anti-dsDNA IgG Research Articles

B-105 Farr Radioimmunoprecipitation Method Enhances Serial Monitoring of Quantitative Anti-dsDNA (double-stranded) Antibody Measurements in Lupus

Abstract Background Anti-double stranded DNA (anti-dsDNA) antibodies are an important criterion in the diagnosis, classification, and management of systemic lupus erythematous (SLE). High avidity anti-dsDNA IgG may be more clinically relevant and specific for SLE. As such, radioimmunoassay according to the Farr technique (Farr-RIPA) is often considered more sensitive and quantitative than enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae immunofluorescence assay (CLIFT) methods. Methods Measurements of anti-dsDNA antibodies in 147,016 serum patient samples were obtained by lab-developed Farr-RIPA. Serum with endogenous antibodies binds to 125I-radiolabelled dsDNA and undergoes subsequent immunoprecipitation under high salt conditions which selects for highly avidity anti-dsDNA antibodies. The analytic measurement range (AMR) of the assay is 5.7 to 85 IU/mL, and dilutional linearity is validated to 68,000 IU/mL for results >85 (ULOQ). Inter-assay precision is <10% CV. Results Of 147,016 measured samples, 5.4% (7800) were positive for anti-ds DNA antibodies (mean 251.7) with numeric values, ranging from 8.0 to 55,399 IU/mL. The numeric distribution of all positive results was such that 76% (5859/7800) were between 8.0 - 60 IU/mL, and the median was 19.4 with an interquartile range (IQR) of 36.1. A small percentage (3.9%) had very high anti-dsDNA levels greater than 1000 IU/mL and 0.4% (35/7800) were greater than 10,000 IU/mL. Serial measurements comprised almost half (45.5%) of all positives, occurring in 1016 unique patients an average of 3.5 levels within a 2-year time frame. A fifth of those patients, 200, were more frequently monitored with at least 4 levels. Of patients with the very highest dsDNA (>10,000), we analyzed the results of 17 unique patients who were frequently monitored an average of 6.1 times. Peak and nadir values yielded a mean delta of 20,751 with a mean of 264 days elapsing between peak and nadir. All except one showed a marked decrease in Anti-dsDNA over time with an average decrease of 15,685 IU/mL. Conclusions Here, we performed 147,016 Anti-dsDNA antibody measurements by Farr-RIPA, a precise method with a broad analytic range as compared to other methods which have a limited upper limit of quantitation or are semi-quantitative. Our 7800 positive Farr-RIPA results exhibited quantitative results from 8.0 to 55,399 IU/mL. The median of 19.4 and IQR of 36.1 showed most results to be <55.5 IU/mL. Very high levels >300 and >1000 IU/mL were seen in 10% and 4% of patient samples, respectively. Serial monitoring was frequent. In patients with the very highest levels >10,000, an-eight fold decrease in peak Anti-dsDNA levels was achieved over a mean of 264 days with more than half (9/17) achieving levels <250. The ability to detect and meaningfully monitor these patients is enhanced by the Farr-RIPA method. In conclusion, in 7800 positive samples, we demonstrated a wide range of inter- and intra-patient numeric Anti-dsDNA concentrations that denote the clinical usefulness of serial monitoring by Farr-RIPA.

Increased autoreactivity and maturity of EBI2+ antibody-secreting cells from nasal polyps.

Elevated numbers of antibody-secreting cells (ASCs) and anti-double-stranded DNA (anti-dsDNA) antibodies are found in nasal polyp (NP) tissue. The presence of anti-dsDNA IgG in tissue prospectively predicts recurrent NP but the characteristics of the source ASCs are unknown. Here, we investigated whether NP B cells expressing the extrafollicular marker EBI2 have increased propensity for autoantibody production and evaluated the molecular characteristics of NP ASCs. NPs showed increased frequencies of anti-dsDNA IgG and total IgG ASCs compared with tonsils, with more pronounced differences among EBI2+ cells. In NPs, EBI2+ cells were frequently double negative (IgD-CD27-) and ASCs. Single-cell RNA-Seq analysis of tonsils and NPs revealed substantial differences in B lineage composition, including differences in percentages of ASCs, germinal centers, proliferative cells, and non-ASCs. NPs exhibited higher expression of specific isotypes (IGHE, IGHA1, IGHA2, and IGHG4) and mature plasma genes, including SDC1 and XBP1, than tonsils. Gene Ontology biological processes indicated upregulated NF-κB and downregulated apoptosis pathways in NP ASCs. Together, these data indicate that NP EBI2+ ASCs secret increased total and anti-dsDNA IgG compared with those from tonsils and had molecular features of mature plasma cell differentiation.

