Cordyceps protein alleviates renal injury by inhibiting T cell infiltration and Th1 cell differentiation in lupus nephritis mice.

BACKGROUND: T cell infiltration and differentiation play a central part in the development of lupus nephritis (LN). Our prior research has indicated that protein, the primary active component of cordyceps (WCP), a traditional Chinese medicine, possesses properties that can enhance renal fibrosis and provide kidney protection. Nonetheless, the connection between WCP and T cell infiltration and differentiation in LN remains poorly understood. OBJECTIVE: The objective of this research was to assess the immunomodulatory impacts of WCP in LN mice and elucidate the underlying mechanism through in vivo and in vitro investigations. METHODS: To investigate the impact and mechanism of WCP in MRL/lpr lupus-prone mice, WCP (1.5 g/kg/d), Bailing capsules (BC, 0.75 g/kg/d), and saline in equivalent quantities were administered to the mice over a period of 8 weeks. The therapeutic effects, T cell infiltration and differentiation of WCP on MRL/lpr mice were verified through ELISA, Hematoxylin-eosin (H&E), Periodic Acid Schiff (PAS) staining, immunofluorescence, Luminex analysis and flow cytometry. The mechanism by which WCP alleviates LN was investigated using tissues of mice, T cells and Mouse Podocyte Clone-5 (MPC-5) cells by transcriptomics, Western blot (WB), and Real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: We found that WCP improved LN in MRL/lpr mice by reducing urinary protein, creatinine, and serum auto antibodies, increasing complement 3 (C3) level, improving renal immunopathology and downregulating serum cytokines, including IFN-γ, IL-12, and RANTES. Notably, the infiltration of CD4+ and CD8+ T cells in the kidney was reduced by WCP. Similarly, the cell transwell co-culturation study showed that the WCP treated MPC-5 cells were weaker in inducing T cell migration. Consistent with this finding, our observations revealed that WCP could inhibit T cell-related chemokine expression in kidney and MPC-5 cells, as well as reduce the levels of TLR4, MYD88, phosphorylated-p38, phosphorylated-ERK, and phosphorylated-JNK. On the other hand, WCP was found to greatly inhibit the Th1 cells differentiation in vivo and in vitro. Cytokine-receptor induced Th1 cell differentiation pathway and PI3K-AKT pathway were the most enriched pathways based on differentially expressed genes (DEGs) enrichment analysis among different cell groups. Results from RT-qPCR and WB showed that WCP notably reduced the levels of IL-12, p-STAT4, IFN-γ, p-STAT1, p-PI3K, and p-AKT in T cells. CONCLUSION: WCP demonstrated positive immunomodulatory effects on LN disease, by decreasing the T cells infiltration through TLR4/MYD88/MAPK signaling pathway and inhibiting Th1 cells differentiation via IL-12-STAT4 and IFN-γ-STAT1 pathways, in addition to the PI3K-AKT pathway.

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.

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.

Double-Negative T (DNT) Cells in Patients with Systemic Lupus Erythematosus.

Double-negative T (DNT) cells are a rare and unconventional T-lymphocyte subpopulation lacking both CD4 and CD8 markers. Their immunopathological roles and clinical relevance have yet to be elucidated. Beyond autoimmune lymphoproliferative syndrome (ALPS), these cells may also play a role in rheumatic disorders, including systemic lupus erythematosus (SLE); indeed, these two diseases share several autoimmune manifestations (including nephritis). Moreover, one of the main experimental murine models used to investigate lupus, namely the MRL/lpr mouse, is characterized by an expansion of DNT cells, which can support the production of pathogenic autoantibodies and/or modulate the immune response in this context. However, lupus murine models are not completely consistent with their human SLE counterpart, of course. In this mini review, we summarize and analyze the most relevant clinical studies investigating the DNT cell population in SLE patients. Overall, based on the present literature review and analysis, DNT cell homeostasis seems to be altered in patients with SLE. Indeed, most of the available clinical studies (which include both adults and children) reported an increased DNT cell percentage in SLE patients, especially during the active phases, even though no clear correlation with disease activity and/or inflammatory parameters has been clearly established. Well-designed, standardized, and longitudinal clinical studies focused on DNT cell population are needed, in order to further elucidate the actual contribution of these cells in SLE pathogenesis and their interactions with other immune cells (also implicated and/or altered in SLE, such as basophils), and clarify whether their expansion and/or immunophenotypic aspects may have any immunopathological relevance (and, then, represent potential disease markers and, in perspective, even therapeutic targets) or are just an unspecific epiphenomenon of autoimmunity.

