulatory cells (Tregs), which are specialized in suppressing TFH-GC response and Ab production, remains elusive in LN. Contrasting reports have shown increased/reduced circulating TFR cells in human lupus that might not accurately reflect their presence in the GCs of relevant lymphoid organs. In this study, we report a progressive reduction in TFR cells and decreased TFR/TFH ratio despite increased Tregs in the renal lymph nodes of NZBWF1/j mice, which correlated with increased GC-B cells and proteinuria onset. Cotreatment with soluble OX40L and Jagged-1 (JAG1) proteins increased Tregs, TFR cells, and TFR/TFH ratio, with a concomitant reduction in TFH cells, GC B cells, and anti-dsDNA IgG Ab levels, and suppressed LN onset. Mechanistic studies showed attenuated TFH functions and diminished GC events such as somatic hypermutation and isotype class-switching in OX40L-JAG1-treated mice. RNA sequencing studies revealed inhibition of hypoxia-inducible factor 1-α (HIF-1a) and STAT3 signaling in T conventional cells from OX40L-JAG1-treated mice, which are critical for the glycolytic flux and differentiation into TFH cell lineage. Therefore, the increased TFR/TFH ratio seen in OX40L-JAG1-treated mice could involve both impaired differentiation of TFH cells from T conventional cells and expansion of TFR cells. We show a key role for GC-TFR/TFH imbalance in LN pathogenesis and how restoring homeostatic balance can suppress LN.

STAT3 inhibition ameliorates renal interstitial inflammation in MRL/lpr mice with diffuse proliferative lupus nephritis.

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) is one of the most common and severe clinical syndromes of diffuse proliferative lupus nephritis (DPLN), of which poor prognosis is indicated by aggravated renal function deterioration. However, the specific therapy and mechanisms of AKI in DPLN remain to be explored. METHODS: The correlation between AKI and clinical pathological changes in DPLN patients was analyzed. Expression of STAT3 signaling was detected in MRL/lpr mice with DPLN using immunohistochemical staining and immunoblotting. Inhibition of STAT3 activation by combination therapy was assessed in MRL/lpr mice. RESULTS: Correlation analysis revealed only the interstitial leukocytes were significantly related to AKI in endocapillary DPLN patients. MRL/lpr mice treated with vehicle, which can recapitulate renal damages of DPLN patients, showed upregulation of STAT3, pSTAT3 and caspase-1 in renal cortex. FLLL32 combined with methylprednisolone therapy significantly inhibited the STAT3 activation, improved acute kidney damage, reduced the interstitial infiltration of inflammatory cells and decreased the AKI incidence in MRL/lpr mice. CONCLUSION: STAT3 activation may play an important role in the pathogenesis of DPLN and the development of AKI. Hence, STAT3 inhibition based on the combination of FLLL32 with methylprednisolone may represent a new strategy for treatment of DPLN with AKI.

The Expression of Toll-like Receptors (TLR7 and TLR9) in Class III and Class IV of Recently Diagnosed Lupus Nephritis with 12-Month Follow-Up.

Renal involvement is an important cause of morbidity and mortality in systemic lupus erythematosus (SLE). The present study included patients with recently diagnosed Class III and Class IV lupus nephritis (LN) treated by Rheumatology who, upon the detection of alterations in their kidney function, were referred to Nephrology for the joint management of both medical specialties. The purpose of this study was to compare the plasma expression of Toll-Like Receptor 7 (TLR7) and TLR9 in healthy control (HC) subjects and newly diagnosed Class III and Class IV LN patients with 12-month follow-ups. The plasma expression of TLR7 and TLR9 proteins was determined by the ELISA method. A significant increase in the expression of TLR7 protein was found in Class III LN in the basal determination compared to the expression in the HC (p = 0.002) and at 12 months of follow-up (p = 0.03) vs. HC. The expression of TLR9 showed a behavior opposite to that of TLR7. TLR9 showed decreased protein expression in LN Class III patients' baseline and final measurements. The result was similar in the basal and final determinations of LN Class IV compared to the expression in HC. A significant decrease in SLEDAI -2K was observed at 12 months of follow-up in patients in Class III (p = 0.01) and Class IV (p = 0.0001) of LN. Complement C3 levels improved significantly at 12-month follow-up in Class IV patients (p = 0.0001). Complement C4 levels decreased significantly at 12-month follow-up in LN Class III compared to baseline (p = 0.01). Anti-DNA antibodies decreased significantly at 12 months of follow-up in Class IV LN (p = 0.01). A significant increase in proteinuria was found at 12 months of follow-up in Class III LN, compared to the baseline determination (p = 0.02). In LN Class IV, proteinuria decreased at 12 months of follow-up compared to baseline (p = 0.0001). Albuminuria decreased at 12 months of follow-up in LN Class IV (p = 0.006). Class IV LN, albuminuria also decreased at 12 months of follow-up (p = 0.009). Hematuria persisted in all patients and the glomerular filtration rate did not change. Three Class IV patients died before 12 months of follow-up from various causes. In conclusion, although the rheumatologic data appeared to improve, the renal function data remained inconsistent. Decreased expression of TLR9 and increased expression of TLR7 could be useful in the early diagnosis of Class III and Class IV LN is correct.