Progranulin mediates the onset of pristane induced systemic lupus erythematosus

BackgroundsProgranulin (PGRN) is a growth factor-like molecule with diverse roles in homeostatic and pathogenic processes including the control of immune and inflammatory responses. Pathogenic inflammation is a hallmark of systemic lupus erythematosus (SLE) and elevated serum levels of PGRN has been evaluated as a biomarker of disease activity in SLE. However, the role of PGRN in SLE has not been fully investigated. This study is aimed to determine the potential involvements of PGRN in SLE.MethodsWild type (WT) and PGRN knockout (PGRN-/-) C57BL/6 mice received intraperitoneal injection of pristane for induction of a murine model of SLE. Sera were collected every biweekly and levels of anti-dsDNA antibody, IgG, and inflammatory factors were measured. Mice were sacrificed 5 months later and the renal lesions, as well as the proportions of T cell subtypes in the spleen were analyzed.ResultsFollowing exposure to pristane, PGRN-/- mice generated significantly lower levels of anti-dsDNA antibody and IgG relative to WT mice. PGRN-/- mouse kidneys had less IgG and collagen deposition compared with WT mice after pristane injection.ConclusionThe results indicate that PGRN participates in inflammatory response and renal damage in pristane induced SLE models, suggesting that PGRN mediates the onset of SLE.

Association of gut commensal translocation with autoantibody production in systemic lupus erythematosus

Abstract Objective Bacterial translocation across the gut barrier has been implicated in the pathogenesis of systemic lupus erythematosus (SLE), though underlying mechanisms remain unclear. This study aimed to investigate the role of translocated bacteria in the context of molecular mimicry by utilizing lupus model mice and blood samples from untreated SLE patients. Methods Bacterial translocation was evaluated using nonselective cultured mesenteric lymph nodes (MLNs) from B6SKG mice, a lupus model characterized by impaired TCR signalling and gut dysbiosis. The relationships of detected pathobionts with autoantibody production were examined using in vivo experiments, enzyme-linked immunosorbent assay, immunoblotting, and epitope mapping. Results Culture-based bacterial profiling in MLNs demonstrated that Lactobacillus murinus was enriched in B6SKG mice with elevated anti-dsDNA IgG levels. Subcutaneous injection of heat-killed L. murinus induced anti-dsDNA IgG production without altering T- or B cell subset composition. Immunoblotting and mass spectrometry analysis identified a peptide ATP-binding cassette (ABC) transporter as a molecular mimicry antigen, with its cross-reactivity in lupus mice confirmed by serological assays and in vivo immunization. The L. murinus ABC transporter exhibited surface epitopes that were cross-reactive with sera from lupus mice and patients. The ABC transporter from R. gnavus, known for its pathogenic role in lupus patients, had a similar epitope sequence to that of the L. murinus ABC transporter and reacted with lupus sera. Conclusion ABC transporters from gut bacteria can serve as cross-reactive antigens that may promote anti-dsDNA antibody production in genetically susceptible mice. These findings underscore the role of commensal-derived molecular mimicry and bacterial translocation in lupus pathogenesis.

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.

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

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

Deletion of the Mitochondrial Membrane Protein Fam210b Is Associated with the Development of Systemic Lupus Erythematosus.

