Mesenchymal stem cells-derived extracellular vesicles ameliorate lupus nephritis by regulating T and B cell responses.

BACKGROUND: Human umbilical cord mesenchymal stem cells-derived extracellular vesicles (hUCMSC-EVs) have potent immunomodulatory properties similar to parent cells. This study investigated the therapeutic effects and immunomodulatory mechanisms of hUCMSC-EVs in an experimental lupus nephritis model. METHODS: The hUCMSC-EVs were isolated by using differential ultracentrifugation. In vivo, the therapeutic effects of hUCMSC-EVs in lupus-prone MRL/lpr mice were investigated, and the mechanisms of treatment were explored according to the abnormal T and B cell responses among both the spleen and kidney. RESULTS: MRL/lpr mice treated with hUCMSC-EVs reduced proteinuria extent, serum creatinine and renal pathological damage; decreased splenic index and serum anti-dsDNA IgG level; and improved survival rate. hUCMSC-EVs lowered the percentage of T helper (Th)1 cells, double-negative T (DNT) cells, and plasma cells among splenocytes; inhibited the infiltration of Th17 cells but promoted regulatory T (Treg) cells in the kidney, followed by a reduction in pro-inflammatory cytokine levels(IFN-γ, IL-2, IL-6, IL-21, and IL-17 A). In addition, hUCMSC-EVs inhibited the activation of STAT3 and down-regulated IL-17 A protein levels in the kidney. CONCLUSION: The results of this study demonstrated that hUCMSC-EVs had therapeutic effects on experimental lupus nephritis (LN) by regulating Th1/Th17/Treg imbalance and inhibiting DNT and plasma cells. Additionally, hUCMSC-EVs inhibited Th17 cell differentiation in kidney by regulating the IL-6/STAT3/IL-17 signal pathway, which might be an important mechanism for alleviating renal injury. Taken together, we demonstrated that hUCMSC-EVs regulating T and B cell immune responses might represent a novel mechanism of hUCMSCs in treating LN, thus providing a new strategy for treating LN.

Partial long-term clinical improvement after a BCG challenge in systemic lupus erythematosus-prone mice.

Systemic Lupus Erythematosus (SLE) is an autoimmune disorder that causes a breakdown of immune tolerance. Current treatments mainly involve general immunosuppression, increasing the risk of infections. On the other hand, Bacillus Calmette-Guérin (BCG) has been investigated as a potential therapy for autoimmune diseases in recent years, prompting an ongoing investigation. This study aimed to evaluate the effect of BCG vaccination on early and late clinical presentation of SLE in a murine disease model. MRL/MPJ-Faslpr mice were immunized with BCG or treated with PBS as a control. The progress of the disease was evaluated at 27 days post-immunization (dpi) (early) and 56 dpi (late). Clinical parameters and proteinuria were monitored. Blood samples were collected for measurement of antinuclear antibodies (ANAs), anti-double-stranded DNA (anti-dsDNA), and cytokine determination was performed using ELISA. Samples collected from mice were analyzed by flow cytometry and histopathology. We observed a clinical improvement in BCG-treated mice, reduced proteinuria in the latter stages of the disease, and decreased TNF-α. However, BCG did not elicit significant changes in ANAs, anti-dsDNA, histopathological scores, or immune cell infiltration. BCG was only partially beneficial in an SLE mouse model, and further research is needed to determine whether the immunity induced by this vaccine can counteract lupus's autoimmune response.

Qinghao-Biejia Herb Pair attenuates SLE atherosclerosis by regulating macrophage polarization via ABCA1/G1-mediated cholesterol efflux.

