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Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs, including the brain. Though 50% of patients may experience neuropsychiatric symptoms (NPSLE), disease mechanisms remain largely unknown. Microglia, the resident innate immune cell of the brain, have recently been implicated in NPSLE. Microglia are heterogenous and may be associated with a homeostatic microglia (CD11clo) or a disease-associated microglia (DAM; CD11chi) state common to neurodegenerative disease and aging. Yet, few studies have examined microglia subsets in NPSLE. We showed that expression of a shared NPSLE transcriptional signature and DAM-associated genes correlates with the severity of behavioral deficits in microglia isolated from two NPSLE models prior to overt systemic disease. Further, our single-cell RNA-seq data identify homeostatic microglia and DAM states in control and NPSLE-prone mice. DAM in NPSLE are enriched for genes associated with antigen presentation but depleted for genes associated with phagocytosis, which is in contrast to DAM in neurodegenerative disease that are critical for phagocytic functions. Moreover, NPSLE CD11clo microglia upregulate genes linked to synapse pruning and phagocytosis, consistent with exacerbated synaptic pruning by microglia in models of NPSLE. We also find that restricted expression of the DAM transcriptional program in NPSLE DAM corresponds to improved behavioral outcomes in NPSLE-prone mice following treatment with fingolimod, a sphingosine-1-phosphate receptor modulator that reduces microglia activation and improves blood brain barrier integrity. These discoveries mark the first to implicate DAM as a potentially pathogenic microglia subset in NPSLE.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that can affect multiple organ systems, including the kidneys, skin, and brain. T cells are an important mediator in this end organ damage. CD6 is a co-stimulatory receptor, predominantly expressed on T cells, which binds with activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. This signaling pathway is vital for T cell activation, proliferation, differentiation and trafficking. We found increased expression of both CD6 and ALCAM in the kidneys of MRL/lpr mice (a spontaneous model of SLE) versus healthy control B6 mice. In a separate experiment, female MRL/lpr mice were aged to 9–10 weeks of age, at which point we began treating with either anti-CD6 antibody (60 ug/dose, intraperitoneally twice per week), isotype control (60 ug/dose, twice per week), or cyclophosphamide (25 mg/kg, once per week). We also included a no treatment group and a group of MRL/MpJ mice, a congenic healthy control strain. Mice treated with anti-CD6 show lower levels of proteinuria and BUN (p

Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting multiple organs, including the brain. Though 50% of patients may experience neuropsychiatric symptoms (NPSLE), disease mechanisms remain largely unknown. Microglia are the resident innate immune cell of the brain and suggested to act as drivers of neurological conditions ranging from neurodevelopmental (autism) to neurodegenerative (Alzheimer’s disease) disorders. The majority of investigations into microglia, particularly the recently discovered disease-associated microglia (DAM) subset, have occurred in models of neurodegenerative disease. However, few studies have examined microglia, particularly DAM, in NPSLE. We performed RNA-seq on microglia isolated from two NPSLE-prone mouse strains to correlate gene signatures with behavioral deficits. Similar to patients, both NPSLE models exhibit cognitive impairment. Of the significantly upregulated genes found in NPSLE microglia compared to their respective controls, a common 18-gene ‘NPSLE signature’ is shared and enriched for genes associated with lipid metabolism, scavenger receptor activity and downregulating inflammatory responses and cell chemotaxis processes. NPSLE microglia are also enriched for DAM-associated genes and expression of ‘NPSLE’ and ‘DAM’ signatures significantly correlates with the severity of behavioral deficits. The discovery of our novel ‘NPSLE signature’ and enrichment of the ‘DAM signature’ represents the first to connect microglia-specific transcriptional signatures with clinical outcomes. In future studies, we will assess the penetrance of these signatures to further interrogate how defective microglial function may incite NPSLE.

Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus (SLE) in which T cells are thought to play an essential role. CD6 is a co-stimulatory receptor on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation and trafficking, and increased levels of CD6 are associated with pathogenic T cell responses. To assess the role of the CD6-ALCAM pathway in LN pathogenesis, we tested a monoclonal antibody against CD6 in a short-term, validated, inducible murine model of lupus nephritis known as nephrotoxic serum nephritis (NTN). NTN mice were treated 3× per week with an anti-CD6 mAb (10D12, 60ug/dose, n=23) or with vehicle control (n=23). Healthy mice were also included as a control (n=12). Mice treated with the anti-CD6 mAb displayed decreased levels of proteinuria (p