Silencing HE4 alleviates the renal fibrosis in lupus nephritis mice by regulating the C3/MMPs/prss axis.

To explore the regulatory effect of human epididymis protein 4 (HE4) on renal fibrosis in mice with lupus nephritis (LN) and the underlying mechanism. Ten-week old MRL/LPR mice were injected with HE4 shRNA adenovirus vector through the renal pelvis for 5 days. Renal tissues were extracted for HE and Masson staining to evaluate pathological changes and fibrosis in lupus nephritis mice. The level of urine protein was measured using a biochemical analyzer, while the expression level of HE4 and p-NF-κB p65 in renal tissues was visualized using an immunofluorescence assay. The level of ß2-microglobulin (ß2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (Kim-1) was determined by the immunohistochemical assay. Western blotting was used to determine the levels of C3, HE4, matrix metalloproteinase-2 (MMP2), MMP9, p-p65, prss23, and prss35 in renal tissues. Compared to wild-type C57BL/6 mice, MRL/LPR mice showed a marked increase in the number of glomeruli, hyperplasic basement membrane, severe infiltration of inflammatory cells in renal tubules and glomeruli, obvious necrosis in glomeruli, elevated fibrosis levels, and increased levels of urine protein, ß2-MG, NGAL, Kim-1, C3, HE4, MMP2, MMP9, and p-p65; and decreased levels of prss23 and prss35 were observed in MRL/LPR mice. After the administration of the HE4 shRNA adenovirus vector, the repaired structure of renal tubules and glomeruli improved infiltration of inflammatory cells, reduced collagen fiber and urine protein, suppressed levels of C3, HE4, MMP2, MMP9, and p-P65, and facilitated the expression of prss23 and prss35 which were observed. Silencing HE4 improved renal fibrosis and inhibited inflammation in mice with lupus nephritis, which may play a role in inhibiting C3/MMPs and promoting prss-related protein expression.

Sirtuin 1 overexpression contributes to the expansion of follicular helper T cells in Systemic lupus erythematosus and serves as an easy-to-intervene therapeutic target.

OBJECTIVE: SIRT1, an NAD+-dependent deacetylase, is up-regulated in CD4+ T cells from SLE patients and MRL/lpr lupus-like mice. This study aimed to explore the role of SIRT1 in Tfh cell expansion and its potential value as a therapeutic target for SLE. METHODS: Frequencies of CD4+CXCR5+PD-1+ Tfh cells in peripheral blood from SLE patients and their expression of SIRT1 and BCL-6 were determined with flow cytometry. Naïve CD4+ T cells were transfected with SIRT1-expressing lentivirus and small interfering RNA (siRNA) targeting SIRT1, respectively, and then cultured in a Tfh-polarizing condition to study the impact of SIRT1 on Tfh cell differentiation. This impact was also evaluated in both CD4+ T cells and naïve CD4+ T cells by treatment with SIRT1 inhibitors (EX527 and nicotinamide) in vitro. MRL/lpr mice and pristane-induced lupus mice were treated with continuous daily intake of nicotinamide, and their lupus phenotypes including skin rash, arthritis, proteinuria and serum anti-dsDNA autoantibodies were compared with controls. RESULTS: Expression of SIRT1 was elevated in Tfh cells from SLE patients and positively correlated with Tfh cell frequencies. SIRT1 expression gradually increased during Tfh cell differentiation. Overexpression of SIRT1 by lentiviral vectors significantly promoted Tfh cell differentiation/proliferation. Reciprocally, suppressing expression of SIRT1 by siRNA and inhibiting SIRT1 activity by EX-527 or nicotinamide hindered Tfh cell expansion. Continuous daily intake of nicotinamide alleviated lupus-like phenotypes and decreased serum CXCL13 in the two mouse models. CONCLUSION: SIRT1 overexpression contributes to the expansion of Tfh cells in SLE and may serve as a potential target for treatment.