ETHNOPHARMACOLOGICAL RELEVANCE: Qinghao-Biejia herb pair (QB) is the core herb pair of "Jieduquyuziyin prescription" and is one of the commonly used herb pairs for the clinical treatment of systemic lupus erythematosus (SLE). Previous studies have shown that QB reduces the expression of inflammatory cytokines like IL-6 and TNF-α in the serum and kidney of MRL/lpr mice. Additionally, it inhibits the expression of TLR4 and MyD88 in the kidney and aorta and reduces the deposition of renal complement C3 and aortic plaque after treatment. These findings suggest that QB has a preventive and therapeutic effect on lupus rats. AIM OF THE STUDY: This study sought to investigate the mechanisms underlying the anti-SLE combined with atherosclerosis activity of the Qinghao-Biejia herb pair. MATERIALS AND METHODS: Drug targets for QB were identified using the HERB database, while targets associated with SLE and atherosclerosis were retrieved from the GeneCards database. The intersection of these drug and disease targets was then analyzed using a protein-protein interaction (PPI) network with GO and KEGG pathway enrichment analysis. In vivo, apolipoprotein E-deficient (ApoE-/-) mice were induced to develop SLE-AS by intraperitoneal injection of pristane and continued feeding of a high-fat diet. The changes in relevant indexes were observed after 12 weeks of gavage treatment with hydroxychloroquine, QB, Q (Qinghao alone), and B (Biejia alone). Bone marrow-derived macrophages from ApoE-/- mice and Raw 264.7 macrophages were used to explore the mechanisms of QB treatment. RESULTS: The levels of inflammatory cytokines in serum and pathological liver changes in mice were improved to varying degrees in the treatment groups. Additionally, there was a reduction in aortic atheromatous plaque formation and some improvement in cholesterol efflux. Furthermore, QB suppressed the expression of inflammatory cytokines in M1 macrophages, suggesting a role in regulating macrophage polarization. CONCLUSION: QB demonstrates clear efficacy for treating SLE-AS, and its therapeutic mechanism may involve the regulation of macrophage phenotypes by promoting cholesterol efflux.

Curcumin attenuates lupus nephritis by inhibiting neutrophil migration via PI3K/AKT/NF-κB signalling pathway.

OBJECTIVE: To investigate the role of curcumin in the treatment of lupus nephritis (LN) by inhibiting the migration of neutrophils and the underlying mechanism involved. METHODS: Two lupus mouse models, MRL/lpr mice and R848-treated mice, were treated with 50 mg/kg curcumin by intraperitoneal injection. H&E and Masson staining were used to estimate histopathological changes in the kidney. Immunofluorescence was used to assess the deposition of immune complexes. The expression of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription polymerase reaction (RT-PCR), and the protein expression was detected by western blotting. RESULTS: We revealed the remarkable potential of curcumin in improving inflammatory conditions in both MRL/lpr mice and R848-induced lupus mice. Curcumin effectively decelerates the progression of inflammation and diminishes the infiltration of neutrophils and their release of pivotal inflammatory factors, thereby reducing inflammation in renal tissues. Mechanistically, curcumin significantly inhibits the expression of p-PI3K, p-AKT and p-NF-κB, which are upregulated by interleukin-8 to induce neutrophil migration and renal inflammation, thereby reducing neutrophil migration and the release of inflammatory factors. CONCLUSION: Curcumin significantly inhibits the recruitment of neutrophils and the release of proinflammatory factors in the kidney by inhibiting the PI3K/AKT/NF-κB signalling pathway, providing new therapeutic targets and medication strategies for the treatment of LN.

The PP2A inhibitor LB-100 mitigates lupus nephritis by suppressing tertiary lymphoid structure formation.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ involvement and autoantibody production. Patients with SLE face a substantial risk of developing lupus nephritis (LN), which imposes a substantial burden on both patients and their families. Protein phosphatase 2A (PP2A) is a widely distributed serine/threonine phosphatase that participates in regulating multiple signaling pathways. Inhibition of PP2A has been implicated in the treatment of various diseases. LB-100, a small molecule inhibitor of PP2A, has demonstrated anti-tumor therapeutic effects and high safety profile in preclinical experiments. However, the role of PP2A and its inhibitor has been insufficiently studied in LN. In this study, we assessed the potential effects of LB-100 in both MRL/lpr mice and R848-induced BALB/c mice. Our findings indicated that LB-100 administration led to reduced spleen enlargement, decreased deposition of immune complexes, ameliorated renal damage, and improved kidney function in both spontaneous and R848-induced lupus mouse models. Importantly, we observed the formation of tertiary lymphoid structures (TLSs) in the kidneys of two distinct lupus mouse models. The levels of signature genes of TLS were elevated in the kidneys of lupus mice, whereas LB-100 mitigated chemokine production and inhibited TLS formation. In addition, we confirmed that inhibition or knockdown of PP2A reduced the production of T cell-related chemokines by renal tubular epithelial cells (RTEC). In summary, our study highlighted the renal protective potential of the PP2A inhibitor LB-100 in two distinct lupus mouse models, suggesting its potential as a novel strategy for treating LN and other autoimmune diseases.