Neuropsychiatric systemic lupus erythematosus (NPSLE) manifests as a range of symptoms, including cognitive abnormalities, mood disorders, and psychosis. The pathogenesis is complex and not yet completely understood. We recently proposed that the choroid plexus (CP) could be an alternative entry site to the blood brain barrier for immune mediators to enter the central nervous system. In the MRL/lpr lupus mouse model, the CP is heavily infiltrated with T and B cells, which is reminiscent of human NPSLE patients whose autopsies revealed infiltrates in the CP. Nevertheless, the role of CP infiltrating T cells in the pathogenesis is not known and is the objective for this study. In two cohorts, we evaluated whether intracerebroventricularly (ICV) delivered MRL/lpr CP T cells accelerate neuropsychiatric disease in young female MRL/lpr mice. Six week old female MRL/lpr mice were injected with MRL/lpr CP CD3+ T cells, MRL/lpr splenic CD3+ T cells, or PBS. The T cells were pooled from the CP and spleen of 16 week old MRL/lpr mice and expanded with anti-CD3/CD28 antibodies (1 ug/mL) and mIL-2 (10 U/mL) for 4 days. About ~200,000 cells were injected into the lateral ventricle from the bregma at AP −0.34 mm, ML −1.0 mm, and depth −2.0 mm. After 4 weeks post-injection, we found that CP T cell recipient mice had increased cognitive deficits compared to PBS controls. Histological assessment revealed an increase in lymphocytic infiltration, the number of proliferating cells, and IgG and complement deposition, in the choroid plexus of CP T recipient mice. Our result indicate that T cells can migrate to and proliferate in the CP following ICV injection, and that brain infiltrating MRL/lpr T cells may be a key contributor to the pathogenesis of NPSLE.

The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) remains unclear, despite the large number of lupus patients affected by neuropsychiatric manifestations. We recently showed that MRL/lpr mice, a classic SLE mouse model with neuropsychiatric symptoms, develop tertiary lymphoid structures (TLS) (a.k.a. ectopic germinal centers). TLS structurally and functionally resemble lymph nodes and form at the site of chronic inflammation to create more localized and specialized immune responses, which may promote a local immune response against self-antigen and thus contribute to the pathogenesis. Since CXCL13 is a key chemokine that drives lymphoid formation, we injected female MRL/lpr mice intraperitoneally with an anti-CXCL13 antibody, an IgG isotype control antibody, or PBS three times weekly for 12 weeks starting at 6–8 weeks of age. Cognitive dysfunction and affective deficits were assessed at the end of treatment. Anti-CXCL13 mAb treated MRL/lpr mice showed significant improvement in spatial memory, recognition memory, and depression-like behavior as compared to the two control groups (p=0.034, p=0.013, and p=0.0087, respectively). Furthermore, mice receiving the antibody by intracerebroventricular delivery showed similar results (p=0.055, p=0.20, and p=0.018, respectively). There were, however, no apparent differences in indicators of systemic disease, including autoantibody titers. Our results suggest that TLS play an important role in the pathophysiology of affective deficits and cognitive dysfunction, and that CXCL13 neutralization may be a potential therapeutic strategy to target NPSLE.

Approximately 40% of lupus patients experience neurological and/or psychiatric disorders. However, the mechanisms contributing to the wide range of neuropsychiatric manifestations (NPSLE) have not yet been fully elucidated. We have demonstrated that the choroid plexus (CP) in the MRL/lpr NPSLE lupus mouse model is heavily infiltrated with effector T cells. We investigated T cell receptor (TCR) usage and repertoire diversity in CP infiltrating T cells, to enhance our understanding of this T cell population and its possible contribution to neuropsychiatric disease. The CP, salivary glands, and spleens were dissected from female MRL/lpr mice (n=9, n=9, n=4 respectively) and the spleens from female MRL/MpJ (n=5) at 17–22 weeks of age, at which time MRL/lpr mice display prominent neurobehavioral deficits. The TCR repertoire was evaluated by isolating genomic DNA, amplifying the CDR3 sequence by multiplex PCR (Adaptive Technologies), and analyzing the sequences with the Adaptive Technologies immunoSeq Analyzer and the R software, LymphoSeq. MRL/lpr CP tissues had increased numbers of T cell sequences, unique productive sequences, and clonality. The top 10 productive rearrangements were more abundant in MRL/lpr CP when compared to the MRL/lpr and MRL/MpJ splenic tissues (p = 0.0673 and p = 0.0003 respectively). The MRL/lpr CP had an increased frequency of TCRBV02, TCRBV04, TCRBV12, and TCRBV26 genes compared to MRL/lpr splenic and salivary gland tissues. Despite the lymphoproliferation found in this lupus strain, the increased oligoclonal expansion and limited TCR V beta gene usage in the MRL/lpr CP T cell repertoire, as compared to other MRL/lpr tissues, implies a distinct and specific infiltration of T cells into the CP.

