Systemic Lupus Erythematosus Mice Research Articles

Glucocorticoids on bone remodeling in systemic lupus erythematosus mice.

Systemic lupus erythematosus requires glucocorticoids for management. This study investigates how glucocorticoids influence bone in a SLE mouse model, focusing on bone mineral density (BMD), microstructure, and remodeling markers. MRL/lpr and C57BL/6 mice were administered dexamethasone or saline as a control for 4-weeks. Bone assessments included analyses of BMD, bone structure, and serum levels of RANKL and OPG. Dexamethasone decreased BMD and altered cortical and trabecular bone thickness in both MRL/lpr and C57BL/6 mice. In C57BL/6 mice, cortical bone exhibited increased catabolism while trabecular bone showed signs of increased anabolism, whereas MRL/lpr mice did not show significant changes in bone turnover. Both strains experienced weight loss, with a significant decrease in femur length observed only in C57BL/6 mice. Dexamethasone exacerbated BMD reduction in MRL/lpr mice and halted its increase in C57BL/6 mice. C57BL/6 mice exhibited notable changes in cortical and trabecular bone structure, while MRL/lpr mice didn't. After receiving dexamethasone, both strains showed higher serum RANKL levels, especially in C57BL/6 mice. OPG decreased in both strains. Both glucocorticoids and SLE contribute to abnormal bone remodeling through RANKL/OPG pathway. Glucocorticoid (GC) treatment in a mouse model of systemic lupus erythematosus (SLE) leads to significant changes in bone parameters, including decreased bone mineral density (BMD) and alterations in bone structure. Those change are associated with the modulation of RANKL and OPG expression. Both GC and inflammation in SLE contribute to BMD reduction, and GC may have a certain protective effect on bone in the early stage of chronic inflammation. GC can upregulate RANKL expression and downregulate OPG expression in vivo. During a state of chronic inflammation, RANKL expression increases. However, OPG may not exert a significant influence on inflammatory stimulation.

Combined injection of pristane and Bacillus Calmette-Guerin Vaccine successfully establishes a lupus model with atherosclerosis.

Cardiovascular disease (CVD) induced by atherosclerosis (AS) is the main fatal complication of systemic lupus erythematosus (SLE). Establishing an appropriate animal model of SLE with AS is of great value for investigating the pathogenesis and therapeutic targets of SLE-CVD. In the present work, pristane was injected intraperitoneally into C57BL/6J mice to establish the SLE model and Bacillus Calmette-Guerin Vaccine (BCG) was injected intradermally one month later to enhance immunity and induce AS. Both pristane or pristane and BCG treated C57BL/6J mice exhibit a classical lupus-like phenotype which manifested as proteinuria and high levels of serum anti-ANA and anti-dsDNA autoantibodies, in conjunction with pathological changes in spleen and kidney, complement C3 deposition in the kidney, overactivation of T and B cells, a decreased proportion of splenic CD19+ B cells, and an increased frequency of CD19-CD138+ plasma cells, CD19+CD27+ memory B cells, CD3+CD4+ helper T (Th) cells, and CD3+CD4+CXCR5+PD-1+ T follicular helper cells. The combined pristane and BCG challenge aggravated AS in SLE mice, which had an enlarged thymus gland, an increased T cell proliferative vitality, an expanded Th cell pool, severe perivascular lymphocytes, and CD68+ macrophage infiltration, in addition to an intimal lipid deposition, and further elevation of Toll-like receptor 2 (TLR2), TLR4, and the transcription factor nuclear factor-κB (NF-κB) expression in kidney tissue and blood vessels when comparing with pristane or BCG injection alone. Pristane combined BCG challenge is able to establish a validated SLE-AS mouse model in C57BL/6J mice.

ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis.

Systemic lupus erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with SLE, we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, NRTKs regulate activation, migration, and proliferation of immune cells. We found that the patients' ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced pluripotent stem cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.

FoxO1 Deficiency in Monocytic Myeloid-Derived Suppressor Cells Exacerbates B Cell Dysfunction in Systemic Lupus Erythematosus.