Intra-bone marrow mesenchymal stem cell transplantation modulates myeloid bias tendency of hematopoietic stem and progenitor cells in severe MRL/lpr lupus mice.

The hematopoietic homeostasis in the bone marrow is inextricably intertwined with the immune milieu in peripheral circulation. Researches investigating the pathogenesis of systemic lupus erythematosus (SLE) have defined considerable secretion of inflammatory mediators and activation of pro-inflammatory cells. However, the impacts of "extrinsic" factors on hematopoietic stem and progenitor cells (HSPCs) remain unclear, and it is uncertain whether treatments can help coordinate the biased differentiation. In this study, we showed differences in the proportions of common myeloid progenitors (CMP) and myeloid output in the bone marrow of premorbid and morbid MRL/lpr mice using flow cytometry. RNA-seq analysis of lineage-affiliated transcriptional factors and dysregulated genes within lin- HSPCs revealed inflammation potentiation during disease progression. Further, intra-bone marrow mesenchymal stem cells transplantation (IBM-MSCT) partially coordinated myeloid generation and counteracted lupus-associated inflammation gene alterations, compared to intravenous injection. Additionally, co-culturing with umbilical cord mesenchymal stem cells (UC-MSCs) intervened in myeloid lineage tendency, as detected by RT-qPCR of myeloid-related genes. Our research demonstrated enhanced tendency toward myeloid differentiation and highlighted the feasibility of IBM-MSCT for lineage-biased HSPCs in MRL/lpr lupus model, providing novel insight into hematopoiesis and MSC-related treatments for SLE.

Combined application of mesenchymal stem cells and different glucocorticoid dosing alleviates osteoporosis in SLE murine models.

OBJECTIVE: Bone mesenchymal stem cells (BMSCs) have been tentatively applied in the treatment of glucocorticoid-induced osteoporosis (GIOP) and systemic lupus erythematosus (SLE). However, the effects of BMSCs on osteoporosis within the context of glucocorticoid (GC) application in SLE remain unclear. Our aim was to explore the roles of BMSCs and different doses of GC interventions on osteoporosis in SLE murine models. METHODS: MRL/MpJ-Faslpr mice were divided into eight groups with BMSC treatment and different dose of GC intervention. Three-dimensional imaging analysis and hematoxylin and eosin (H&E) staining were performed to observe morphological changes. The concentrations of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in serum were measured by enzyme-linked immunosorbent assay (ELISA). The subpopulation of B cells and T cells in bone marrows and spleens were analyzed by flow cytometry. Serum cytokines and chemokines were assessed using Luminex magnetic bead technology. RESULTS: BMSCs ameliorated osteoporosis in murine SLE models by enhancing bone mass, improving bone structure, and promoting bone formation through increased bone mineral content and optimization of trabecular morphology. BMSC and GC treatments reduced the number of B cells in bone marrows, but the effect was not significant in spleens. BMSCs significantly promoted the expression of IL-10 while reducing IL-18. Moreover, BMSCs exert immunomodulatory effects by reducing Th17 expression and rectifying the Th17/Treg imbalance. CONCLUSION: BMSCs effectively alleviate osteoporosis induced by SLE itself, as well as osteoporosis resulting from SLE combined with various doses of GC therapy. The therapeutic effects of BMSCs appear to be mediated by their influence on bone marrow B cells, T cell subsets, and associated cytokines. High-dose GC treatment exerts a potent anti-inflammatory effect but may hinder the immunotherapeutic potential of BMSCs. Our research may offer valuable guidance to clinicians regarding the use of BMSC treatment in SLE and provide insights into the judicious use of GCs in clinical practice.

The interferon-rich skin environment regulates Langerhans cell ADAM17 to promote photosensitivity in lupus.