BACKGROUND Cell-bound complement activation products (CB-CAPs) are sensitive and specific diagnostic markers of systemic lupus erythematosus (SLE). We compared the performance of CB-CAPs to low serum complement C3 or C4 in distinguishing SLE from other rheumatic diseases and healthy individuals. METHODS Adult subjects (n=1200) were enrolled from multiple academic centers, including SLE (498), healthy individuals (252) and subjects with other rheumatic diseases (450). Erythrocyte bound C4d [EC4d] and B-Lymphocyte bound C4d [BC4d] were quantitated using flow cytometry. Serum C3 and C4 levels were determined using immunoturbidimetry. Measurements included sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic curve (ROC) and Youden Index, for each marker as well as combinations. RESULTS Abnormal CB-CAPs status yielded 62% sensitivity with 88% specificity in distinguishing SLE from the group with other diseases compared to low C3/C4 status − 38% sensitivity, 93% specificity. Youden index was 0.492±0.03 for CB-CAPs compared to 0.313±0.03 for low C3/C4 (p The cumulative complement scoring system yielded higher AUC (0.81). A score with greater than 1 complement abnormality yielded 45% sensitivity and 98% specificity. CONCLUSION Our data suggests that CB-CAPs have greater diagnostic performance than low serum complement C3/C4. The combination of these complement abnormalities in a composite complement score is superior in distinguishing SLE from other rheumatic diseases and healthy individuals.

IFN-α is a potent activator of innate and adaptive immunity, and its administration to preautoimmune (NZB×NZW)F1 mice promotes virulent systemic lupus erythematosus (SLE) disease. Given the known contributions of B cells and BAFF to SLE, we evaluated the ability of IFN-α administration to induce disease in wild-type (WT), B cell-deficient, and BAFF-deficient NZM 2328 mice. Whereas WT mice rapidly developed proliferative glomerulonephritis, marked proteinuria, and increased mortality in response to IFN-α administration, B cell-deficient mice developed neither renal pathology nor clinical disease. Moreover, BAFF-deficient mice, despite developing limited glomerular IgG and C3 deposition, also remained free of histological glomerulonephritis and clinical disease. Strikingly, similar T cell expansion and serum IgG responses were observed in adenovirus (Adv)-IFN–treated WT and BAFF-deficient mice despite their disparate pathological and clinical responses, whereas numbers of activated B cells increased in WT mice but not in BAFF-deficient mice. Nonetheless, B cell, plasma cell, and T cell infiltration of the kidneys in Adv-IFN–treated WT mice was similar to that in WT mice treated with Adv-control. Its ability to promote SLE disease in WT mice notwithstanding, IFN-α administration failed to drive the preferential expansion of CD4+ memory T cells that occurs during the natural course of disease, and glomerular infiltration of macrophages failed to associate with development of disease. These results collectively suggest that therapeutic targeting in SLE of BAFF and/or B cells in SLE could be successful even in states of IFN-α overexpression. Moreover, our results document important biological differences between IFN-α–driven and spontaneous natural SLE disease.

Neuropsychiatric manifestations of systemic lupus erythematosus (NPSLE) affect approximately 40% of patients. Lipocalin-2 (LCN2), an acute-phase reactant protein, is an established urinary biomarker in lupus patients. Previous studies using LCN2-deficient mice have demonstrated its role in glial migration and chemokine regulation in the brain. We therefore hypothesized that LCN2 is involved in NPSLE pathogenesis. We investigated the lupus prone B6.Sle1.Sle3 (Sle1,3) mouse and the effects of LCN2 deficiency on the development of the neuropsychiatric phenotype exhibited by this strain. Sle1,3, B6.LCN2KO, B6, and Sle1,3-LCN2KO mice (6–10 month old; n=5–10/group) underwent comprehensive neurobehavioral assessment, and brains were evaluated by flow cytometry and RNA sequencing. Sle1,3 mice exhibited significant impairment in spatial (p Our findings establish the Sle1,3 mouse as an NPSLE model and demonstrate that LCN2 deficiency attenuates neurobehavioral deficits and regulates microglial expression of genes essential to NPSLE development.