Myeloid-derived suppressor cells (MDSCs) contribute to the pathogenesis of systemic lupus erythematosus (SLE), in part due to promoting the survival of plasma cells. FoxO1 expression in monocytic MDSCs (M-MDSCs) exhibits a negative correlation with the SLE Disease Activity Index score. This study aimed to investigate the hypothesis that M-MDSC-specific FoxO1 deficiency enhances aberrant B cell function in aggressive SLE. We used GEO data sets and clinical cohorts to verify the clinical significance of FoxO1 expression and circulating M-MDSCs. Using Cre-LoxP technology, we generated myeloid FoxO1 deficiency mice (mFoxO1-/-) to establish murine lupus-prone models. The transcriptional stage was assessed by integrating chromatin immunoprecipitation (ChIP)-sequencing with transcriptomic analysis, luciferase reporter assay, and ChIP-quantitative polymerase chain reaction. Methylated RNA immunoprecipitation sequencing, RNA sequencing, and CRISPR-dCas9 were used to identify N6-adenosine methylation (m6A) modification. In vitro B cell coculture experiments, capmatinib intragastric administration, m6A-modulated MDSCs adoptive transfer, and sample validation of patients with SLE were performed to determine the role of FoxO1 on M-MDSCs dysregulation during B cell autoreacted with SLE. We present evidence that low FoxO1 is predominantly expressed in M-MDSCs in both patients with SLE and lupus mice, and mice with myeloid FoxO1 deficiency (mFoxO1-/-) are more prone to B cell dysfunction. Mechanically, FoxO1 inhibits mesenchymal-epithelial transition factor protein (Met) transcription by binding to the promoter region. M-MDSCs FoxO1 deficiency blocks the Met/cyclooxygenase2/prostaglandin E2 secretion pathway, promoting B cell proliferation and hyperactivation. The Met antagonist capmatinib effectively mitigates lupus exacerbation. Furthermore, alkB homolog 5 (ALKBH5) targeting catalyzes m6A modification on FoxO1 messenger RNA in coding sequences and 3' untranslated regions. The up-regulation of FoxO1 mediated by ALKBH5 overexpression in M-MDSCs improves lupus progression. Finally, these correlations were confirmed in untreated patients with SLE. Our findings indicate that effective inhibition of B cells mediated by the ALKBH5/FoxO1/Met axis in M-MDSCs could offer a novel therapeutic approach to manage SLE.

IL-10 mediates pleural remodeling in systemic lupus erythematosus.

Interleukin-10 (IL-10), a pivotal anti-inflammatory cytokine, has gotten attention for its involvement in tissue remodeling and organ fibrosis. Pleurisy and subsequent pleural remodeling are recognized as quantifiable indicators of systemic lupus erythematosus (SLE) activity. However, the role of IL-10 in SLE-associated pleural remodeling remains unknown. In this study, we investigated role of IL-10 in SLE-associated pleural remodeling and the underlying mechanism. Clinical data and serum specimens were obtained from SLE patients, while pleural mesothelial cells and mouse models served as primary experimental subjects. The protein expression-related technologies, histopathological staining, and other experimental methods were used in the study. Our investigation got several key findings. Firstly, serum obtained from SLE patients with pleural thickening was found to induce pleural mesothelial cell remodeling. Subsequently, heightened levels of IL-10 were found in serum from SLE patients with pleural thickening compared to that of SLE patients without pleural thickening. Secondly, administration of recombinant IL-10 was confirmed its ability to induce pleural mesothelial cell remodeling, on the contrary, this remodeling was effectively mitigated by IL-10 inhibition. Notably, blockade of IL-10 significantly prevented collagen deposition and prevented thickening in pleura of SLE mouse models. Lastly, the IL-10/JAK2/STAT3/HIF1α/TMEM45A/P4HA1 signaling axis was elucidated to mediate pleural remodeling and thickening. Our study uncovered that IL-10 mediated pleural remodeling in SLE. We suggested that serum IL-10 level exceeding 6.32 pg/mL was a potential reference threshold for predicting pleural thickening in SLE patients.

Ponatinib exacerbate renal injury in systemic lupus erythematosus mouse model through PDGFR-PI3K/AKT pathway

Lupus nephritis (LN) is a common clinical complication of systemic lupus erythematosus (SLE). Proliferative lupus nephritis represents the gravest form of LN, and since effective drugs for its treatment are still lacking, tyrosine kinase inhibitors (TKIs) find extensive clinical utility due to their notable impact on suppressing cell proliferation and may serve as potential drugs for LN treatment. However, previous studies on the effects of TKI on LN have been controversial. Ponatinib, a third-generation TKI, lacks studies on its role in LN. This study aimed to investigate the impact of the ponatinib on LN. MRL/lpr mice were evaluated for renal function, autoimmune markers and histopathological changes after oral administration of ponatinib. RNA-seq analysis was performed to explore the molecular pathways involved in ponatinib-induced kidney injury. Ponatinib uniquely exacerbated renal damage in MRL/lpr mice, evidenced by a decline in renal function and acute pathological changes, without affecting lupus-related autoimmune markers. Differential expressed genes analysis and functional enrichment implicate ponatinib-induced renal damage in MRL/lpr mice associated with adiponectin. Furthermore, we verified ponatinib signaling the PI3K/AKT pathway through PDGFRα, potentially influencing high molecular weight adiponectin (HMW ADIPOQ) expression and exacerbating renal damage. In conclusion, this study demonstrates that ponatinib can up-regulate HMW ADIPOQ expression via the PI3K/AKT pathway by inhibiting PDGFRα phosphorylation, highlighting the potential nephrotoxic effects of ponatinib in lupus-prone mice, and underscoring the importance of monitoring renal function in systemic autoimmune diseases patients receiving ponatinib.