The autoimmune disease lupus erythematosus (lupus) is characterized by photosensitivity, where even ambient ultraviolet radiation (UVR) exposure can lead to development of inflammatory skin lesions. We have previously shown that Langerhans cells (LCs) limit keratinocyte apoptosis and photosensitivity via a disintegrin and metalloprotease 17 (ADAM17)-mediated release of epidermal growth factor receptor (EGFR) ligands and that LC ADAM17 sheddase activity is reduced in lupus. Here, we sought to understand how the lupus skin environment contributes to LC ADAM17 dysfunction and, in the process, differentiate between effects on LC ADAM17 sheddase function, LC ADAM17 expression, and LC numbers. We show through transcriptomic analysis a shared IFN-rich environment in non-lesional skin across human lupus and three murine models: MRL/lpr, B6.Sle1yaa, and imiquimod (IMQ) mice. IFN-I inhibits LC ADAM17 sheddase activity in murine and human LCs, and IFNAR blockade in lupus model mice restores LC ADAM17 sheddase activity, all without consistent effects on LC ADAM17 protein expression or LC numbers. Anti-IFNAR-mediated LC ADAM17 sheddase function restoration is associated with reduced photosensitive responses that are dependent on EGFR signaling and LC ADAM17. Reactive oxygen species (ROS) is a known mediator of ADAM17 activity; we show that UVR-induced LC ROS production is reduced in lupus model mice, restored by anti-IFNAR, and is cytoplasmic in origin. Our findings suggest that IFN-I promotes photosensitivity at least in part by inhibiting UVR-induced LC ADAM17 sheddase function and raise the possibility that anifrolumab ameliorates lupus skin disease in part by restoring this function. This work provides insight into IFN-I-mediated disease mechanisms, LC regulation, and a potential mechanism of action for anifrolumab in lupus.

Dexamethasone-Integrated Mesenchymal Stem Cells for Systemic Lupus Erythematosus Treatment via Multiple Immunomodulatory Mechanisms.

The therapeutic application of mesenchymal stem cells (MSCs) has good potential as a treatment strategy for systemic lupus erythematosus (SLE), but traditional MSC therapy still has limitations in effectively modulating immune cells. Herein, we present a promising strategy based on dexamethasone liposome-integrated MSCs (Dexlip-MSCs) for treating SLE via multiple immunomodulatory pathways. This therapeutic strategy prolonged the circulation time of dexamethasone liposomes in vivo, restrained CD4+T-cell proliferation, and inhibited the release of proinflammatory mediators (IFN-γ and TNF-α) by CD4+T cells. In addition, Dexlip-MSCs initiated cellular reprogramming by activating the glucocorticoid receptor (GR) signaling pathway to upregulate the expression of anti-inflammatory factors such as cysteine-rich secretory protein LCCL-containing domain 2 (CRISPLD2) and downregulate the expression of proinflammatory factors. In addition, Dexlip-MSCs synergistically increased the anti-inflammatory inhibitory effect of CD4+T cells through the release of dexamethasone liposomes or Dex-integrated MSC-derived exosomes (Dex-MSC-EXOs). Based on these synergistic biological effects, we demonstrated that Dexlip-MSCs alleviated disease progression in MRL/lpr mice more effectively than Dexlip or MSCs alone. These features indicate that our stem cell delivery strategy is a promising therapeutic approach for clinical SLE treatment.

CD4+CD57+ senescent T cells as promoters of systemic lupus erythematosus pathogenesis and the therapeutic potential of senolytic BCL-2 inhibitor.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by persistent activation of immune cells and overproduction of autoantibodies. The accumulation of senescent T and B cells has been observed in SLE and other immune-mediated diseases. However, the exact mechanistic pathways contributing to this process in SLE remain incompletely understood. In this study, we found that in SLE patients: (1) the frequency of CD4+CD57+ senescent T cells was significantly elevated and positively correlated with disease activity; (2) the expression levels of B-lymphoma-2 (BCL-2) family and interferon-induced genes (ISGs) were significantly upregulated; and (3) in vitro, the cytokine IL-15 stimulation increased the frequency of senescent CD4+ T cells and upregulated the expression of BCL-2 family and ISGs. Further, treatment with ABT-263 (a senolytic BCL-2 inhibitor) in MRL/lpr mice resulted in decreased: (1) frequency of CD4+CD44hiCD62L-PD-1+CD153+ senescent CD4+ T cells; (2) frequency of CD19+CD11c+T-bet+ age-related B cells; (3) level of serum antinuclear antibody; (4) proteinuria; (5) frequency of Tfh cells; and (6) renal histopathological abnormalities. Collectively, these results indicated a dominant role for CD4+CD57+ senescent CD4+ T cells in the pathogenesis of SLE and senolytic BCL-2 inhibitor ABT-263 may be the potential treatment in ameliorating lupus phenotypes.