Nano-encapsulated anandamide reduces inflammatory cytokines in vitro and lesion severity in a murine model of cutaneous lupus erythematosus.

Cutaneous lupus erythematosus (CLE) is a heterogeneous autoimmune skin disease which occurs independently and in conjunction with systemic lupus erythematosus. Drug development for CLE is severely lacking. Anandamide (AEA) is a primary endocannabinoid which exhibits immunomodulatory effects through mixed cannabinoid receptor agonism. We evaluated AEA as topical treatment for CLE and assessed benefits of nanoparticle encapsulation (AEA-NP) on cutaneous drug penetration, delivery and biological activity. Compared to untreated controls, AEA-NP decreased IL-6 and MCP-1 in UVB-stimulated keratinocytes (p < 0.05) in vitro. In BALB/c mice, AEA-NP displayed improved cutaneous penetration, extended release and persistence of AEA in the follicular unit extending to the base after 24 h. Utilizing the MRL-lpr lupus murine model, twice weekly treatment of lesions with topical AEA-NP for 10 weeks led to decreased clinical and histologic lesion scores compared to unencapsulated AEA and untreated controls (p < 0.05). Prophylactic application of AEA-NP to commonly involved areas on MRL-lpr mice similarly resulted in decreased clinical and histologic scores when compared to controls (p < 0.05), and reduced C3 and IBA-1 in lesional tissue (p < 0.05). The demonstrated clinical and immunomodulatory effects of treatment with AEA support its potential as therapy for CLE. This work also suggests that encapsulation of AEA improves penetration and treatment efficacy. Future studies will be conducted to assess full therapeutic potential.

Anti-prolactin treatment alleviates lupus conditions by regulating the JAK2-STAT3 pathway.

OBJECTIVES: We previously revealed the role of prolactin (PRL) in antibody production and disease activity in patients with systemic lupus erythematosus. In this study, we sought to determine whether inhibition of PRL could improve lupus-like disease in MRL/lpr mice. METHODS: The expression levels of PRL in various cell types of lupus patients were measured by flow cytometry. The effects of anti-PRL on animal survival, renal histopathology, creatinine, proteinuria, anti-dsDNA antibody, cytokine production, splenomegaly and lymphadenopathy were assessed. The effect of anti-PRL on the Jak2-Stat3 signalling pathway was detected by western blotting. RESULTS: Prolactin was upregulated in B cells, neutrophils, CD4+ T cells, and monocytes isolated from patients with lupus. Furthermore, inhibition of PRL by anti-PRL treatment around the time of onset prolonged the survival of MRL/lpr mice, significantly reduced anti-dsDNA antibody production, and alleviated symptoms of lupus nephritis, splenomegaly, and lymphadenopathy. In addition, anti-PRL-treated mice showed a decrease in the levels of pathogenic cytokines such as IL-21 and IL-6. Furthermore, mechanistically, anti-PRL treatment significantly reduced the levels of p-Jak2 and p-Stat3 in MRL/lpr mice. CONCLUSIONS: In summary, these data suggest that PRL inhibition alleviates lupus-like disease in MRL/lpr mice by modulating the Jak2-Stat3 signalling cascade. More importantly, our results imply the potential of PRL inhibitors and may provide a novel therapeutic approach for lupus.

TIGIT-Fc fusion protein alleviates murine lupus nephritis through the regulation of SPI-B-PAX5-XBP1 axis-mediated B-cell differentiation.