Mitochondrial Dysfunction in Systemic Lupus Erythematosus with a Focus on Lupus Nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting mostly women of child-bearing age. Immune dysfunction in SLE results from disrupted apoptosis which lead to an unregulated interferon (IFN) stimulation and the production of autoantibodies, leading to immune complex formation, complement activation, and organ damage. Lupus nephritis (LN) is a common and severe complication of SLE, impacting approximately 30% to 40% of SLE patients. Recent studies have demonstrated an alteration in mitochondrial homeostasis in SLE patients. Mitochondrial dysfunction contributes significantly to SLE pathogenesis by enhancing type 1 IFN production through various pathways involving neutrophils, platelets, and T cells. Defective mitophagy, the process of clearing damaged mitochondria, exacerbates this cycle, leading to increased immune dysregulation. In this review, we aim to detail the physiopathological link between mitochondrial dysfunction and disease activity in SLE. Additionally, we will explore the potential role of mitochondria as biomarkers and therapeutic targets in SLE, with a specific focus on LN. In LN, mitochondrial abnormalities are observed in renal cells, correlating with disease progression and renal fibrosis. Studies exploring cell-free mitochondrial DNA as a biomarker in SLE and LN have shown promising but preliminary results, necessitating further validation and standardization. Therapeutically targeting mitochondrial dysfunction in SLE, using drugs like metformin or mTOR inhibitors, shows potential in modulating immune responses and improving clinical outcomes. The interplay between mitochondria, immune dysregulation, and renal involvement in SLE and LN underscores the need for comprehensive research and innovative therapeutic strategies. Understanding mitochondrial dynamics and their impact on immune responses offers promising avenues for developing personalized treatments and non-invasive biomarkers, ultimately improving outcomes for LN patients.

Characterizing Glomerular Barrier Dysfunction with Patient-Derived Serum in Glomerulus-on-a-Chip Models: Unveiling New Insights into Glomerulonephritis.

Glomerulonephritis (GN) is characterized by podocyte injury or glomerular filtration dysfunction, which results in proteinuria and eventual loss of kidney function. Progress in studying the mechanism of GN, and developing an effective therapy, has been limited by the absence of suitable in vitro models that can closely recapitulate human physiological responses. We developed a microfluidic glomerulus-on-a-chip device that can recapitulate the physiological environment to construct a functional filtration barrier, with which we investigated biological changes in podocytes and dynamic alterations in the permeability of the glomerular filtration barrier (GFB) on a chip. We also evaluated the potential of GN-mimicking devices as a model for predicting responses to human GN. Glomerular endothelial cells and podocytes successfully formed intact monolayers on opposite sides of the membrane in our chip device. Permselectivity analysis confirmed that the chip was constituted by a functional GFB that could accurately perform differential clearance of albumin and dextran. Reduction in cell viability resulting from damage was observed in all serum-induced GN models. The expression of podocyte-specific marker WT1 was also decreased. Albumin permeability was increased in most models of serum-induced IgA nephropathy (IgAN) and membranous nephropathy (MN). However, sera from patients with minimal change disease (MCD) or lupus nephritis (LN) did not induce a loss of permeability. This glomerulus-on-a-chip system may provide a platform of glomerular cell culture for in vitro GFB in formation of a functional three-dimensional glomerular structure. Establishing a disease model of GN on a chip could accelerate our understanding of pathophysiological mechanisms of glomerulopathy.