Farnesyl pyrophosphate potentiates dendritic cell migration in autoimmunity through mitochondrial remodelling.

Cellular metabolism modulates dendritic cell (DC) maturation and activation. Migratory dendritic cells (mig-DCs) travelling from the tissues to draining lymph nodes (dLNs) are critical for instructing adaptive immune responses. However, how lipid metabolites influence mig-DCs in autoimmunity remains elusive. Here, we demonstrate that farnesyl pyrophosphate (FPP), an intermediate of the mevalonate pathway, accumulates in mig-DCs derived from mice with systemic lupus erythematosus (SLE). FPP promotes mig-DC survival and germinal centre responses in the dLNs by coordinating protein geranylgeranylation and mitochondrial remodelling. Mechanistically, FPP-dependent RhoA geranylgeranylation promotes mitochondrial fusion and oxidative respiration through mitochondrial RhoA-MFN interaction, which subsequently facilitates the resolution of endoplasmic reticulum stress in mig-DCs. Simvastatin, a chemical inhibitor of the mevalonate pathway, restores mitochondrial function in mig-DCs and ameliorates systemic pathogenesis in SLE mice. Our study reveals a critical role for FPP in dictating mig-DC survival by reprogramming mitochondrial structure and metabolism, providing new insights into the pathogenesis of DC-dependent autoimmune diseases.

Effect of Spermidine on Endothelial Function in Systemic Lupus Erythematosus Mice.

Endothelial dysfunction is common in Systemic Lupus Erythematosus (SLE), even in the absence of cardiovascular disease. Evidence suggests that impaired mitophagy contributes to SLE. Mitochondrial dysfunction is also associated with impaired endothelial function. Spermidine, a natural polyamine, stimulates mitophagy by the PINK1-parkin pathway and counters age-associated endothelial dysfunction. However, the effect of spermidine on mitophagy and vascular function in SLE has not been explored. To address this gap, 9-week-old female lupus-prone (MRL/lpr) and healthy control (MRL/MpJ) mice were randomly assigned to spermidine treatment (lpr_Spermidine and MpJ_Spermidine) for 8 weeks or as control (lpr_Control and MpJ_Control). lpr_Control mice exhibited impaired endothelial function (e.g., decreased relaxation to acetylcholine), increased markers of inflammation, and lower protein content of parkin, a mitophagy marker, in the thoracic aorta. Spermidine treatment prevented endothelial dysfunction in MRL-lpr mice. Furthermore, aortas from lpr_Spermidine mice had lower levels of inflammatory markers and higher levels of parkin. Lupus phenotypes were not affected by spermidine. Collectively, these results demonstrate the beneficial effects of spermidine treatment on endothelial function, inflammation, and mitophagy in SLE mice. These results support future studies of the beneficial effects of spermidine on endothelial dysfunction and cardiovascular disease risk in SLE.

Abstract 42: Fecal Microbiota Transplantation Decreases Autoantibody Production and Lowers Blood Pressure in an Experimental Model of Autoimmune Disease