OBJECTIVES: T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is a newly discovered immune checkpoint (IC) that exhibits immunosuppressive function in the regulation of immune system. Activation of TIGIT signaling has emerged as a promising approach for autoimmune disease immunotherapy, such as systemic lupus erythematosus (SLE). METHODS: We generated a chimeric protein, TIGIT-immunoglobulin (Ig), by fusing the extracellular domain of murine TIGIT to the Fc region of mouse IgG2a, which was used to investigated the effect of activating the TIGIT signaling in murine lupus models (MRL/lpr and chronic graft-versus-host disease mice). Treated mice were harvested, and samples of serum, kidney, and spleen were collected for outcome evaluation. In vitro treatment of TIGIT-Ig in B cells was used for exploring the roles of TIGIT in toll-like receptor 7 (TLR7)-mediated B cell differentiation and antibody production. RESULTS: TIGIT-Ig treatment delayed disease progression in both lupus models, accompanied by a decrease in the production of anti-double stranded DNA antibodies (anti-dsDNA), proteinuria, proteinuria/creatinine, and Ig kidney deposition. Additionally, the group treated with TIGIT-Ig displayed a decreased proportion of T helper cell (Th)1 cells, T follicular helper (Tfh) cells, and B-cell subsets, including germinal center B cells (GC B), plasmablasts, and plasma cells, compared to the group treated with control IgG. Interestingly, we also observed an increased proportion of Tregs in the spleen of the TIGIT-Ig group. We have discovered a new way in which activating the TIGIT pathway can regulate B-cell differentiation through the SPI-B-PAX5-XBP1 pathway, resulting in a reduction in autoantibodies. CONCLUSION: Together, TIGIT may be a promising IC target for SLE treatment.

Knockdown of KLF5 ameliorates renal fibrosis in MRL/lpr mice via inhibition of MX1 transcription.

OBJECTIVE: This study aims to elucidate the role of Kruppel-like factor (KLF5) and myxovirus resistance 1 (MX1) in the progression of renal fibrosis in lupus nephritis (LN). METHODS: First, the expression of KLF5 and MX1 was assessed in the peripheral blood of LN patients and healthy participants. Next, the pathological changes in renal tissues were evaluated and compared in BALB/c and MRL/lpr mice, by detecting the expression of fibrosis marker proteins (transforming growth factor-ß [TGF-ß] and CTGF) and α-SMA, the content of urine protein, and the levels of serum creatinine, blood urea nitrogen, and serum double-stranded DNA antibody. In TGF-ß1-induced HK-2 cells, the messenger RNA levels of KLF5 and MX1 were tested by qRT-PCR, and the protein expression of α-SMA, type I collagen (Col I), fibronectin (FN), and matrix metalloproteinase 9 (MMP9) was measured by western blot analysis. Moreover, the relationship between KLF5 and MX1 was predicted and verified. RESULTS: In renal tissues of MRL/lpr mice and the peripheral blood of LN patients, KLF5 and MX1 were highly expressed. Pearson analysis revealed that KLF5 was positively correlated with MX1. Furthermore, KLF5 bound to MX1 promoter and promoted its transcription level. MRL/lpr mice showed substantial renal injury, accompanied by increased expression of α-SMA, TGF-ß, CTGF, Col I, FN, and MMP9. Injection of sh-KLF5 or sh-MX1 alone in MRL/lpr mice reduced renal fibrosis in LN, while simultaneous injection of sh-KLF5 and ad-MX1 exacerbated renal injury and fibrosis. Furthermore, we obtained the same results in TGF-ß1-induced HK-2 cells. CONCLUSION: Knockdown of KLF5 alleviated renal fibrosis in LN through repressing the transcription of MX1.

Rosuvastatin treatment alone cannot alleviate lupus in murine model: a pilot study.