Mitochondrial dysfunction has been increasingly recognized as a trigger for systemic lupus erythematosus (SLE). Recent bioinformatics studies have suggested Fam210b as a significant candidate for the classification and therapeutic targeting of SLE. To experimentally prove the role of Fam210b in SLE, we constructed Fam210b knockout (Fam210b-/-) mice using the CRISPR-Cas9 method. We found that approximately 15.68% of Fam210b-/- mice spontaneously developed lupus-like autoimmunity, which was characterized by skin ulcerations, splenomegaly, and an increase in anti-double-stranded DNA (anti-dsDNA) IgG antibodies and anti-nuclear antibodies(ANA). Single-cell sequencing showed that Fam210b was mainly expressed in erythroid cells. Critically, the knockout of Fam210b resulted in abnormal erythrocyte differentiation and development in the spleens of mice. Concurrently, the spleens exhibited an increased number of CD71+ erythroid cells, along with elevated levels of reactive oxygen species (ROS) in the erythrocytes. The co-culture of CD71+ erythroid cells and lymphocytes resulted in lymphocyte activation and promoted dsDNA and IgG production. In summary, Fam210b knockout leads to a low probability of lupus-like symptoms in mice through the overproduction of ROS in CD71+ erythroid cells. Thus, Fam210b reduction may serve as a novel key marker that triggers the development of SLE.

#525 SGLT2 inhibition in non-diabetic CKD: results in experimental lupus nephritis

Abstract Background and Aims Sodium glucose cotransporter type 2 inhibitors (SGLT2i) potently attenuate cardiovascular morbidity and progression of CKD in patients with type 2 diabetes. It has been proposed that SGLT2 inhibition may also elicit renoprotective effects in non-diabetic CKD. The aim of this work is to evaluate whether the treatment with an SGLT2i improves kidney disease progression in a mouse model of lupus nephritis (LN). Method We performed a single center randomized controlled preclinical trial using female MRLlpr/lpr mice. The animals were randomly assigned to (1) vehicle or (2) empagliflozin (EMP, 10 mg/kg/day) treatment once signs of active systemic lupus erythematosus (SLE) appeared: proteinuria >100 mg/dL (++ in reactive strips) or lymphadenopathy in minimum 2 bilateral sites with proteinuria >30 mg/dL (+ in reactive strips). We included 13 mice per group that were treated during 4 weeks. Empagliflozin was given as an admix into standard chow diet. Reduction of urinary albumin to creatine ratio (UACR) was considered the primary endpoint whereas glomerular filtration rate (GFR), renal histology (LN activity/chronicity index, glomerulosclerosis, interstitial fibrosis) and SLE markers (total serum IgG, anti-dsDNA IgG, glomerular IgG deposits, lymph node weight, spleen weight) were considered secondary endpoints. Results After 4 weeks of treatment, no significant differences in fasting blood glucose levels were found between the experimental groups. As expected, empagliflozin administration notably inhibited SGLT2 in the tubular cells demonstrated by a significant increase of urinary glucose (p < 0.0001). This is consistent with the increased water intake observed in MRLlpr/lpr mice treated with empagliflozin (p < 0.0001). At the end of the experiment, vehicle treated MRLlpr/lpr mice showed a significant increase in UACR (p = 0.0012) consistent with LN progression that was not attenuated by empagliflozin. In line, empagliflozin did not modify GFR and LN activity/chronicity indexes, glomerulosclerosis or interstitial fibrosis in kidney. Finally, regarding SLE markers, 4-weeks empagliflozin treatment was not able to decrease total serum IgG, anti-dsDNA IgG, glomerular IgG deposits or lymph node weight. Only spleen weight was significantly decreased in empagliflozin treated MRLlpr/lpr mice (p = 0.003). Conclusion In our experimental setting, empagliflozin treatment reduced spleen weight suggesting a protective role in SLE in MRLlpr/lpr mice. However, empagliflozin did not modify LN progression in MRLlpr/lpr mice probably because the acute LN phase. It is expected that the positive effect in terms of reducing proteinuria will be observed in patients with LN and residual proteinuria

#752 Ponatinib exacerbates renal damage in MRL/lpr lupus model mice through the lipid metabolism pathway