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder that disproportionately affects women. It is characterized by a breach of immunological self-tolerance and the expansion of autoreactive T and B lymphocytes, leading to the production of autoantibodies. Autoantibody production leads to downstream chronic inflammation resulting in high rates of hypertension, renal injury, and cardiovascular disease. Gut dysbiosis is associated with many autoimmune diseases, including SLE. In the present study, we hypothesized that gut dysbiosis in SLE may play a role in the development of immune system dysfunction and hypertension in an experimental model of SLE, and that fecal microbiota transplantation (FMT) with microbes from control mice may improve these parameters. To test this hypothesis, we treated 26-week-old female control (NZW, n=10) and SLE (NZBWF1, n=9) mice with broad spectrum antibiotics in drinking water for one week to deplete the existing microbiota. Subsequently, mice were given 0.1 mL of fecal microbes from either control or SLE mice via oral gavage two times per week for four weeks in the following groups (n=9-10 mice/group): Control-vehicle, Control-SLE microbes, SLE-vehicle, SLE-Control microbes. Changes in the gut microbiome after FMT were assessed by metagenomic sequencing of fecal pellets from all four groups. Mean arterial pressure (MAP, mmHg) was higher in SLE mice compared to control mice (Control: 112±3 vs. SLE: 140±2 mmHg, p<0.0001). SLE mice that received FMT with control microbes had lower blood pressure (SLE-FMT: 126±4 mmHg, p=0.03). While there were no changes in circulating and splenic B cell percentages, SLE mice had significantly higher percentages of CD11b + CD11c + autoimmune-associated B cells compared to control mice (Control: 0.41±0.04% vs. SLE: 1.2±0.1%, p=0.0006), which were significantly decreased in SLE-FMT mice (SLE-FMT: 0.76±0.1%, p=0.02). Circulating autoantibodies were also measured at the conclusion of the study. Anti-dsDNA IgG was elevated in SLE mice compared to control (Control: 149±38 vs. SLE: 527±114 kU/mL, p=0.02), and were significantly lower in SLE mice treated with control microbes (SLE-Control microbes: 126±4 mmHg, p=0.03). These data suggest that gut microbiota present in NZBWF1 mice contribute to the accumulation of activated B cells and increased autoantibody production, which are known pathogenic factors in the development of hypertension in SLE.

Abstract P180: Female mice with systemic lupus erythematosus produce antibodies to mitochondrial antigens and have impaired renal mitochondrial function

Patients with autoimmune disorders like systemic lupus erythematosus (SLE) are at significant risk for renal and cardiovascular mortality. The pathology of SLE is driven by a loss of immune tolerance with the subsequent production of autoantibodies (IgG), and our laboratory previously demonstrated a mechanistic role for autoantibodies in autoimmune mediated hypertension. While anti-double stranded DNA (dsDNA) antibodies are pathognomonic for SLE, a breadth of autoantibodies may contribute to disease pathology. The goal of this study is to identify autoantibodies that may contribute to the renal and cardiovascular consequences associated with SLE. To accomplish this goal, an antigen array was performed (University of Texas Southwestern Genomics & Microarray Core Facility) using plasma samples collected from an established experimental mouse model of SLE (30 week old female NZBWF1, n=8) and healthy controls (30 week old female NZWLacJ, n=7). As anticipated, autoantibodies raised to nuclear antigens were prominently represented in the array and were significantly increased relative to control mice. This includes antibodies to dsDNA, ssRNA, chromatin, and histones (total) (p<0.02). In addition, SLE mice produce antibodies to mitochondrial antigens including cardiolipin and cytochrome C (p<0.02 vs control mice), critical components of the inner mitochondrial membrane and respiratory chain, respectively. In a separate study, renal mitochondrial function was tested using high resolution respirometry (Oroboros Instruments) in whole kidney homogenates from 30 week old female SLE (n=6) and control mice (n=5). Results show that coupled respiration, an estimation of oxidative phosphorylation, is significantly impaired at Complex I in kidneys from SLE mice compared to healthy controls (62±9 vs. 90±7 pmol/s/mg tissue, p<0.05). Uncoupled respiration, an estimation of maximal electron transport, is also reduced in the kidneys of female SLE mice compared to controls (157±23 vs. 212±14 pmol/s/mg tissue, p=0.07). These data provide insight for new mechanistic pathways to pursue in understanding the underlying mechanisms promoting cardiovascular risk factors like hypertension during SLE.

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.

Cellular spermine targets JAK signaling to restrain cytokine-mediated autoimmunity

Prolonged activation of the type I interferon (IFN-I) pathway leads to autoimmune diseases such as systemic lupus erythematosus (SLE). Metabolic regulation of cytokine signaling is critical for cellular homeostasis. Through metabolomics analyses of IFN-β-activated macrophages and an IFN-stimulated-response-element reporter screening, we identified spermine as a metabolite brake for Janus kinase (JAK) signaling. Spermine directly bound to the FERM and SH2 domains of JAK1 to impair JAK1-cytokine receptor interaction, thus broadly suppressing JAK1 phosphorylation triggered by cytokines IFN-I, IFN-II, interleukin (IL)-2, and IL-6. Peripheral blood mononuclear cells (PBMCs) from individuals with SLE showing decreased spermine concentrations exhibited enhanced IFN-I and lupus gene signatures. Spermine treatment attenuated autoimmune pathogenesis in SLE and psoriasis mice and reduced IFN-I signaling in monocytes from individuals with SLE. We synthesized a spermine derivative (spermine derivative 1 [SD1]) and showed that it had a potent immunosuppressive function. Our findings reveal spermine as a metabolic checkpoint for cellular homeostasis and a potential immunosuppressive molecule for controlling autoimmune disease.

ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with systemic lupus erythematosus (SLE) we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, non-receptor tyrosine kinases (NRTKs) regulate activation, migration, and proliferation of immune cells. We found that the patients' ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced Pluripotent Stem Cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.

#1843 Deletion of IL17-producing follicular helper T cells restrains the germinal center reaction in systemic lupus erythematosus

Abstract Background and Aims Systemic lupus erythematosus (SLE) is an autoimmune disorder in which autoantibody formation can lead to severe kidney pathology. Interleukin (IL)17-producing T cells have been suggested to play an important role in the pathogenesis of SLE, and IL-17 inhibition was shown to ameliorate SLE pathology in lupus-prone mice. Moreover, the frequency of circulating follicular helper T (Tfh) cells, a T cell subset that orchestrates the maturation of high-affinity antibodies in the germinal center of secondary lymphoid organs, is associated with disease activity in SLE. Increased frequencies of IL17-producing Tfh have been reported in SLE patients compared to controls, but their role in SLE pathogenesis is still unknown. Method By leveraging the B6.Sle1NZM2410/AegYaa strain, we generated lupus-prone mice in which IL17-producing Tfh cells could be either fate-mapped (SLE Tfh17-FM, Il17aCRERosaLoxSTOPLox-YFPSle1+/+Yaa+) or fate-mapped and selectively depleted by intraperitoneal diphtheria toxin (DT) administration (SLE Tfh17-DTR, Il17aCRERosaLoxSTOPLox-YFPCxcr5LoxSTOPLox-DTRSle1+/+Yaa+). Tfh17-FM and Tfh17-DTR mice (both Sle1−/−Yaa−) were used as controls. Results Tfh17 comprised approximately 0.4% of total splenic Tfh in SLE mice at 6 weeks of life, and DT administration caused potent and selective depletion of these cells in the SLE Tfh17-DTR strain (Fig. 1A). Early Tfh17 deletion from week 4 to week 9 did not modify the increased Tfh frequency observed at 20 weeks in SLE mice, but resulted in long-term reduction of splenic germinal center (GC) B cells and plasmacells (Fig. 1B-C). IL17 receptor A was overexpressed in non-GC B cells and plasmacells of SLE mice compared to controls, suggesting a possible increased susceptibility of these subsets to IL17 (Fig. 1D). SLE Tfh17-DTR mice treated with DT displayed a trend towards decreased anti-DNA levels compared to SLE Tfh17-FM (Fig. 1E), but both glomerular immunocomplex deposition and renal pathology were not significantly impacted (Fig. 1F-G). Conclusion The germinal center reaction is affected by selective depletion of IL17-producing Tfh cells in SLE mice, with reduced differentiation of GC B cells and plasmacells. The modest effect of Tfh17 depletion on autoantibody levels and lupus nephritis pathology suggests that other Tfh subsets may play a role in GC-derived autoantibody generation. In addition, extrafollicular sources may also contribute to autoantibody production in SLE.

Autoimmune Disease Pathogenesis Exacerbates Inflammation Following Myocardial Infarction in a Rodent Model of Systemic Lupus Erythematosus