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by the production of autoantibodies and high cholesterol levels. HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors have exhibited anti-inflammatory effects in several clinical trials. We conducted this study to evaluate the effect of rosuvastatin on inflammatory responses in lupus-prone mice. Methods: MRL/lpr mice were intraperitoneally injected with rosuvastatin (10 mg/kg, n=4) or vehicle (2% dimethyl sulfoxide, n=4) five times a week from 13 to 17 weeks of age. The serum levels of low-density lipoprotein (LDL) cholesterol and autoantibodies were measured, as well as the urine levels of albumin. Renal tissues were stained for histopathological analysis. Concentrations of key inflammatory cytokines were measured in the serum, and messenger RNA (mRNA) levels in target organs (kidney, spleen, and lymph nodes) were evaluated. Results: Rosuvastatin treatment significantly decreased serum LDL cholesterol concentration in MRL/lpr mice. However, the clinical manifestations and autoantibody titres did not improve with rosuvastatin treatment. In addition, serum inflammatory cytokines and proteinuria did not change. Histopathological analysis of the kidneys revealed no improvement. When assessing the expression of mRNA, treatment with rosuvastatin decreased tumor necrosis alpha and interleukin-17 concentration in spleen and kidney tissue and in the kidneys and lymph nodes of MRL/lpr mice, respectively. Conclusion: Although it can decrease inflammatory cytokines in the lymphoid organs and kidneys of MRL/lpr mice, treatment with rosuvastatin is insufficient to alleviate SLE.

Impaired dynamic X-chromosome inactivation maintenance in T cells is a feature of spontaneous murine SLE that is exacerbated in female-biased models.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a highly female-biased systemic autoimmune disease, but the molecular basis for this female bias remains incompletely elucidated. B and T lymphocytes from patients with SLE and female-biased mouse models of SLE exhibit features of epigenetic dysregulation on the X chromosome which may contribute to this strong female bias. We therefore examined the fidelity of dynamic X-chromosome inactivation maintenance (dXCIm) in the pathogenesis of two murine models of spontaneous lupus-NZM2328 and MRL/lpr-with disparate levels of female-bias to determine whether impaired dXCIm contributes to the female bias of disease. METHODS: CD23+ B cells and CD3+ T cells were purified from age-matched C57BL/6 (B6), MRL/lpr, and NZM2328 male and female mice, activated in vitro, and processed for Xist RNA fluorescence in situ hybridization, H3K27me3 immunofluorescence imaging, qPCR, and RNA sequencing analyses. RESULTS: The dynamic relocalization of Xist RNA and the canonical heterochromatin mark, H3K27me3, to the inactive X chromosome was preserved in CD23+ B cells, but impaired in activated CD3+ T cells from the MRL/lpr model (p < 0.01 vs. B6), and even more impaired in the heavily female-biased NZM2328 model (p < 0.001 vs. B6; p < 0.05 vs. MRL/lpr). RNAseq of activated T cells from NZM2328 mice revealed the female-biased upregulation of 32 X-linked genes distributed broadly across the X chromosome, many of which have roles in immune function. Many genes encoding Xist RNA-interacting proteins were also differentially expressed and predominantly downregulated, which may account for the observed mislocalization of Xist RNA to the inactive X chromosome. CONCLUSIONS: Although evident in T cells from both the MRL/lpr and NZM2328 models of spontaneous SLE, impaired dXCIm is more severe in the heavily female-biased NZM2328 model. The aberrant X-linked gene dosage in female NZM2328 mice may contribute towards the development of female-biased immune responses in SLE-prone hosts. These findings provide important insights into the epigenetic mechanisms contributing to female-biased autoimmunity.

Deficiency of Pink1 promotes the differentiation of Th1 cells.