Abstract Background and Aims The role of tyrosine kinase inhibitors (TKIs) in lupus nephritis is controversial. Ponatinib is a third-generation tyrosine kinase inhibitor that targets multiple receptors. This study aims to explore the effect of ponatinib in lupus nephritis, with the hope of providing insights for the clinical application of ponatinib. Method At 8 weeks of age, mice were randomly divided into four groups. One group received no treatment, while the other three groups were respectively administered Ponatinib, prednisolone acetate, and a combination of Ponatinib and prednisolone acetate. The treatment was concluded when the mice reached 16 weeks of age. ELISA was employed to measure the levels of anti-dsDNA IgG, ANA, and C3 in mouse serum. DEGs and DELs were identified using the DESeq2 package in R. Pathway and gene ontology enrichment analyses were performed using the MetaCore database. Results Compared to the negative control, those mice receiving ponatinib showed a significant increase in the urinary protein/creatinine ratio (P < 0.01), along with notable elevations in blood creatinine and blood urea nitrogen. Pathological examination of kidney tissue suggested predominant acute lesions, including transparent thrombi, cellulose-like necrosis, cellular/fibrous crescents, and interstitial inflammation. Interestingly, serum testing in MRL/lpr mice revealed no significant differences in lupus activity-related indicators, including autoantibodies ANA, double-stranded DNA, and complement C3, between the negative control group and the ponatinib group. Furthermore, it was discovered that the kidney damage caused by ponatinib in MRL/lpr mice could be reversed by prednisone. RNA-Seq of kidney tissue from MRL/lpr mice showed a total of 44 differentially expressed genes when comparing the negative control with mice receiving ponatinib, including 23 upregulated and 21 downregulated genes. Pathway analysis revealed that the top four pathways were all related to lipid metabolism. Conclusion Ponatinib significantly exacerbated kidney damage in MRL/lpr mice and did not induce changes in lupus activity.

Clinical and laboratory characteristics of early-onset and delayed-onset lupus nephritis patients: A single-center retrospective study

BackgroundLupus nephritis (LN) manifests systemic lupus erythematosus (SLE) and is characterized by various clinical and laboratory features. This study aimed to comprehensively evaluate the characteristics of LN patients according to the time of LN diagnosis: early-onset (LN diagnosed within one year from SLE diagnosis) vs. delayed-onset (LN diagnosed more than one year after SLE diagnosis).MethodsWe conducted a retrospective analysis of medical records from all SLE patients treated at the University Hospital in Kraków, Poland, from 2012 to 2022. We collected data on demographic, clinical, and laboratory characteristics, including histological findings, treatment modalities, and disease outcomes. Statistical analyses were performed to identify factors impacting LN development and prognosis.ResultsAmong 331 LN patients, early-onset was diagnosed in 207 (62.54%) and delayed-onset was documented in 122 cases (36.86%). In 2 (0.6%) LN cases, the time of first kidney manifestation in the SLE course was unknown. Delayed-onset LN had a higher female-to-male ratio and younger age at SLE diagnosis. This group was associated with more severe clinical manifestations. In turn, studied subgroups did not differ in internist comorbidities, kidney histopathology, and family history regarding autoimmune diseases. Delayed-onset LN exhibited a higher frequency of anti-dsDNA, anti-Smith, anti-Ro, anti-RNP, and anti-cardiolipin IgG autoantibodies. During a 14-year follow-up period, 16 patients died. Mortality rate and causes of death were comparable in both analyzed subgroups.ConclusionsMore severe clinical manifestations in delayed-onset LN prompt strict monitoring of non-LN SLE patients to diagnose and treat kidney involvement early. Also, recognizing the higher frequency of autoantibodies such as anti-dsDNA or anti-Smith in delayed-onset LN underscores the potential value of autoantibody profiling as a diagnostic and prognostic tool.

Novel mitophagy inducer alleviates lupus nephritis by reducing myeloid cell activation and autoantigen presentation

Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE), but its mechanism of onset remains unclear. Since impaired mitophagy has been implicated in multiple organs in SLE, we hypothesized that mitophagy dysfunction is critical in the development of LN and that pharmacologically targeting mitophagy would ameliorate this disease. Therefore, lupus-prone MRL/MpJ-Faslpr (MRL/lpr) and NZBWF1/J mice were treated with a novel mitophagy inducer, UMI-77, during their onset of LN. This treatment effectively mitigated kidney inflammation and damage as assessed by histology and flow cytometry. Furthermore, dendritic cell (DC)-T cell coculture assay indicated that UMI-77 treatment attenuated DC function that would drive T cell proliferation but did not directly influence the potent T cell proliferation in lupus mice. UMI-77 also restored mitochondrial function and attenuated proinflammatory phenotypes in lupus DCs. Adoptive transfer of DCs from MRL/lpr mice augmented serum anti-dsDNA IgG, urine protein and T cell infiltration of the kidney in MRL/MpJ mice, which could be prevented by either treating lupus donors in vivo or lupus DCs directly with UMI-77. UMI-77 also restored mitochondrial function in myeloid cells from patients with LN in vitro as evidenced by increased ATP levels. Thus, enhancing mitophagy in SLE restrains autoimmunity and limits kidney inflammation for LN development. Hence, our findings suggest targeting mitophagy as a tangible pathway to treat LN.