Patients with systemic autoimmune diseases such as systemic lupus erythematosus (SLE) have an increased risk of suffering a myocardial infarction (MI), and are also more likely to die, develop heart failure, or suffer a second cardiac event than patients without an autoimmune disease. SLE primarily affects women and is characterized by autoreactive T and B lymphocytes that lead to widespread production of autoantibodies to a variety of nuclear and cytoplasmic antigens. Autoantibody-antigen immune complexes deposit in tissues and activate downstream inflammatory pathways leading to end-organ damage to tissues such as the heart, lungs, kidneys, and vasculature. However, the mechanisms whereby SLE disease may exacerbate MI and lead to poor outcomes in autoimmune patients is not well understood. The purpose of the present study was to test the hypothesis that SLE disease pathogenesis contributes to worsened outcomes after MI because of systemic inflammatory processes taking place. To test this hypothesis, saline (control, n=7) and pristane-treated (SLE, n=8) C57BL/6 mice were subjected to permanent coronary artery ligation (MI) and studied at 7 days post MI. Left ventricular (LV) function was examined at baseline (D0) and D7 using echocardiography (VEVO 3100). Control and SLE mice had similar LV parameters at baseline, but SLE mice had significantly more wall thinning in the infarct area (Control: 0.54±0.05 vs. SLE: 0.36±0.03 mm, p=0.005) as well as in the remote area (Control: 0.59±0.07 vs. SLE: 0.38±0.03 mm, p=0.01). SLE mice also displayed increased LV dilation (Control: 61.7±5.3 vs. SLE: 78.9±3.8 μL, p=0.02) at 7 days post MI. Ejection fraction was significantly lower in SLE mice as well (Control: 26.3±2.0% vs. 19.0±2.5%, p=0.045). We examined expression of inflammatory cytokines and collagen subunit genes in infarcted heart tissue using qPCR. SLE mice had increased expression of several inflammatory cytokines, including IL-6 and IL-18, while other cytokines such as IL-1β and TNF-α were not significantly different between the groups. In addition, SLE mice had decreased expression of the Col1a1, which encodes the alpha subunit of Type I Collagen, and Cd31, an endothelial cell marker, suggesting impairment of scar formation and angiogenesis post MI in SLE mice. Flow cytometric analyses of infiltrating LV immune cells showed increased total CD45+ cells in the infarcted area of SLE mice (Control: 10.6±1.2% vs. SLE: 13.8±0.6%, p=0.03), and a trend for an increase in CD45R+ B cells (Control: 7.5±1.1% vs. SLE: 12.5±2.1%, p=0.05). Taken together, these data indicate that SLE disease impacted recovery post MI, possibly due to increased B cell infiltration in the heart. Future studies will assess outcomes at later time points and also examine the progression of renal injury post MI. NIH/NHLBI R00 HL146888 and U54 HL169191 to Taylor NHLBI R01 HL166737 and AHA CDA856365 to Mouton NIGMS P20 GM104357 and P30 GM149404 to Department of Physiology and Biophysics. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early Life Stress in the Pristane Model of Systemic Lupus Erythematosus increases cardiovascular risk but not kidney disease severity earlier in disease progression

Experiencing Early Life Stress (ELS), such as physical/verbal abuse before the age of 18, has become an increasingly apparent concern in the context of lifelong health. Systemic Lupus Erythematosus (SLE) is one autoimmune condition linked with ELS, predominantly affects women, and has high CVD burden. Exposure to ELS independently increases the risk of CVD and SLE, so we have developed a model to study this gap. The mouse model of ELS, maternal separation with early weaning (MSEW), combined with the pristane model of SLE mimics the accelerated disease (shown by earlier production of autoantibodies, increased vascular stiffness, and vascular dysfunction at 4 months post injection (p.i.) opposed to 7 months p.i.) in ELS. We hypothesized that the accelerated disease and CVD burden in MSEW+SLE mice, as opposed to normally reared (NR)+SLE mice, arose from severe kidney damage and increased blood pressure. MSEW mice were separated from dams for 4 hours/day (postnatal day 2 to 5) and 8 hours/day before weaning at day 17. NR litters were weaned at postnatal day 21. MSEW or NR female mice were randomly administered PBS or pristane through i.p. injection. Urine was collected at 4 months and 7 months p.i. to test for kidney damage markers and telemetry was utilized. MSEW animals develop increased Anti-Smith autoantibodies at 4 months p.i while SLE NR animals had no difference in titer levels compared to NR PBS mice. However, we found no difference in urine NGAL (Neutrophil Gelatinase-Associated Lipocalin) at 4 (p=0.32) or 7 months p.i. (p=0.6) or urine proteinuria 7 months p.i. (p=0.9) between groups. Blood pressure between MSEW+SLE and NR+SLE mice at 4, 5, and 6 months p.i. compared to control mice was not different. At 7 months p.i., SBP in NR+SLE (140.3 ± 10.22, n=4), MSEW+SLE (140.28 ±9.32, n=4) was not statistically greater than the NR/MSEW controls (128.1 ± 3.16, n=5 NR, n=7 MSEW). Ejection fraction in NR+SLE (66.04 ± 3.24, n=3), MSEW+SLE (75.17 ±3.19, n=2) compared to NR/MSEW controls (64.36 ± 2.49, n=5 NR, n=4 MSEW) was slightly changed over time. We see that at 6 months p.i. PWV and adventitial wall thickness is greater in MSEW+SLE (2.89 mm/sec ± 0.11) mice compared to both NR+SLE mice (2.047 mm/sec ± 0.16) and NR control mice (1.49 mm/sec ± 0.12, p=<0.001, F= 26.02), but at seven months PWV is not different between MSEW+SLE (2.94 mm/sec ± 0.13) mice compared to both NR+SLE mice (2.63 mm/sec ± 0.32, p=0.30). The acceleration of autoantibodies and PWV in MSEW+SLE mice, but not kidney or heart function indicates a specific impact on larger vessels and autoimmune cells in SLE. This work furthers the understanding of how ELS accelerates cardiovascular burden in autoimmune diseases and early identifying targets to prevent worsened outcomes in patients exposed to childhood trauma. Crohn’s and Colitis Foundation NIH R25 PROmoTE NIH T32 PRIME F31HL165863-0 NIH P01 HL158500. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Efficacy evaluation of B cell targeting drugs and the involved mechanism of action study in humanized BAFF transgenic SLE mice model