Previous studies have found that Pink1 is crucial for T cell activation and the function of Treg cells. However, the effect of Pink1 on inflammatory Th1 cells is largely unknown. In the process of Th1 differentiation from human naïve T cells, we found a reduction of Pink1 and Parkin. We then focused our attention on the Pink1 KO mice. Although there was no difference in the baseline of the T cell subset of Pink1 KO mice, Th1 differentiation from Pink1 KO naïve T cells in vitro showed a significant increase. Subsequently, we transferred naïve CD4+ T cells into Rag2 KO mice to establish a T-cell colitis mouse model and found that CD4+ T cells in mesentery lymph nodes of mice receiving Pink1 KO cells increased significantly, especially Th1 cells. Intestinal IHC staining also showed that the transcription factor T-bet of Th1 increased. Treatment of CD4+ T cells from lupus-like mice with mitophagy agonist urolithin A, a reduction of Th1 cells was observed, suggesting the clinical value of using mitophagy agonists to suppress Th1-dominated disease in the future.

Gut microbiota mediated the effects of high relative humidity on lupus in female MRL/lpr mice.

INTRODUCTION: The relationship between humidity and systemic lupus erythematosus (SLE) has yielded inconsistent results in prior research, while the effects of humidity on lupus in animal experiments and its underlying mechanism remain inadequately explored. METHODS: The present study aimed to investigate the impact of high humidity (80 ± 5%) on lupus using female and male MRL/lpr mice, with a particular focus on elucidating the role of gut microbiota in this process. To this end, fecal microbiota transplantation (FMT) was employed to transfer the gut microbiota of MRL/lpr mice under high humidity to blank MRL/lpr mice under normal humidity (50 ± 5%), allowing for an assessment of the effect of FMT on lupus. RESULTS: The study revealed that high humidity exacerbated lupus indices (serum anti-dsDNA, ANA, IL-6, and IFN- g, and renal pathology) in female MRL/lpr mice but had no significant effect on male MRL/lpr mice. The aggravation of lupus caused by high humidity may be attributed to the increased abundances of the Rikenella, Romboutsia, Turicibacter, and Escherichia-Shigella genera in female MRL/lpr mice. Furthermore, FMT also exacerbated lupus in female MRL/lpr mice but not in male MRL/lpr mice. CONCLUSION: In summary, this study has demonstrated that high humidity exacerbated lupus by modulating gut microbiota in female MRL/lpr mice. The findings underscore the importance of considering environmental factors and gut microbiota in the development and progression of lupus, particularly among female patients.

Cognitive impairment of MRL mice is related to NMDA receptor-mediated inflammatory response and production of adhesion molecules in MRL/lpr mice-derived micro-vascular endothelial cells.

Systemic lupus erythematosus (SLE) is a chronic recurrent autoimmune disease affecting almost all organs. This study was conducted to investigate cognitive impairment of SLE mice (MRL/lpr mice), and explore associated pathological mechanism. Behavior tests (open-field test, elevated plus-maze test, forced swimming test, sucrose preference test, and Morris water maze test) were conducted in MRL/MPJ and MRL/lpr mice. ELISA test was performed to determine levels of antibodies (anti-dsDNA, anti-RPA, anti-ACA, and anti-NR2a/b) and inflammatory factors [tumour necrosis factor a (TNF-a), interleukin (IL)-6, IL-8, and IL-10]. Micro-vascular endothelial cells (MVECs) were isolated, identified, and divided into MVECs (NC), anti-NR2a/2b, memantine, glycine, dexamethasone, and IL-1b groups. Cell proliferation was measured using cell counting kit-8 (CCK-8) assay, and Western blotting was applied to evaluate ELAM-1, VCAM-1, ICAM-1, IKBa, p-IKBa expression. MRL/lpr mice demonstrated lower locomotion/exploration ability, higher anxiety, obvious depression symptoms, lower learning/memory capability compared with MRL/MPJ mice. MRL/lpr mice demonstrated high levels of anti-NR2a/b antibody and auto-antibodies. NMDA receptor antagonist (memantine) significantly increased, and NMDA receptor agonist (glycine) significantly decreased MVECs proliferation compared with NC group ( p < 0.05). Memantine significantly reduced and glycine predominantly enhanced TNF-a, IL-6, IL-8, and IL-10 levels compared with NC group ( p < 0.05). NMDA receptor antagonist and agonist modulated adhesion molecules expression in MVECs. ELAM-1, VCAM-1, and ICAM-1 expressions were significantly down-modulated in memantine group, and remarkably up-modulated in glycine group compared with NC group ( p < 0.05). NMDA receptor antagonist and agonist regulated phosphorylation of p-IKBa. The above effects of memantine evenly equaled to dexamethasone, and glycine evenly equaled to IL-1b. In conclusion, cognitive impairment of MRL mice might be associated with NMDA receptor-mediated inflammatory response and production of adhesion molecules in MRL/lpr mice-derived MVECs.