Lupus mice derived mesenchymal stromal cells: Beneficial or detrimental on SLE disease outcome

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies against nuclear genes, deposition of immune complexes, and autoimmune T cells, through which, tissue damage would ultimately occur. Furthermore, loss of immune tolerance and imbalance of Th1/Th2 cells in addition to Th17/Treg are contributed to the pathogenesis of SLE. Mesenchymal stromal cells (MSCs) infusion is a potential therapy for SLE disease. Despite a majority of SLE patients achieving clinical remission after allogeneic MSC infusion from healthy individuals, SLE patients have less benefited from autologous MSC infusion, justifying the probable compromised function of SLE patients-derived MSCs. In this study, we aim to further investigate the potential immunoregulatory mechanisms in which mesenchymal stromal cells derived from pristane-induced lupus mice, following injection into healthy and lupus mice, exert their possible effects on the lupus process. Method40 female Balb/c mice aged 3 weeks were purchased and randomly divided into six groups. First, lupus disease was induced into the lupus groups by intraperitoneal injection of pristane and then the mice were surveyed for 6 months. The body weight, anti-dsDNA autoantibody levels, serum creatinine, and Blood Urea Nitrogen (BUN) levels were measured in two-month intervals. After 6 months, the group of lupus mice was sacrificed, and lupus MSCs were isolated. Two months later, cultured lupus MSCs were intravenously injected into two groups of healthy and lupus mice. After two months, the mice were euthanized and the kidneys of each group were examined histologically by hematoxylin & eosin (H&E) staining and the immunofluorescence method was also performed to evaluate IgG and C3 deposition. The frequency of splenic Th1, Th2, Th17, and Treg cells was measured by flow cytometry. Moreover, the cytokine levels of IFN-γ, IL-4, IL-17, and TGF-β in sera were measured by ELISA method. ResultsOur results showed that the induction of lupus disease by pristane in Balb/c mice caused the formation of lipogranuloma, increased levels of anti-dsDNA autoantibodies, and impaired renal function in all pristane-induced lupus groups. In addition, the injection of lupus mesenchymal stromal cells (L-MSC) into healthy and lupus mice led to a further rise in anti-dsDNA serum levels, IgG and C3 deposition, and further dysfunction of mice renal tissue. Also, the flow cytometry results implicated that compared to the control groups, splenic Th1, Th2, and Th17 inflammatory cell subtypes and their secreted cytokines (IFN-γ, IL-4, and IL-17) in the sera of healthy and lupus mice were increased after the intake of L-MSC. Additionally, the splenic Treg cells were also significantly increased in the lupus mice receiving L-MSC. However, a decrease in serum levels of TGF-β cytokine was observed in healthy and lupus mice following L-MSC injection. In contrast, the lupus mice receiving healthy mesenchymal stem cells (H-MSC) manifested opposite results. ConclusionIn a nutshell, our results suggest that although allogeneic MSCs are encouraging candidates for SLE treatment, syngeneic MSCs may not be eligible for treating SLE patients due to their defects in regulating the immune system in addition to their capability in promoting inflammation which would consequently worsen the SLE disease status.

Allogeneic cord blood regulatory T cells decrease dsDNA antibody and improve albuminuria in systemic lupus erythematosus.