IFN-α affects Th17/Treg cell balance through c-Maf and associated with the progression of EBV- SLE.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease, of which the pathogens is remains obscure. Viral infection, particularly Epstein Barr viru (EBV) infection, has been considered a common pathogenic factor. This study suggests that c-Maf may be an important target in T cell differentiation during SLE progression, providing a potentially new perspective on the role of viral infection in the pathogenesis of autoimmune diseases. METHODS: Cytokines of EBV-infected SLE patients were measured by ELISA and assessed in conjunction with their clinical data. IFN-α, c-Maf, and the differentiation of Th17/Treg cells in SLE patients and MRL/LPR mice were analyzed using FCM, WB, RT-PCR, etc. Following the infection of cells and mice with EBV or viral mimic poly (dA:dT), the changes of the aforementioned indicators were investigated. The relationship among IFN-α, STAT3, c-Maf and Th17 cells was determined by si-RNA technique. RESULTS: Many SLE patients are found to be complicated by viral infections; Further, studies have demonstrated that viral infection, especially EBV, is involved in SLE development. This study showed that viral infections might promote IFN-α secretion, inhibit c-Maf expression by activating STAT3, increase Th17 cell differentiation, and lead to the immune imbalance of Th17/Treg cells, thus playing a role in the onset and progression of SLE. CONCLUSION: This study demonstrates that EBV infections may contribute to SLE development by activating STAT3 through IFN-α, inhibiting c-Maf, and causing Th17/Treg immune imbalance. Our work provided a new insight into the pathogenesis and treatment of SLE.

Blockade of OX40/OX40L signaling using anti-OX40L alleviates murine lupus nephritis.

Genetic variants of the OX40 ligand (OX40L) locus are associated with the risk of systemic lupus erythematosus (SLE), it is unclear how the OX40L blockade delays the lupus phenotype. Therefore, we examined the effects of an anti-OX40L antibody in MRL/Lpr mice. Next, we investigated the effect of anti-OX40L on immunosuppression in keyhole limpet hemocyanin-immunized C57BL/6J mice. In vitro treatment of anti-OX40L in CD4+ T and B220+ B cells was used to explore the role of OX40L in the pathogenesis of SLE. Anti-OX40L alleviated murine lupus nephritis, accompanied by decreased production of anti-dsDNA and proteinuria, as well as lower frequencies of splenic T helper (Th) 1 and T-follicular helper cells (Tfh). In keyhole limpet hemocyanin-immunized mice, decreased levels of immunoglobulins and plasmablasts were observed in the anti-OX40L group. Anti-OX40L reduced the number and area of germinal centers. Compared with the control IgG group, anti-OX40L downregulated CD4+ T-cell differentiation into Th1 and Tfh cells and upregulated CD4+ T-cell differentiation into regulatory T cells in vitro. Furthermore, anti-OX40L inhibited toll-like receptor 7-mediated differentiation of antibody-secreting cells and antibody production through the regulation of the SPIB-BLIMP1-XBP1 axis in B cells. These results suggest that OX40L is a promising therapeutic target for SLE.

Constitutive knockout of interleukin-6 ameliorates memory deficits and entorhinal astrocytosis in the MRL/lpr mouse model of neuropsychiatric lupus.