Fecal microbiota from MRL/lpr mice exacerbates pristane-induced lupus.

BACKGROUND: The roles of gut microbiota in the pathogenesis of SLE have been receiving much attention during recent years. However, it remains unknown how fecal microbiota transplantation (FMT) and microbial metabolites affect immune responses and lupus progression. METHODS: We transferred fecal microbiota from MRL/lpr (Lpr) mice and MRL/Mpj (Mpj) mice or PBS to pristane-induced lupus mice and observed disease development. We also screened gut microbiota and metabolite spectrums of pristane-induced lupus mice with FMT via 16S rRNA sequencing, metagenomic sequencing, and metabolomics, followed by correlation analysis. RESULTS: FMT from MRL/lpr mice promoted the pathogenesis of pristane-induced lupus and affected immune cell profiles in the intestine, particularly the plasma cells. The structure and composition of microbial communities in the gut of the FMT-Lpr mice were different from those of the FMT-Mpj mice and FMT-PBS mice. The abundances of specific microbes such as prevotella taxa were predominantly elevated in the gut microbiome of the FMT-Lpr mice, which were positively associated with functional pathways such as cyanoamino acid metabolism. Differential metabolites such as valine and L-isoleucine were identified with varied abundances among the three groups. The abundance alterations of the prevotella taxa may affect the phenotypic changes such as proteinuria levels in the pristane-induced lupus mice. CONCLUSION: These findings further confirm that gut microbiota play an important role in the pathogenesis of lupus. Thus, altering the gut microbiome may provide a novel way to treat lupus.

Vitamin D Activates miR-126a-5p to Target GSK-3ß and Alleviates Systemic Lupus Erythematosus in MRL/LPR Mice.

BACKGROUND: The etiology of systemic lupus erythematosus (SLE) is complex, and the disease is thus difficult to cure. In this regard, it has been established that SLE patients are characterized by differing levels of vitamin D-hydroxylation; however, the direct effects of vitamin D (VitD) in these patients remain unknown. OBJECTIVE: Therefore, we investigated the effects and mechanisms of action of VitD in the context of SLE. METHODS: The effects of VitD on MRL/LPR mice were studied by synthesizing glycogen synthase kinase-3ß (GSK-3ß)-interfering lentiviruses and transfecting with miR-126a-5p mimics. Changes in the body weight of mice were recorded for 6 weeks. Western blotting was performed to determine the levels of T-bet, GATA3, and GSK-3ß protein expression, and qRT-PCR was performed to determine the levels of miR-126a-5p and GSK-3ß mRNA expression. ELISA was performed to determine the levels of ANA, dsDNA, and snRNP/Sm in mice serum. RESULTS: GSK-3ß and miR-126a-5p were expressed at high and low levels, respectively, in MRL/LPR mice. VitD (30 ng/kg) was found to reduce the expression of GSK-3ß and increase miR-126a-5p expression, which targets GSK-3ß. T-bet and GATA3 were found to be positively regulated by miR-126a-5p and VitD and negatively regulated by GSK-3ß. The body weight of mice was not altered by VitD. ANA, dsDNA, and snRNP/Sm were positively regulated by miR- 126a-5p and VitD and negatively regulated by GSK-3ß. The effects of GSK-3ß were enhanced in response to the inhibition of miR-126a-5p expression. CONCLUSION: VitD upregulated miR-126a-5p to target GSK-3ß expression, thereby alleviating the SLE in MRL/LPR mice.