Lupus nephritis (LN) constitutes the most severe organ manifestations of systemic lupus erythematosus (SLE), where pathogenic T cells have been identified to play an essential role in 'helping' B cells to make autoantibodies and produce inflammatory cytokines that drive kidney injury in SLE. Regulatory T cells (Tregs), responsible for decreasing inflammation, are defective and decreased in SLE and have been associated with disease progression. We hypothesize that treatment with allogeneic, healthy Tregs derived from umbilical cord blood (UCB) may arrest such an inflammatory process and protect against kidney damage. UCB-Tregs function was examined by their ability to suppress CellTrace Violet-labeled SLE peripheral blood mononuclear cells (PBMCs) or healthy donor (HD) conventional T cells (Tcons); and by inhibiting secretion of inflammatory cytokines by SLE PBMCs. Humanized SLE model was established where female Rag2-/-γc-/- mice were transplanted with 3 × 106 human SLE-PBMCs by intravenous injection on day 0, followed by single or multiple injection of UCB-Tregs to understand their impact on disease development. Mice PB was assessed weekly by flow cytometry. Phenotypic analysis of isolated cells from mouse PB, lung, spleen, liver and kidney was performed by flow cytometry. Kidney damage was assessed by quantifying urinary albumin and creatinine secretion. Systemic disease was evaluated by anti-dsDNA IgG Ab analysis as well as immunohistochemistry analysis of organs. Systemic inflammation was determined by measuring cytokine levels. In vitro, UCB-Tregs are able to suppress HD Tcons and pathogenic SLE-PBMCs to a similar extent. UCB-Tregs decrease secretion of several inflammatory cytokines including IFN-γ, IP-10, TNF-α, IL-6, IL-17A, and sCD40L by SLE PBMCs in a time-dependent manner, with a corresponding increase in secretion of suppressor cytokine, IL-10. In vivo, single or multiple doses of UCB-Tregs led to a decrease in CD8+ T effector cells in different organs and a decrease in circulating inflammatory cytokines. Improvement in skin inflammation and loss of hair; and resolution of CD3+, CD8+, CD20+ and Ki67+ SLE-PBMC infiltration was observed in UCB-Treg recipients with a corresponding decrease in plasma anti-double stranded DNA IgG antibody levels and improved albuminuria. UCB-Tregs can decrease inflammatory burden in SLE, reduce auto-antibody production and resolve end organ damage especially, improve kidney function. Adoptive therapy with UCB-Tregs should be explored for treatment of lupus nephritis in the clinical setting.

Pathogenetic associations of anti-ribosomal P protein antibody titres and their subclasses in patients with systemic lupus erythematosus.

We evaluated the association between anti-ribosomal P antibody (anti-RibP) titres and disease activity in Japanese SLE patients. Eighty patients admitted and treated in Niigata University Hospital for new-onset or flare-up of SLE were included in this retrospective cross-sectional study. Clinical data were obtained from medical records at admission. The anti-RibP index, and cytokine and tryptophan metabolite levels were determined by ELISA. Of the 80 SLE patients, 30 had anti-RibP. Anti-RibP presence was associated with a greater prevalence of skin rash and more severe inflammatory responses, demonstrated by higher inflammatory cytokine levels, hypocomplementemia, and accelerated tryptophan metabolism, in younger patients. The serum anti-RibP index was correlated with age at diagnosis, clinical indicators, initial prednisolone dose, and cytokines and tryptophan metabolite levels in univariate analysis. Multivariate analysis showed that the anti-RibP index was independently associated with the initial prednisolone dose and the prevalence of skin rash. The anti-RibP IgGs were mainly the IgG2 and IgG3 subclasses, and anti-RibP IgG3 was associated with hypocomplementemia, higher DAS, accelerated kynurenine pathway activity, and higher proinflammatory cytokine production. The coexistence of anti-dsDNA IgG and anti-RibP IgG2 or IgG3 accompanied higher IL-10 and IFN-α2 levels; furthermore, anti-RibP IgG3 coexistence with anti-dsDNA antibody contributed to the requirement for higher initial prednisolone doses and accelerated kynurenine pathway activity. Anti-RibP was associated with clinical manifestations and parameters in SLE, and its index might be a useful indicator of disease severity. Anti-RibP IgG3 was the IgG subclass most strongly associated with the pathogenesis of SLE.

T-bet-expressing B cells contribute to the autoreactive plasma cell pool in Lyn-/- mice.

