Animal models of studying the pathogenesis of multi-organ tissue damage in lupus.

Moderate-to-severe systemic lupus erythematosus (SLE) is characterized by extensive autoantibody deposition and persistent autoinflammation. As the existing animal models are limited in accurately reproducing the pathological characteristics of human SLE, we introduced a novel animal model simulating multi-organ autoinflammation through intra-organ injections. The model closely mimicked key features of SLE, including IgG deposition, inflammation, and tissue damage. The model could be used to assess the roles of IgG, immune cells, cytokines, and Fc gamma receptor (FcγR) in the pathogenesis of autoinflammation. The results obtained from this model could be confirmed by lupus MRL/lpr mice. The review suggested that the diagnostic criteria should be reconsidered to incorporate IgG deposition in tissues and highlighted the limitations of current T-cell and B-cell-focused treatments. To summarize, the IgG deposition model can be used to investigate the pathogenesis and treatment of multi-organ tissue damage associated with SLE.

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.

Emerging Molecular and Synaptic Targets for the Management of Chronic Pain Caused by Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (MRL/lpr lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1ß, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1ß, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1ß and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.

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.

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.

Ectopic CD4+ T cells in choroid plexus mediate neuropsychiatric lupus symptoms in mice via interferon-γ induced microglia activation.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a disabling and potentially life-threatening complication of SLE. This study aims to investigate whether ectopic CD4+ T cells in the choroid plexus mediate NPSLE in mice. Intracerebroventricular (ICV) injection of anti-CD4 antibody effectively depleted CP-resident CD4+ T cells and alleviated NPSLE-like symptoms in MRL/lpr mice. Following ICV injection, the majority of isolated lupus CD4+ T cells from donor MRL/lpr mice predominantly stayed in the CP for at least 28 days in recipient C57BL/6 mice, while nearly all isolated CD4+ T cells from MRL/MpJ mice disappeared within 7 days. ICV injection of lupus CD4+ T cells resulted in NPSLE-like symptoms, including impaired behavioral performances, increased microglial activation, and abnormal microstructure changes. Flow cytometry analysis revealed that the majority of isolated lupus CD4+ T cells were positive for IFN-γ. Neutralizing intracerebral IFN-γ alleviated NPSLE-like symptoms in MRL/lpr mice. Moreover, ICV injection of anti-IFN-γ antibody or microglial depletion by PLX3397 benefited most NPSLE-like symptoms in lupus CD4+ T-treated mice, while ICV injection of IFN-γ mimicked most NPSLE-like symptoms. In conclusion, CP-resident lupus CD4+ T cells contribute to NPSLE-like symptoms in mice via Interferon-γ induced microglia activation. Depleting CP-resident lupus CD4+ T cells, interferon-γ, or activated microglia may be potential therapeutic targets for NPSLE.

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.

Protopanaxadiol improves lupus nephritis by regulating the PTX3/MAPK/ERK1/2 pathway.

Lupus nephritis (LN) is a kidney disease that occurs after systemic lupus erythematosus (SLE) affects the kidneys. Pentraxin 3 (PTX3) is highly expressed in the serum of patients with LN. Renal PTX3 deposition is directly related to clinical symptoms such as proteinuria and inflammation. The excessive proliferation of mesangial cells (MCs) is one of the representative pathological changes in the progression of LN, which is closely related to its pathogenesis. Protopanaxadiol (PPD) is the main component of ginsenoside metabolism and has not been reported in LN. The aim of this study was to investigate the relationship between PTX3 and mesangial cell proliferation and to evaluate the potential role and mechanism of PPD in improving LN. PTX3 is highly expressed in the kidneys of LN patients and LN mice and is positively correlated with renal pathological indicators, including proteinuria and PCNA. The excessive expression of PTX3 facilitated the proliferation of MCs, facilitated the activation of the MAPK/ERK1/2 signaling pathway, and increased the expression of HIF-1α. Further studies showed that PPD can effectively inhibit the abnormal proliferation of MCs with high expression of PTX3 and significantly improve LN symptoms such as proteinuria in MRL/lpr mice. The mechanism may be related to the inhibition of the PTX3/MAPK/ERK1/2 pathway. In this study, both in vitro, in vivo, and clinical sample results show that PTX3 is involved in the regulation of MCs proliferation and the early occurrence of LN. Natural active compound PPD can improve LN by regulating the PTX3/MAPK/ERK1/2 pathway.

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.

Microglia orchestrate synaptic and neuronal stripping: Implication in neuropsychiatric lupus.