Distinct RBC alloantibody responses in type 1 interferon-dependent and -independent lupus mouse models.

During transfusion of red blood cells (RBCs), recipients are exposed to both ABO and non-ABO 'minor' antigens. RBC donor units and recipient RBCs are not routinely matched for non-ABO antigens. Thus, recipients are exposed to many RBC alloantigens that can lead to RBC alloantibody production and subsequent clinically significant hemolysis. RBC alloantibodies also significantly limit the provision of compatible RBC units for recipients. Prior studies indicate that the frequency of RBC alloimmunization is increased during inflammatory responses and in patients with autoimmune diseases. Still, mechanisms contributing to alloimmune responses in patients with autoimmunity are not well understood. More than half of adult patients with systemic lupus erythematosus (SLE) produce type 1 interferons (IFNα/ß) and express IFNα/ß stimulated genes (ISGs). Previously, we reported that IFNα/ß promote RBC alloimmune responses in the pristane mouse model, which develops a lupus-like phenotype that is dependent on IFNα/ß signaling. However, it is unclear whether IFNα/ß or the lupus-like phenotype induces alloimmunization in lupus models. Therefore, we tested the hypothesis that IFNα/ß promotes RBC alloimmune responses in lupus by examining alloimmune responses in IFNα/ß-independent (MRL-lpr) and IFNα/ß-dependent (pristane) lupus models. Whereas pristane treatment significantly induced interferon-stimulated genes (ISGs), MRL-lpr mice produced significantly lower levels that were comparable to levels in untreated WT mice. Transfusion of murine RBCs that express the KEL antigen led to anti-KEL IgG production by pristane-treated WT mice. However, MRL-lpr mice produced minimal levels of anti-KEL IgG. Treatment of MRL-lpr mice with recombinant IFNα significantly enhanced alloimmunization. Collectively, results indicate that a lupus-like phenotype in pre-clinical models is not sufficient to induce RBC alloantibody production, and IFNα/ß gene signatures may be responsible for RBC alloimmune responses in lupus mouse models. If these findings are extended to alternate pre-clinical models and clinical studies, patients with SLE who express an IFNα/ß gene signature may have an increased risk of developing RBC alloantibodies and may benefit from more personalized transfusion protocols.

Gut microbiota mediated the effects of high relative humidity on lupus in female MRL/lpr mice

Abstract Introduction The relationship between humidity and systemic lupus erythematosus (SLE) has yielded inconsistent results in prior research, while the effects of humidity on lupus in animal experiments and its underlying mechanism remain inadequately explored. Methods The present study aimed to investigate the impact of high humidity (80 ± 5%) on lupus using female and male MRL/lpr mice, with a particular focus on elucidating the role of gut microbiota in this process. To this end, fecal microbiota transplantation (FMT) was employed to transfer the gut microbiota of MRL/lpr mice under high humidity to blank MRL/lpr mice under normal humidity (50 ± 5%), allowing for an assessment of the effect of FMT on lupus. Results The study revealed that high humidity exacerbated lupus indices (serum anti-dsDNA, ANA, IL-6, and IFN- g, and renal pathology) in female MRL/lpr mice but had no significant effect on male MRL/lpr mice. The aggravation of lupus caused by high humidity may be attributed to the increased abundances of the Rikenella, Romboutsia, Turicibacter, and Escherichia-Shigella genera in female MRL/lpr mice. Furthermore, FMT also exacerbated lupus in female MRL/lpr mice but not in male MRL/lpr mice. Conclusion In summary, this study has demonstrated that high humidity exacerbated lupus by modulating gut microbiota in female MRL/lpr mice. The findings underscore the importance of considering environmental factors and gut microbiota in the development and progression of lupus, particularly among female patients.