Systemic Lupus Erythematosus (SLE) is characterized by pathogenic autoantibodies against nucleic acid-containing antigens. Understanding which B-cell subsets give rise to these autoantibodies may reveal therapeutic approaches for SLE that spare protective responses. Mice lacking the tyrosine kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune diseases characterized by increased autoreactive plasma cells (PCs). We used a fate-mapping strategy to determine the contribution of T-bet+ B cells, a subset thought to be pathogenic in lupus, to the accumulation of PCs and autoantibodies in Lyn-/- mice. Approximately, 50% of splenic PCs in Lyn-/- mice originated from T-bet+ cells, a significant increase compared to WT mice. In vitro, splenic PCs derived from T-bet+ B cells secreted both IgM and IgG anti-dsDNA antibodies. To determine the role of these cells in autoantibody production in vivo, we prevented T-bet+ B cells from differentiating into PCs or class switching in Lyn-/- mice. This resulted in a partial reduction in splenic PCs and anti-dsDNA IgM and complete abrogation of anti-dsDNA IgG. Thus, T-bet+ B cells make an important contribution to the autoreactive PC pool in Lyn-/- mice.

Fecal microbiota transplantation attenuates hypertension in a female mouse model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a prototypic multisystem autoimmune disorder that disproportionately affects women. SLE 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. Alterations in the gut microbiota, or 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=19) and SLE (NZBWF1, n=19) mice with broad spectrum antibiotics (0.4 g/L ampicillin, 0.4 g/L neomycin sulfate, 0.4 g/L metronidazole, and 0.2 g/L vancomycin) in drinking water for one week to deplete the existing microbiota. Subsequently, mice were given 0.1 mL of fecal microbes 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 before and after FMT were assessed by 16S rRNA analysis of the microbiome. Mean arterial pressure (MAP, mmHg), measured in conscious mice by carotid artery catheter was higher in SLE mice that received SLE microbes (Control: 112±3 vs. SLE: 140 ±2 mmHg, p<0.0001). SLE mice that received FMT with control microbes had lower blood pressure (SLE-Control microbes: 126±4 mmHg, p=0.03). Renal injury, as assessed by urinary albumin excretion at the conclusion of the study, was not significantly different among the groups (p=0.3523). Circulating autoantibodies and immunoglobulins 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). Circulating IgM was also elevated in SLE mice (Control: 0.053±0.008 vs. SLE: 0.32±0.04 mg/mL, p<0.0001), and FMT lowered IgM in SLE mice that received FMT (0.19±0.03 mg/mL, p=0.0069). These data suggest that gut microbiota present in NZBWF1 mice contribute to both autoantibody production and the development of hypertension, and that interventions to alter the microbiora could have a beneficial impact on disease activity in SLE patients. NIH NIGMS P20GM104357 to UMMC Department of Physiology and Biophysics, NIH NHLBI R00HL146888 to Erin Taylor This is the full abstract presented at the American Physiology Summit 2023 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.

CXCR3 deficiency decreases autoantibody production by inhibiting aberrant activated T follicular helper cells and B cells in lupus mice

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a high level of autoantibody production. T follicular helper (Tfh) cells and B cells participate in the development of SLE. Several studies have shown that CXCR3+ cells are increased in SLE patients. However, the mechanism through which CXCR3 influences lupus development remains unclear. In this study, we established lupus models to determine the role of CXCR3 in lupus pathogenesis. The concentration of autoantibodies was detected using the enzyme-linked immunosorbent assay (ELISA), and the percentages of Tfh cells and B cells were measured using flow cytometry. RNA sequencing (RNA-seq) was performed to detect the differentially expressed genes in CD4+ T cells from wild-type (WT) and CXCR3 knock-out (KO) lupus mice. Migration of CD4+ T cells in spleen section was assessed using immunofluorescence. CD4+ T cell function in helping B cells produce antibodies was determined using a co-culture experiment and supernatant IgG ELISA. Lupus mice were treated with a CXCR3 antagonist to confirm the therapeutic effects. We found that the expression of CXCR3 was increased in CD4+ T cells from lupus mice. CXCR3 deficiency reduced autoantibody production with decreased proportions of Tfh cells, germinal center (GC) B cells, and plasma cells. Expression of Tfh-related genes was downregulated in CD4+ T cells from CXCR3 KO lupus mice. Migration to B cell follicles and T-helper function of CD4+ T cells were reduced in CXCR3 KO lupus mice. CXCR3 antagonist AMG487 decreased the level of serum anti-dsDNA IgG in lupus mice. We clarify that CXCR3 may play an important role in autoantibody production by increasing the percentages of aberrant activated Tfh cells and B cells and promoting the migration and T-helper function of CD4+ T cells in lupus mice. Thus, CXCR3 may be a potential target for lupus therapy.