Systemic lupus erythematosus (SLE) can affect multiple different organ systems, including the kidneys (lupus nephritis, LN), brain (neuropsychiatric SLE, NPSLE), and skin (cutaneous lupus, CLE). B cells and macrophages are implicated in the pathogenesis of these disease manifestations. We have previously shown the importance of Bruton’s tyrosine kinase (BTK), an enzyme important in B cell and macrophage signaling, in reversing disease in a model of immune nephritis induced by the passive transfer of nephrotoxic antibodies. To extend our findings to a more severe type of nephritis more similar to human disease and examine the effects of BTK inhibition on extra-renal lupus manifestations, we treated a spontaneous model of lupus, MRL/lpr mice, with the novel BTK inhibitor, BI-BTK-1. Early treatment with BI-BTK-1 normalized proteinuria (p MRL/lpr mice develop brain and skin disease that model the pathology of NPSLE and CLE, respectively. Early BI-BTK-1 treatment significantly improved cognitive function and decreased inflammatory cell infiltration into the brain choroid plexus. Moreover, skin disease was also significantly improved as determined by attenuated macroscopic and histologic skin scores. Our results further highlight the therapeutic potential of BI-BTK-1 in SLE, not only in treatment of LN, but potentially also for skin and brain disease as well.

Although the pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is still not well understood, autoantibodies, loss of brain barrier integrity, and pro-inflammatory cytokines have all been implicated in disease progression. Fingolimod (FL), an approved treatment for multiple sclerosis, improves neurologic disease through S1PR modulation and reducing passage of autoaggressive lymphocytes into the CNS. In initial studies, we found that FL treatment reduces lymphocytic infiltration of the choroid plexus in MRL/lpr lupus mice and attenuates memory impairment and depressive-like behavior. Here, we determined the effects of FL on systemic disease and pro-inflammatory cytokine expression in the CNS. Brain, spleen, lymph node, and serum samples were evaluated in MRL/lpr mice treated with FL three times weekly for 10 weeks starting at 10 weeks of age and from untreated controls (n=16/group). FL-treated mice exhibited a significant reduction in splenomegaly and lymphadenopathy. However, there was no change in serum titers of anti-double stranded DNA antibodies. Moreover, immunofluorescence staining revealed no difference in IgG deposition between treated and control mice, suggesting that autoantibody reduction is not critical for FL-mediated disease improvement. Surprisingly, quantification of brain cortical cytokine levels revealed a significant increase in IL-1a, IL-1b, IFNg, IFNb and IL-12p70 in FL-treated mice, while IL-1a, IL-1b and MCP-1 were increased in the hippocampus. These findings suggest that FL attenuates NPSLE, but the mechanism of improvement is not mediated by a reduction of autoantibodies or pro-inflammatory cytokine signaling.

Classification and treatment decisions in lupus nephritis (LN) are largely based on renal histology. Single-cell RNAseq (scRNAseq) analysis may accurately differentiate types of renal involvement at the transcriptomic level, and better inform treatment decisions and prognosis. scRNAseq was performed on kidney and non-lesional skin tissue collected from clinically indicated renal biopsies, and skin biopsies obtained at the time of renal biopsies, in 20 systemic lupus erythematosus (SLE) patients. Cell-types were determined using principal component analysis and tSNE plotting, resulting in the definitive identification of keratinocytes, tubular cells, mesangial cells, fibroblasts, endothelial cells, and leukocytes. Tubular cells in patients with proliferative nephritis demonstrated upregulated TNF signaling compared with membranous nephritis. Interestingly, keratinocytes of patients with proliferative nephritis also demonstrated upregulated TNF signaling. Furthermore, tubular cells of patients who did not respond to standard immunosuppressive treatment showed upregulation of extracellular matrix proteins and fibrotic markers at the time of biopsy. Using logistic regression analysis, a 4-gene tubular fibrosis score was created which predicted response to treatment, with an area under curve of 0.9. We conclude that scRNAseq using small amounts of renal biopsy tissue in SLE can differentiate between the different classes of LN, and provide important insights into potential pathogenic mechanisms. Further, changes in the skin of LN patients can provide a useful source of biomarkers and may reflect important information concerning concurrent kidney pathological events.