BACKGROUND: Neuropsychiatric lupus (NPSLE) describes the cognitive, memory, and affective emotional burdens faced by many lupus patients. While NPSLE's pathogenesis has not been fully elucidated, clinical imaging studies and cerebrospinal fluid (CSF) findings, namely elevated interleukin-6 (IL-6) levels, point to ongoing neuroinflammation in affected patients. Not only linked to systemic autoimmunity, IL-6 can also activate neurotoxic glial cells the brain. A prior pre-clinical study demonstrated that IL-6 can acutely induce a loss of sucrose preference; the present study sought to assess the necessity of chronic IL-6 exposure in the NPSLE-like disease of MRL/lpr lupus mice. METHODS: We quantified 1308 proteins in individual serum or pooled CSF samples from MRL/lpr and control MRL/mpj mice using protein microarrays. Serum IL-6 levels were plotted against characteristic NPSLE neurobehavioral deficits. Next, IL-6 knockout MRL/lpr (IL-6 KO; n = 15) and IL-6 wildtype MRL/lpr mice (IL-6 WT; n = 15) underwent behavioral testing, focusing on murine correlates of learning and memory deficits, depression, and anxiety. Using qPCR, we quantified the expression of inflammatory genes in the cortex and hippocampus of MRL/lpr IL-6 KO and WT mice. Immunofluorescent staining was performed to quantify numbers of microglia (Iba1 +) and astrocytes (GFAP +) in multiple cortical regions, the hippocampus, and the amygdala. RESULTS: MRL/lpr CSF analyses revealed increases in IL-17, MCP-1, TNF-α, and IL-6 (a priori p-value < 0.1). Serum levels of IL-6 correlated with learning and memory performance (R2 = 0.58; p = 0.03), but not motivated behavior, in MRL/lpr mice. Compared to MRL/lpr IL-6 WT, IL-6 KO mice exhibited improved novelty preference on object placement (45.4% vs 60.2%, p < 0.0001) and object recognition (48.9% vs 67.9%, p = 0.002) but equivalent performance in tests for anxiety-like disease and depression-like behavior. IL-6 KO mice displayed decreased cortical expression of aif1 (microglia; p = 0.049) and gfap (astrocytes; p = 0.044). Correspondingly, IL-6 KO mice exhibited decreased density of GFAP + cells compared to IL-6 WT in the entorhinal cortex (89 vs 148 cells/mm2, p = 0.037), an area vital to memory. CONCLUSIONS: The inflammatory composition of MRL/lpr CSF resembles that of human NPSLE patients. Increased in the CNS, IL-6 is necessary to the development of learning and memory deficits in the MRL/lpr model of NPSLE. Furthermore, the stimulation of entorhinal astrocytosis appears to be a key mechanism by which IL-6 promotes these behavioral deficits.

Isolation and Single-Cell Transcriptomic Analysis of Murine Regulatory B Cells.

Regulatory B (Breg) cells have been demonstrated to play an important role in the inhibition of a wide range of immunological responses, and they are absent or malfunction in autoimmune diseases like lupus. Breg cells can control immunological responses and keep the immune system in a balanced state by releasing immunosuppressive cytokines such as transforming growth factor-beta (TGF-ß) and interleukin-10 (IL-10), which in turn promote regulatory T (Treg) cells and reduce effector T cell responses. Breg cells have also been linked to the modulation of cancer immunity. Due to their immunosuppressive role, in the context of cancer, Breg cells aid in tumor immune evasion and promote tumor progression. Nonetheless, it has been established that Breg cells are involved in both cancer immunity and autoimmunity, and their characterizations beyond surface markers, for example, on the transcriptomic level, are essential for our understanding of Breg biology in health and disease. In this chapter, using lupus-prone MRL/lpr mice, we describe a Breg cell isolation protocol for the purpose of single-cell RNA sequencing analysis.

LATS2 degradation promoted fibrosis damage and rescued by vitamin K3 in lupus nephritis.

BACKGROUND: Lupus nephritis (LN) is the most common complication of systemic lupus erythematosus (SLE). The limited treatment options for LN increase the economic burdens on patients. Because fibrotic progression leads to irreversible renal damage in LN patients and further progresses to chronic kidney disease (CKD) and the end stage of renal disease (ESRD), developing new targets to prevent LN fibrotic progression could lead to a feasible treatment strategy for LN patients. METHODS: In this study, we examined YAP activation and LATS2 downregulation in LN kidney biopsy samples (LN: n = 8, normal: n = 2) and lupus-prone MRL/lpr mice (n = 8 for each disease stage). The function of LATS2 was further investigated by in situ injection of Ad-LATS2 into mice with LN (n = 6 mice per group). We examined the role of SIAH2-LATS2 regulation by IP-MS and co-IP, and the protective effect of the SIAH2 inhibitor was investigated in mice with LN. RESULTS: Restoring LATS2 by an adenovirus in vivo alleviated renal fibrotic damage in mice with LN. Moreover, we found that LATS2 was degraded by a K48 ubiquitination-proteasome pathway mediated by SIAH2 and promoted YAP activation to worsen fibrosis progression in LN. The H150 region of the substrate binding domain (SBD) is an important site for SIAH2-LATS2 binding. The SIAH2-specific inhibitor vitamin K3 protected against LN-associated fibrotic damage in vivo. CONCLUSION: In summary, we identified the SIAH2-LATS2 axis as an attractive intervention target in LN to alter the resistance to fibrosis.