Abstract The B cell activating factor (BAFF) functions as a crucial factor in B cell survival, and heightened BAFF expression can augment the presence of autoreactive B cells, thereby fostering autoimmunity like Systemic Lupus Erythematosus (SLE). This study introduces transgenic mouse strains with human BAFF overexpression, offering insights into SLE mechanisms and B cell-targeting therapies. Human BAFF overexpression in B6-hBAFF mice led to a substantial rise in Igs and anti-dsDNA levels from 6 weeks of age, persistently escalating with time compared to wild-type mice. A heightened population of B cells but a reduced ratio of T cells was observed. At week 25 of age, B6-hBAFF mice developed lupus nephritis. Belimumab, a BAFF antibody administrated to BAFF transgenic mice demonstrated the effectively reduced serum antibody levels after 16 weeks of dosing. Belimumab ameliorated kidney function by decreasing urine albumin-to-creatinine ratio and IgA deposition after 20 weeks of treatment. Mice were also treated with Enpatoran (TLR7/8 inhibitors) and BTK inhibitor Orelabrutinib. Only the BTK inhibitor Orelabrutinib significantly eliminated serum Igs levels. Enpatoran exhibited a similar ability to Orelabrutinib in reducing T follicular helper cell and plasma cell populations spleen and lymph nodes. In summary, the humanized BAFF transgenic SLE model proves to be a valuable tool for preclinical efficacy studies in SLE treatment and understanding pathogenesis.

Galantamine Reduces Blood Pressure and Renal Injury in Female Angiotensin II-induced Hypertensive Mice

Inflammation is a known culprit in the development and progression of hypertension. The autoimmune disease, systemic lupus erythematosus (SLE), is an important model of hypertension in the setting of chronic kidney inflammation. We have shown that administration of an acetylcholinesterase inhibitor, galantamine, attenuates the hypertension, kidney inflammation, and kidney injury in female SLE mice by activating the vagally-mediated cholinergic anti-inflammatory pathway. Although SLE hypertension represents a specific population of patients, we hypothesized that galantamine would have similar anti-inflammatory effects in the angiotensin II (AngII)-induced hypertensive mouse, which more commonly models essential hypertension. Female C57BL/6 mice were implanted with minipumps at 9 weeks of age for subcutaneous delivery of either AngII (1000 ng/kg/min) or saline (controls), and galantamine hydrobromide (4 mg/kg/day) or vehicle, for 21 days. At 12 weeks, carotid catheters were implanted and mean arterial pressure (MAP) was measured in conscious mice for two consecutive days. Mice were then euthanized, and kidneys collected. Markers of kidney inflammation and injury, including interleukin (IL)-6 and kidney injury molecule (KIM)-1, were measured by ELISA and normalized to total kidney protein. MAP (mmHg) was higher in AngII-treated mice compared to controls (174±4 vs. 133±3; n=4-5, p=0.0023). Galantamine lowered MAP in AngII mice (157±8; n=7, p=0.2567) without altering MAP in controls (133±4; n=5, p=0.9999). Renal IL-6 (pg/μg) was not different between AngII and control mice (0.004±0.0001 vs. 0.005±0.0006; p=0.6685). Although galantamine lowered IL-6 in both groups, it was only significant in controls (0.003±0.0002; p=0.0134) and not AngII mice (0.003±0.0003; p=0.4677). Renal cortical KIM-1 (pg/μg) trended higher in AngII mice compared to controls (2.53±0.50 vs 0.63±0.16; p=0.0741). Galantamine lowered KIM-1 in AngII mice (1.18±0.39, p=0.2064), but not controls (1.45 ± 0.59; p=0.6285). In summary, galantamine protected female mice from hypertension and renal injury in a model of AngII hypertension. This suggests that acetylcholinesterase inhibitor therapy may be effective in patients with hypertension. Studies funded by NIH R01HL153703 and K01HL139859 for KWM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prednisone combined with Dihydroartemisinin attenuates systemic lupus erythematosus by regulating M1/M2 balance through the MAPK signaling pathway