Systemic lupus erythematosus (SLE), a multifactorial autoimmune disease, can affect the brain and cause neuropsychiatric dysfunction, also named neuropsychiatric lupus (NPSLE). Microglial activation is observed in NPSLE patients. However, the mechanisms regulating microglia-mediated neurotoxicity in NPSLE remain elusive. Here, we showed that M1-like proinflammatory cytokine levels were increased in the cerebrospinal fluid (CSF) of SLE patients, especially those with neuropsychiatric symptoms. We also demonstrated that MRL/lpr lupus mice developed anxiety-like behaviours and cognitive deficits in the early and active phases of lupus, respectively. An increase in microglial number was associated with upregulation of proinflammatory cytokines in the MRL/lpr mouse brain. RNA sequencing revealed that genes associated with phagocytosis and M1 polarization were upregulated in microglia from lupus mice. Functionally, activated microglia induced synaptic stripping in vivo and promoted neuronal death in vitro. Finally, tofacitinib ameliorated neuropsychiatric disorders in MRL/lpr mice, as evidenced by reductions in microglial number and synaptic/neuronal loss and alleviation of behavioural abnormalities. Thus, our results indicated that classically activated (M1) microglia play a crucial role in NPSLE pathogenesis. Minocycline and tofacitinib were found to alleviate NPSLE by inhibiting micrglial activation, providing a promising therapeutic strategy.

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.

Treatment with lysophosphatidic acid improves glomerulonephritis through the suppression of macrophage activation in a murine model of systemic lupus erythematosus.

OBJECTIVES: Several therapeutic agents have been developed and used for the clinical treatment of systemic lupus erythematosus (SLE). In cases where SLE is accompanied by severe organ failures, such as neuropsychiatric lupus erythematosus (NPSLE) and acute onset of lupus nephritis, the use of potent immunosuppressive drugs, such as cyclophosphamide, is necessary. However, potent immunosuppressive drugs are known to increase infection risks. Thus, the development of therapeutic agents with novel mechanisms is urgently required. Previously, we reported that treatment with lysophosphatidic acid (LPA) prevents depression-like behaviours by suppressing microglial activation in MRL/lpr mice. In this study, we examined whether the treatment with LPA improves glomerulonephritis by affecting systemic immunity in MRL/lpr mice. METHODS: Eighteen-week-old MRL/lpr mice were treated with a vehicle or LPA for 3 weeks. After treatment, the glomerular inflammation and damage parameters were compared between the 2 groups. Moreover, we examined the effects of LPA on immune cells by flow cytometry using isolated splenocytes. RESULTS: LPA treatment in MRL/lpr mice significantly reduced the daily urinary albumin content and suppressed the CD68-positive cells and Periodic acid-Schiff (PAS)-positive areas in the glomeruli. The treatment also suppressed plasma anti-dsDNA antibodies and inflammatory cytokines in MRL/lpr mice. Although LPA did not significantly affect the total number of splenocytes, the treatment significantly reduced CD11b+Ly6G-Ly6C- cells (mature macrophages), as well as CD11b+Ly6G-Ly6C-CD68+ cells (activated mature macrophages). CONCLUSIONS: These results suggest that LPA may improve glomerulonephritis by suppressing macrophage activation in MRL/lpr mice.

Rutin alleviates lupus nephritis by inhibiting T cell oxidative stress through PPARγ.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by complex clinical symptoms and multi-organ damage. One of the most prevalent complications of SLE is lupus nephritis (LN). Rutin, a natural flavonoid compound found in various plants used in traditional Chinese medicine, has shown promising anti-inflammatory, antioxidant, and renal protective effects. In our study, we treated MRL/lpr mice, a model known for spontaneously developing LN, with Rutin. Our findings reveal that Rutin markedly reduced serum cytokine and autoantibody levels and decreased inflammatory cell infiltration in renal tissues, thereby ameliorating kidney pathology. In vitro experiments indicated that Rutin's therapeutic effect on LN is linked to its significant reduction of oxidative stress in T cells. Further investigations suggest that Rutin enhances oxidative stress management through the modulation of Peroxisome proliferator-activated receptor gamma (PPARγ). We observed that Rutin modulates PPARγ activity, leading to reduced transcriptional activity of NF-κB and STAT3, which in turn inhibits the secretion of inflammatory cytokines such as IL-6, TNF-α, and IL-17. In summary, Rutin can exert an antioxidant effect by regulating PPARγ and shows therapeutic action against LN.

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.

Tlr5 deficiency exacerbates lupus-like disease in the MRL/lpr mouse model.

Introduction: Leaky gut has been linked to autoimmune disorders including lupus. We previously reported upregulation of anti-flagellin antibodies in the blood of lupus patients and lupus-prone mice, which led to our hypothesis that a leaky gut drives lupus through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5). Methods: We created MRL/lpr mice with global Tlr5 deletion through CRISPR/Cas9 and investigated lupus-like disease in these mice. Result: Contrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus with Tlr5 deficiency in female MRL/lpr mice. Remarkably higher levels of proteinuria were observed in Tlr5 -/- MRL/lpr mice suggesting aggravated glomerulonephritis. Histopathological analysis confirmed this result, and Tlr5 deletion significantly increased the deposition of IgG and complement C3 in the glomeruli. In addition, Tlr5 deficiency significantly increased renal infiltration of Th17 and activated cDC1 cells. Splenomegaly and lymphadenopathy were also aggravated in Tlr5-/- MRL/lpr mice suggesting impact on lymphoproliferation. In the spleen, significant decreased frequencies of regulatory lymphocytes and increased germinal centers were observed with Tlr5 deletion. Notably, Tlr5 deficiency did not change host metabolism or the existing leaky gut; however, it significantly reshaped the fecal microbiota. Conclusion: Global deletion of Tlr5 exacerbates lupus-like disease in MRL/lpr mice. Future studies will elucidate the underlying mechanisms by which Tlr5 deficiency modulates host-microbiota interactions to exacerbate lupus.