MDSC-derived S100A8/9 contributes to lupus pathogenesis by promoting TLR7-mediated activation of macrophages and dendritic cells.

Toll-like receptors (TLRs), especially TLR7, play an important role in systemic lupus erythematosus (SLE) pathogenesis. However, the regulatory mechanism underlying the abnormal activation of TLR pathways in patients with SLE has not been elucidated. Notably, accumulating evidence indicates that myeloid-derived suppressor cells (MDSCs) are important regulators of inflammation and autoimmune diseases. Compared with healthy control subjects, patients with SLE have a greater proportion of MDSCs among peripheral blood mononuclear cells (PBMCs); however, the effect of MDSCs on TLR7 pathway activation has not been determined. In the present study, lupus MDSCs significantly promoted TLR7 pathway activation in macrophages and dendritic cells (DCs), exacerbating the imiquimod-induced lupus model. RNA-sequencing analysis revealed significant overexpression of S100 calcium-binding protein A8 (S100A8) and S100A9 in MDSCs from diseased MRL/lpr mice. In vitro and in vivo studies demonstrated that S100A8/9 effectively promoted TLR7 pathway activation and that S100A8/9 deficiency reversed the promoting effect of MDSCs on TLR7 pathway activation in lupus. Mechanistically, MDSC-derived S100A8/9 upregulated interferon gamma (IFN-γ) secretion by macrophages and IFN-γ subsequently promoted TLR7 pathway activation in an autocrine manner. Taken together, these findings suggest that lupus MDSCs promote TLR7 pathway activation and lupus pathogenesis through the S100A8/9-IFN-γ axis. Our study identified an important target for SLE therapy.

Celastrol ameliorates lupus by promoting apoptosis of autoimmune T cells and preventing autoimmune response in MRL/lpr mice.

OBJECTIVE: Celastrol is a bioactive constituent extracted from Tripterygium wilfordii (thunder god vine). It has been demonstrated to have a therapeutic effect on experimental disease models for chronic inflammatory and immune disorders. In the present study, we investigated whether and how celastrol exerts a regulatory effect on the autoimmune response in MRL/lpr mice. METHODS: We performed an in vivo study to determine the therapeutic effects of celastrol in MRL/lpr mice and then further investigated the underlying mechanism of celastrol in the regulation of the autoimmune response in MRL/lpr mice. RESULTS: Celastrol showed a therapeutic effect in MRL/lpr mice by preventing the enlargement of the spleen and lymph nodes, alleviating renal injury, and reducing the levels of ANA and anti-double-stranded DNA antibodies. Furthermore, celastrol suppressed the in vivo inflammatory response in MRL/lpr mice by reducing the serum levels of multiple cytokines, including interleukin (IL)-6, tumour necrosis factor (TNF) and interferon (IFN)-γ, and the production of multiple antibody subsets, including total IgG, IgG1 and IgG2b. In vitro, celastrol reduced anti-CD3 antibody stimulation-induced T helper 1 and TNF-producing cells in CD4+ T cells of MRL/lpr mice. In addition, celastrol significantly affected B cell differentiation and prevented the generation of plasma cells from B cells in MRL/lpr mice by reducing the frequency of activated and germinal centre B cells. Celastrol treatment also affected T cell differentiation and significantly reduced central memory T cell frequencies in MRL/lpr mice. Importantly, celastrol treatment specifically promoted apoptosis of CD138+ but not CD138- T cells to suppress autoimmune T cell accumulation in MRL/lpr mice. CONCLUSIONS: Celastrol exerted therapeutic effects on lupus by specifically promoting apoptosis of autoimmune T cells and preventing the progression of autoimmune response.

Modulation of PKM2 inhibits follicular helper T cell differentiation and ameliorates inflammation in lupus-prone mice.