Immune-mediated glomerulonephritis is a serious end organ pathology that affects patients with systemic lupus erythematosus (SLE). A common murine model used to study lupus nephritis (LN) is nephrotoxic serum nephritis (NTN) in which mice are passively transferred nephrotoxic antibodies. We have previously shown that macrophages are important for the pathogenesis of LN. To further investigate the mechanism and determine if the contribution of macrophages is mediated via the NF-κB pathway we created B6 mice which had RelA knocked out in myeloid cells, thus inhibiting classical NF-κB signaling in this cell lineage. We induced NTN in this strain to assess the importance of macrophage derived NF-κB signaling in contributing to disease progression. Myeloid cell RelA knock out (KO) mice injected with nephrotoxic serum had significantly ameliorated proteinuria (p Taken together, our studies indicate that macrophage NF-κB signaling is instrumental in the contribution of this cell type to the pathogenesis of NTN. Our results suggest that while approaches which decrease macrophage numbers can be effective in immune mediated nephritis, more targeted treatments directed at modulating macrophage signaling and/or function could be beneficial, at least in the early stages of disease.

To identify potential biomarkers in immune-mediated nephritis, urine from mice subjected to an augmented passive model of anti-glomerular basement membrane (GBM)-induced experimental nephritis was resolved using two-dimensional gels. The urinary proteome in these diseased mice was comprised of at least 71 different proteins. Using orthogonal assays, several of these molecules, including serum amyloid P (SAP), PG D synthase, superoxide dismutase, rennin, and total protease were validated to be elevated in the urine and kidneys of mice during anti-GBM disease, as well as in mice with spontaneously arising lupus nephritis. Among these, urinary protease was the only marker that appeared to be exclusively renal in origin, whereas the others were partly serum-derived. Longitudinal studies in murine lupus demonstrated that total urinary protease had better predictive value for histologically active nephritis (r = 0.78) compared with proteinuria (r = −0.04), azotemia (r = 0.28), or the other markers examined, whereas urine SAP emerged as the single most predictive marker of histological glomerulonephritis. Collectively, these studies uncover total urinary protease, PG D synthase, SAP, and superoxide dismutase as novel biomarkers of anti-GBM disease and lupus nephritis, with stronger correlation to renal disease compared with currently employed biomarkers. These findings could have important diagnostic and prognostic ramifications in the management of these renal diatheses.

In otherwise non-autoimmune-prone C57BL/6 (B6) mice rendered genetically deficient in CD152 (CTLA-4), polyclonal hypergammaglobulinemia with increased levels of systemic lupus erythematosus (SLE)-associated IgG autoantibodies, glomerular IgG and C3 deposition, and interstitial nephritis all developed by 3–5 wk of age. Remarkably, superimposing genetic deficiency of BAFF (B cell-activating factor belonging to the TNF family) onto CD152 deficiency did not substantially attenuate humoral autoimmunity and immunopathology in these mice, despite the resulting marked reduction in B-lineage cells. Although superimposing a BAFF transgene (resulting in constitutive BAFF overexpression) onto CD152-deficient mice did lead to increases in B-lineage cells and serum levels of certain SLE-associated IgG autoantibodies, renal immunopathology remained largely unaffected. Taken together, these results demonstrate that global T cell dysregulation, even in an otherwise non-autoimmune-prone host, can promote systemic humoral autoimmunity and immunopathology in a BAFF-independent manner. Moreover, supraphysiologic expression of BAFF in the setting of ongoing autoimmunity does not necessarily lead to greater immunopathology. These findings may help explain the limited clinical efficacy appreciated to date of BAFF antagonists in human SLE.

Target Ag display is a necessary requirement for the expression of certain immune-mediated kidney diseases. We previously had shown that anti-DNA Abs that cross-react with α-actinin may be important in the pathogenesis of murine and human lupus nephritis; in murine models, we had found that a significant proportion of pathogenic serum and kidney-deposited Igs are α-actinin reactive. Furthermore, a pathogenic anti-DNA/α-actinin Ab showed enhanced binding to immortalized mesangial cells (MCs) derived from a lupus prone MRL-lpr/lpr mouse as compared with MCs from BALB/c mice which are not susceptible to spontaneous lupus, suggesting that kidney α-actinin expression may be contributing to nephritis. In the current study, we established that two isoforms of α-actinin that are present in the kidney, α-actinin 1 and α-actinin 4, can both be targeted by anti-α-actinin Abs. We found novel sequence polymorphisms between MRL-lpr/lpr and BALB/c in the gene for α-actinin 4. Moreover, α-actinin 4 and a splice variant of α-actinin 1 were both expressed at significantly higher levels (mRNA and protein) in MCs from the lupus prone MRL-lpr/lpr strain. Significantly, we were able to confirm these differences in intact kidney by examining glomerular Ig deposition of anti-α-actinin Abs. We conclude that enhanced α-actinin expression may determine the extent of Ig deposition in the Ab-mediated kidney disease in lupus. Modulation of Ag expression may be a promising approach to down-regulate immune complex formation in the target organ in individuals with circulating pathogenic Abs.