Proteomics-Based Identification of Potential Therapeutic Targets of Artesunate in a Lupus Nephritis MRL/lpr Mouse Model.

This study aimed to identify potential therapeutic targets of artesunate in an MRL/lpr lupus nephritis mouse model by quantitative proteomics. We detected serum autoimmune markers and proteinuria in 40 female mice that were divided into 4 groups (n = 10): normal C57BL/6 control group; untreated MRL/lpr lupus; 9 mg/kg/day prednisone positive control MRL/lpr lupus; and 15 mg/kg/day artesunate-treated MRL/lpr lupus groups. Renal pathology in the untreated MRL/lpr lupus and artesunate groups was examined by Periodic acid-Schiff (PAS) staining. Artesunate treatment in lupus mice decreased serum autoantibody levels and proteinuria while alleviating lupus nephritis pathology. Through tandem mass tag-tandem mass spectrometry (TMT-MS/MS) analyses, differentially expressed proteins were identified in the artesunate group, and subsequent functional prediction suggested associations with antigen presentation, apoptosis, and immune regulation. Data are available via ProteomeXchange with the identifier PXD046815. Parallel reaction monitoring (PRM) analysis of the top 19 selected proteins confirmed the TMT-MS/MS results. Immunohistochemistry, immunofluorescence, and Western blotting of an enriched protein from PRM analysis, cathepsin S, linked to antigen presentation, highlighted its upregulation in the untreated MRL/lpr lupus group and downregulation following artesunate treatment. This study suggests that artesunate holds potential as a therapeutic agent for lupus nephritis, with cathepsin S identified as a potential target.

Drug upgrade: A complete methodology from old drug to new chemical entities using Nematic Protein Organization Technique.

Drug repurposing is used to propose new therapeutic perspectives. Here, we introduce "Drug Upgrade", that is, characterizing the mode of action of an old drug to generate new chemical entities and new therapeutics. We proposed a novel methodology covering target identification to pharmacology validation. As an old drug, we chose hydroxychloroquine (HCQ) for its well-documented clinical efficacy in lupus and its side effect, retinal toxicity. Using the Nematic Protein Organization Technique (NPOT®) followed by liquid chromatography-tandem mass spectrometry analyses, we identified myeloperoxidase (MPO) and alpha-crystallin ß chain (CRYAB) as primary and secondary targets to HCQ from lupus patients' peripheral blood mononuclear cells (PBMCs) and isolated human retinas. Surface plasmon resonance (SPR) and enzymatic assays confirmed the interaction of HCQ with MPO and CRYAB. We synthesized INS-072 a novel analog of HCQ that increased affinity for MPO and decreased binding to CRYAB compared to HCQ. INS-072 delayed cutaneous eruption significantly compared to HCQ in the murine MRL/lpr model of spontaneous lupus and prevents immune complex vasculitis in mice. In addition, long-term HCQ treatment caused retinal toxicity in mice, unlike INS-072. Our study illustrates a method of drug development, where new applications or improvements can be explored by fully characterizing the drug's mode of action.

Effects of autoimmune abnormalities on fertility and placental morphology in mice.

Autoimmune diseases (AIDs) alter the placental immune environment leading to fetal loss. This study investigated the effects of AIDs on pregnancy and the placenta in AID-prone MRL/MpJ-Faslpr/lpr mice and wild-type MRL/MpJ, which were mated with male MRL/MpJ and MRL/MpJ-Faslpr/lpr at five months and defined as moLpr and moMpJ, respectively. AID indices (spleen weight and serum autoantibody levels) and fertility status (number and size of fetuses, morphology, and comprehensive gene expression of placentas) were evaluated on gestational day 15.5. Both strains showed equivalent fertility, but moLpr showed lighter placentas and fetuses than moMpJ, and decreased fertility with AID severity. moLpr placentas had a higher number of T cells, higher expression of genes associated with T helper 2 and T follicular helper functions, and altered expression of genes (Krt15, Slc7a3, Sprr2a3) that significantly regulate pregnancy or immunity. The gene expression of T cell migration-associated chemokines (Ccl5, Cxcl9) was significantly increased in moLpr placentas, and CCL5 and CXCL9 were detected in moLpr placentas, particularly in T cells and placenta-component cells, respectively. Thus, AID altered placental morphofunction and fertility in mice; however, fertility was maintained at the examined time points. This study enhances our understanding of placental alterations and gestational risk due to AIDs.