ObjectiveDihydroartemisinin (DHA) plays a very important role in various diseases. However, the precise involvement of DHA in systemic lupus erythematosus (SLE), relation to the equilibrium between M1 and M2 cells, remains uncertain. Therefore, we aimed to investigate the role of DHA in SLE and its effect on the M1/M2 cells balance. MethodsSLE mice model was established by pristane induction. Flow cytometry was employed to measure the abundance of M1 and M2 cells within the peripheral blood of individuals diagnosed with SLE. The concentrations of various cytokines, namely TNF-α, IL-1β, IL-4, IL-6, and IL-10, within the serum of SLE patients or SLE mice were assessed via ELISA. Immunofluorescence staining was utilized to detect the deposition of IgG and complement C3 in renal tissues of the mice. We conducted immunohistochemistry analysis to assess the expression levels of Collagen-I, a collagen protein, and α-SMA, a fibrosis marker protein, in the renal tissues of mice. Hematoxylin-eosin staining, Masson’s trichrome staining, and Periodic acid Schiff staining were used to examine histological alterations. In this study, we employed qPCR and western blot techniques to assess the expression levels of key molecular markers, namely CD80 and CD86 for M1 cells, as well as CD206 and Arg-1 for M2 cells, within kidney tissue. Additionally, we investigated the involvement of the MAPK signaling pathway. The Venny 2.1 online software tool was employed to identify shared drug-disease targets, and subsequently, the Cytoscape 3.9.2 software was utilized to construct the “disease-target-ingredient” network diagram. Protein-protein interactions of the target proteins were analyzed using the String database, and the network proteins underwent enrichment analysis for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. ResultsThe results showed that an increase in M1 cells and a decrease in M2 cells within the peripheral blood of individuals diagnosed with SLE. Further analysis revealed that prednisone (PDN) combined with DHA can alleviate kidney damage and regulate the balance of M1 and M2 cells in both glomerular mesangial cells (GMC) and kidney. The MAPK signaling pathway was found to be involved in SLE kidney damage and M1/M2 balance in the kidney. Furthermore, PDN and/or DHA were found to inhibit the MAPK signaling pathway in GMC and kidney. ConclusionWe demonstrated that PDN combined with DHA attenuates SLE by regulating M1/M2 balance through MAPK signaling pathway. These findings propose that the combination of PDN and DHA could serve as a promising therapeutic strategy for SLE, as it has the potential to mitigate kidney damage and reinstate the equilibrium of M1 and M2 cells.

Vitamin D improves autoimmune diseases by inhibiting Wnt signaling pathway.

In this study, we investigated the development of the Wnt signaling pathway in vitamin D (VitD) to improve systemic lupus erythematosus in mice to breakthrough clinical treatment approaches. Body weight changes were recorded during rearing. Antinuclear antibodies (ANA), anti-dsDNA, and anti-snRNP were detected in the mouse serum using an enzyme-linked immunosorbent assay. Apoptosis of Th1 and Th2 immune cells in mice was detected using flow cytometry. Reverse transcription polymerase chain reaction was used to detect the expression of T-bet, GATA3, and Wnt3a mRNA in the spleens of each group. Western blot analysis was performed to detect the expression of Wnt1, p-β-catenin, β-catenin, glycogen synthase kinsase3β (GSK-3β), Wnt3a, c-myc, and cyclin D1 protein in mice spleens. β-catenin in mice spleen was visualized using immunohistochemistry. VitD did not substantial reduce the body weight of MRL/LPR mice, whereas the inhibitor did. VitD notably decreased the concentrations of ANA, anti-double-stranded DNA, and anti-snRNP in the serum of MRL/LPR mice and alleviated apoptosis of Th1 and Th2 cells. VitD markedly increased the expression of T-bet and GATA mRNA in the spleen of MRL/LPR mice and consequently increased the levels of Wnt3a and β-catenin. Western blot analysis revealed that the levels of GSK-3β, p-β-catenin, Wnt1, Wnt3a, c-myc, and cyclin D1 could be reduced by VitD, compared with MRL/LPR. Immunohistochemistry demonstrated that the expression of β-catenin was the most pronounced in the spleen of MRL/LPR mice, and the expression level of β-catenin decreased substantially after VitD intervention. VitD can further inhibit the nuclear translocation of β-catenin by downregulating the expression of Wnt ligands (Wnt1 and Wnt3a), which reduces the expression of the downstream target gene cyclin D1. Systemic lupus erythematosus in mice was improved by inhibiting the activation of Wnt/β-catenin signal pathway.