OBJECTIVES: Expansion of follicular helper T (Tfh) cells and abnormal glucose metabolism are present in patients with systemic lupus erythematosus (SLE). Pyruvate kinase M2 (PKM2) is one of the key glycolytic enzymes, and the underlying mechanism of PKM2-mediated Tfh cell glycolysis in SLE pathogenesis remains elusive. METHODS: We analyzed the percentage of Tfh cells and glycolysis in CD4+ T cells from SLE patients and healthy donors and performed RNA sequencing analysis of peripheral blood CD4+ T cells and differentiated Tfh cells from SLE patients. Following Tfh cell development in vitro and following treatment with PKM2 activator TEPP-46, PKM2 expression, glycolysis, and signaling pathway proteins were analyzed. Finally, diseased MRL/lpr mice were treated with TEPP-46 and assessed for treatment effects. RESULTS: We found that Tfh cell percentage and glycolysis levels were increased in SLE patients and MRL/lpr mice. TEPP-46 induced PKM2 tetramerization, thereby inhibiting Tfh cell glycolysis levels. On the one hand, TEPP-46 reduced the dimeric PKM2 entering the nucleus and reduced binding to the transcription factor BCL6. On the other hand, TEPP-46 inhibited the AKT/GSK-3ß pathway and glycolysis during Tfh cell differentiation. Finally, we confirmed that TEPP-46 effectively alleviated inflammatory damage in lupus-prone mice and reduced the expansion of Tfh cells in vivo. CONCLUSIONS: Our results demonstrate the involvement of PKM2-mediated glycolysis in Tfh cell differentiation and SLE pathogenesis, and PKM2 could be a key therapeutic target for the treatment of SLE.

Anti-DNA antibody-targeted D-peptide nanoparticles ameliorate lupus nephritis in MRL/lpr mice.

Peptide ALW (ALWPPNLHAWVP) targeting anti-dsDNA antibodies has shown promising therapeutic effects in alleviating lupus nephritis, but is potentially limited by poor stability and non-kidney targeting. We recently developed a D-form modified ALW, called D-ALW, which has the capacity to widely inhibit pathogenic polyclonal anti-dsDNA antibody reactions. Further modification of D-ALW using PEG-PLGA nanoparticles to enhance good kidney-targeting ability and extend half-life. Here, we demonstrate that the D-form modified ALW maintains higher binding and inhibition efficiencies and achieves higher stability. Most importantly, D-ALW nanoparticles exhibit excellent kidney-targeting ability and prolong the half-life of the peptides in BALB/c mice. Additionally, compared to D-ALW, D-ALW nanoparticles significantly reduce the glomerular deposition of IgG and C3, improve renal histopathologies, such as glomerular proliferation and inflammatory cells infiltration, and markedly prolong lifespan in MRL/lpr lupus-prone mice. Overall, these results establish that the D-ALW nanoparticles offer synergistic benefits in both safety and efficacy, providing long-term renal preservation and treatment advantages in lupus nephritis.

Dysregulated CXCL12 expression in osteoblasts promotes B-lymphocytes preferentially homing to the bone marrow in MRL/lpr mice.

Objective: Todetect the abnormal distribution of B-lymphocytes between peripheral and bone marrow (BM) compartments and explore the mechanism of abnormal chemotaxis of B-lymphocytes in lupus subjects. Methods: The proportions of CXC chemokine receptor (CXCR)4+ B cells and CFDA-labeled MRL/lpr-derived B cells were detected by flow cytometry. The levels of CXC chemokine ligand (CXCL)12in peripheral blood (PB)were measured by ELISA. The migrated B cells to osteoblasts (OBs) was measured by transwell migration assay. The relative spatial position of B cells, OBs and CXCL12 was presented by Immunofluorescence assay. Results: Firstly, we found that the percentage of CXCR4+ B cells was lower in PB and higher in the BM from both MRL/lpr mice and patientswith Systemic lupus erythematosus (SLE). Secondly, OBs from MRL/lpr mice produced more CXCL12 than that from C57BL/6 mice. Besides, MRL/lpr-derived OBs demonstrated more potent chemotactic ability toward B-lymphocytes than control OBs by vitro an vivo. Additionally, more B-lymphocytes were found to co-localize with OBs within the periosteal zone of bone in MRL/lpr mice. Lastly, the percentages of CXCR4+B cells were found to be negatively correlated with serum Immunoglobulin (Ig) G concentration, moreover, BM CXCL12 levels were found to be positively correlated with SLE disease activity index Score and negatively correlated with serum Complement3 (C3) concentration. Conclusions: our results indicated that there is a shifted distribution of B-lymphocytes between BM and peripheral compartments in both SLE patients and MRL/lpr mice. Besides, the up-regulated levels of CXCL12 in OBs was indicated to contribute to the enhanced chemotactic migration and anchorage of B-lymphocytes to OBs.