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.

Spatial proteomics landscape and immune signature analysis of renal sample of lupus nephritis based on laser-captured microsection.

OBJECTIVE: We aimed to reveal a spatial proteomic and immune signature of kidney function regions in lupus nephritis (LN). MATERIAL AND METHODS: The laser capture microdissection (LCM) was used to isolate the glomerulus, tubules, and interstitial of the kidney from paraffin samples. The data-independent acquisition (DIA) method was used to collect proteomics data. The bioinformatic analysis was performed. RESULTS: A total of 49,658 peptides and 4056 proteins were quantitated. Our results first showed that a high proportion of activated NK cells, naive B cells, and neutrophils in the glomerulus, activated NK cells in interstitial, and resting NK cells were accumulated in tubules in LN. The immune-related function analysis of differential expression proteins in different regions indicated that the glomerulus and interstitial were major sites of immune disturbance and regulation connected with immune response activation. Furthermore, we identified 7, 8, and 9 hub genes in LN's glomerulus, renal interstitial, and tubules. These hub genes were significantly correlated with the infiltration of immune cell subsets. We screened out ALB, CTSB, LCN2, A2M, CDC42, VIM, LTF, and CD14, which show higher performance as candidate biomarkers after correlation analysis with clinical indexes. The function within three regions of the kidney was analyzed. The differential expression proteins (DEGs) between interstitial and glomerulus were significantly enriched in the immune-related biological processes, and myeloid leukocyte-mediated immunity and cellular response to hormone stimulus. The DEGs between tubules and glomerulus were significantly enriched in cell activation and leukocyte-mediated immunity. While the DEGs between tubules and interstitial were enriched in response to lipid, antigen processing, and presentation of peptide antigen response to oxygen-containing compound, the results indicated a different function within kidney regions. CONCLUSIONS: Collectively, we revealed spatial proteomics and immune signature of LN kidney regions by combined using LCM and DIA.

The star target in SLE: IL-17.

PURPOSE: The purpose of this review is to discuss the significance of IL-17 in SLE and the potential of IL-17-targeted therapy. BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease that can affect many organs and tissues throughout the body. It is characterized by overactive B and T cells and loss of immune tolerance to autoantigens. Interleukin-17 (IL-17) is a cytokine that promotes inflammation and has been implicated in the pathogenesis of several autoimmune diseases as well as inflammatory diseases. In in vitro cellular experiments in lupus susceptible mice or SLE patients, there is substantial evidence that IL-17 is a highly promising therapeutic target. METHODS: We searched papers from PubMed database using the search terms, such as interleukin-17, systemic lupus erythematosus, treatment targets, T cells, lupus nephritis, and other relevant terms. RESULTS: We discuss in this paper the molecular mechanisms of IL-17 expression, Th17 cell proliferation, and the relationship between IL-17 and Th17. The significance of IL-17 in SLE and the potential of IL-17-targeted therapy are further discussed in detail. CONCLUSION: IL-17 has a very high potential for the development as a star target in SLE.

Assessment of microvascular involvement in lupus nephritis patients by retinal OCT-angiography and kidney biopsies.

OBJECTIVES: Ocular and renal microvascular damage in lupus nephritis (LN) share similar physiopathological pathways that have investigated using traditional fundus examination and high-resolution colour electroretinography. Optical coherence tomography angiography (OCTA) is a recent, non-invasive technique for imaging the microvasculature of retina and choroid. Aim of the study was to investigate through OCTA analysis the relationship between retinal microvascular alterations and renal functional and histologic features. METHODS: Systemic lupus erythematosus (SLE) patients with LN, SLE without renal involvement and healthy controls were recruited and accomplished an ophthalmological evaluation, including OCTA. SLE-LN patients underwent a rheumatological evaluation, including disease-related clinical and laboratory features collection and kidney biopsy examination. RESULTS: This cross-sectional study enrolled forty-six eyes of 23 LN patients, thirty-two eyes of 16 SLE patients and forty-two eyes of 21 controls. Thirteen SLE-LN patients (56.5%) displayed lupus retinopathy, 10 at moderate (77%) and 3 at severe stage (23%) by fundus oculi examination. Analysis of OCTA data showed with high/moderate accuracy a reduction of retinal capillary vessel density in both SLE and SLE-LN patients compared to controls in superficial and deep plexi. A reduction in fovea thickness and an increase in foveal avascular zone were also detected. OCTA data of LN patients correlated with LN duration, disease activity, kidney function and the presence of LN-vascular lesions at kidney biopsy. CONCLUSIONS: Our results suggest the role of OCTA in early detection of systemic vascular involvement in SLE-LN patients and related kidney functional-histological impairment.