Ophiopogonin D attenuates the progression of murine systemic lupus erythematosus by reducing B cell numbers.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune abnormalities leading to multi-organ damage. The activation of autoreactive B cell differentiation will lead to the production of pathogenic autoantibodies, contributing to the development of SLE. However, the effects of Ophiopogonin D (OP-D) on B cell activation and autoantibody production as well as renal injury in the pathogenesis of SLE remain unclear. MRL/lpr mice, one of the most commonly used animal models of SLE, were intragastrically administered with 5 mg/kg/d OP-D at 17 weeks of age for 3 weeks. The survival rates of mice in each group were monitored for 6 weeks until 23 weeks of age. Proteinuria and serum creatinine levels were measured. Serum levels of immunoglobulin (Ig)G, IgM, and anti-dsDNA autoantibodies were detected by enzyme-linked immunosorbent assay. Numbers of CD19+ B cells in the blood, spleen and bone marrow and numbers of splenic germinal center (GC) B cells were calculated by using flow cytometry. OP-D treatment prolonged survival in MRL/lpr mice. OP-D treatment reduced proteinuria and serum creatinine levels as well as mitigated renal pathological alternation in MRL/lpr mice. Furthermore, serum levels of IgG, IgM, and anti-dsDNA autoantibodies were reduced by OP-D treatment. OP-D lessened not only CD19+ B cells in the spleen and bone marrow but also plasma cells that secreted anti-dsDNA autoantibodies, IgG and IgM in the spleen and bone marrow. OP-D ameliorated the progression of SLE by inhibiting the secretion of autoantibodies though reducing B cell numbers.

A Case of Severe Lupus Myocarditis Preceded by Mesalazine-induced Lupus.

In drug-induced lupus (DIL), symptoms similar to those of systemic lupus erythematosus (SLE) usually resolve after discontinuation of the offending drug. A 41-year-old-woman with a history of ulcerative colitis presented with polyarthritis and myositis and was positive for anti-dsDNA IgG antibody. After discontinuation of mesalazine, the symptoms resolved, and the antibody titer decreased. The patient was diagnosed with DIL. Six months later, lupus myocarditis developed. After treatment with glucocorticoids, cyclophosphamide, intravenous immunoglobulin, and an intra-aortic balloon pump, she showed dramatic improvement. Patients with DIL and an immunological predisposition, such as anti-dsDNA antibodies, may have SLE and should be carefully monitored.

Monitoring of Serum TWEAK Level Guides Glucocorticoid Dosages in the Treatment of Systemic Lupus Erythematosus

Objective: Accurate assessment of systemic lupus erythematosus (SLE) disease activity is critical. This study explored the relationship between the serum level of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score as well as the role of TWEAK in guiding the glucocorticoid dosage in SLE treatment. Methods: This study involved 131 patients with SLE, 34 with subacute cutaneous lupus erythematosus (SCLE), 22 with discoid lupus erythematosus (DLE), and 32 healthy volunteers. The serum and urinary TWEAK levels were determined. Monomeric C-reactive protein (mCRP), anti-dsDNA IgG, antinuclear antibody (ANA), complements in serum and urinary albumin level were measured. The SLEDAI 2000 (SLEDAI-2K) was used to evaluate disease activity. The correlation of the SLEDAI-2K score with all biomarkers was determined. Methylprednisolone was orally administered to patients with SLE depending on the serum level of TWEAK. Results: TWEAK levels were higher in patients with SLE or SCLE than in patients with DLE or healthy controls. The serum TWEAK level was positively correlated with the SLEDAI-2K score in patients with SLE (P < 0.001), and was more strongly correlated with the SLEDAI-2K score than other parameters (P < 0.001). Moreover, TWEAK-based glucocorticoid therapy was associated with lower SLEDAI-2K scores, better tapering of glucocorticoid doses, and fewer lupus flares in patients with SLE. Conclusions: Serum TWEAK is a useful biomarker reflecting SLE disease activity. Monitoring of the serum TWEAK level can improve the outcomes of glucocorticoid therapy in patients with SLE.