GBP5 Inhibition Ameliorates the Progression of Lupus Nephritis by Suppressing NLRP3 Inflammasome Activation.

BACKGROUND: The inflammatory response and NLRP3 inflammasome activation are typical characteristics of lupus nephritis (LN). Guanylate-binding protein 5 (GBP5) has effects on the release of proinflammatory cytokines and the activation of NLRP3 inflammasome. However, it is largely unknown whether and how GBP5 contributes to the progression of LN. METHODS: To detect the role of GBP5 in LN, MRL/lpr mice were administrated with the lentiviral vectors that knockdown GBP5 via tail vein. Proximal tubular epithelial HK-2 cells were treated with LPS and ATP to mimic the inflammatory response of LN in vitro. RESULTS: GBP5 expression was increased in the renal cortical tissues of LN mice. The in vivo results showed that GBP5 inhibition prevented the progression of LN, as evidenced by the decreased levels of 24-hour proteinuria, blood urea nitrogen and creatinine, accompanied by the ameliorated renal pathological damages. The increased mRNA and protein levels of proinflammatory factors (IL-6, TNF-α, iNOS and COX-2) in the renal cortex of LN mice were suppressed by GBP5 knockdown. In vitro, we demonstrated that the treatment of LPS combined with ATP induced an increase in GBP5 mRNA and protein expression in HK-2 cells. Mechanically, knockdown of GBP5 inhibited the activation of NLRP3 inflammasome and the secretion of IL-1ß and IL-18 both in vivo and in vitro. CONCLUSION: Our findings reveal that GBP5 inhibition prevents the progression of LN, most likely by suppressing NLRP3 inflammasome activation. It provides a novel insight into the therapeutic interventions for LN.

Activation of BZW1 by CEBPB in macrophages promotes eIF2α phosphorylation-mediated metabolic reprogramming and endoplasmic reticulum stress in MRL/lpr lupus-prone mice.

BACKGROUND: Lupus nephritis (LN) is associated with significant mortality and morbidity, while effective therapeutics and biomarkers are limited since the pathogenesis is complex. This study investigated the roles of the CEBPB/BZW1/eIF2α axis in metabolic reprogramming and endoplasmic reticulum stress in LN. METHOD: The differentially expressed genes in LN were screened using bioinformatics tools. The expression of CEBPB in the renal tissue of patients with LN and its correlation with the levels of creatinine and urinary protein were analyzed. We used adenoviral vectors to construct LN mice with knockdown CEBPB using MRL/lpr lupus-prone mice and analyzed the physiological and autoimmune indices in mice. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and dual-luciferase reporter assays were conducted to explore the regulation of BZW1 by CEBPB, followed by glycolytic flux analysis, glucose uptake, and enzyme-linked immunosorbent assay (ELISA). Finally, the role of eIF2α phosphorylation by BZW1 in bone marrow-derived macrophages (BMDM) was explored using eIF2α phosphorylation and endoplasmic reticulum stress inhibitors. RESULTS: CEBPB was significantly increased in renal tissues of patients with LN and positively correlated with creatinine and urine protein levels in patients. Downregulation of CEBPB alleviated the autoimmune response and the development of nephritis in LN mice. Transcriptional activation of BZW1 by CEBPB-mediated glucose metabolic reprogramming in macrophages, and upregulation of BZW1 reversed the mitigating effect of CEBPB knockdown on LN. Regulation of eIF2α phosphorylation levels by BZW1 promoted endoplasmic reticulum stress-amplified inflammatory responses in BMDM. CONCLUSION: Transcriptional activation of BZW1 by CEBPB promoted phosphorylation of eIF2α to promote macrophage glycolysis and endoplasmic reticulum stress in the development of LN.