Lupus nephritis (LN) patients currently lack highly effective and safe treatment options. Bruton’s tyrosine kinase (BTK) is a tyrosine kinase which is important for B cell and macrophage function. Based on preliminary studies in an inducible model of nephritis that highlighted the importance of BTK dependent macrophage effector function in the pathogenesis of disease, we extended our studies to explore the effect of BTK inhibition on remission induction of established, proliferative nephritis. Nephritis was induced in female 129 sv/J mice (10 weeks of age) via the passive transfer of nephrotoxic serum (NTS). Once a mouse developed severe nephritis (>300 mg/dl proteinuria for two consecutive days, or a single measurement of >2000 mg/dl), treatment begun via daily oral gavage with 3 mg/kg of BI-BTK-1, a novel, highly selective and potent BTK inhibitor. Each treated mouse was matched to a control mouse that received daily gavage of the vehicle alone. Within two days of beginning treatment, mice treated with BI-BTK-1 had a significant reversal of the proteinuria compared to vehicle control treated mice (p Therapeutic treatment with BI-BTK-1 reversed severe, established nephritis induced by pathogenic antibodies. These exciting results indicate the novel therapeutic potential of BTK inhibition for treating LN patients with established disease.

Approximately 40% of SLE patients suffer from neuropsychiatric manifestations, including cognitive dysfunction and depression. While the pathogenesis of neuropsychiatric lupus (NPSLE) is not fully understood, critical aspects of disease development include neuroinflammation and the loss of blood-brain barrier (BBB) integrity. Fingolimod is an FDA-approved sphingosine-1 phosphate (S1P) receptor modulator that has been shown to reduce CNS lymphocytic infiltration and prevent BBB disruption in multiple sclerosis. In this study, we investigated the effects of fingolimod in MRL-lpr/lpr mice, a classic spontaneous SLE model. Ten-week-old mice were treated prophylactically three times weekly with vehicle alone or fingolimod (3mg/kg, n=16/group). After 4 weeks of treatment, mice were assessed for alterations in cognitive function or affective behaviors. Fingolimod-treated mice exhibited significantly improved preference for novel objects (p=0.019) and objects in novel placement (p=0.007) when compared with control-treated mice, thereby demonstrating maintenance of recognition memory and visual memory, respectively. Mice treated with fingolimod also displayed less despair-induced immobility during the forced swim test (p=0.017), which indicates that fingolimod may mitigate depression. Preliminary histological assessment suggests decreased perivascular and periventricular leakage of serum albumin in fingolimod-treated mice. A reduction in lymphocytic infiltration of the choroid plexus in the lateral ventricles was also observed. Our results highlight the important role of S1P in the pathogenesis of neurocognitive manifestations of SLE and receptor modulation as a potential therapeutic target for NPSLE.

Systemic lupus erythematosus is a polygenic autoimmune disease with a broad spectrum of clinical manifestations including organ-threatening kidney inflammation associated with immune complex (IC) deposition. The kidney is populated by a rich network of tissue-resident mononuclear phagocytes that express Fc receptors (FcRs) and internalize ICs. TL1A (TNFSF15) is a tumor necrosis factor (TNF) superfamily cytokine that can be induced in myeloid cells following ligation of TLR’s and FcRs by IgG IC’s and co-stimulates T cells through its receptor DR3. We have found that DR3 deficiency protects MRL/lpr lupus-prone mice from the renal inflammation but not systemic autoimmunity that develops in these mice, suggesting that TL1A/DR3 interactions could play a role in activating autoimmune T cells and antigen presentation in the kidney. We identified four distinct populations of renal phagocytes based on the expression of F4/80 and CD11b. One population of cells constitutively expressed high levels of surface class II and the CD28 ligand B7-2(CD86), suggesting that they would be potent antigen presenting cells. A fifth population expressing similar markers was present only in inflamed kidneys, both in the MRL/lpr mouse model of lupus and nephrotoxic serum-induced nephritis. Both of these populations constitutively expressed surface TL1A. The new population increases with the severity of nephritis and is unequally distributed between the cortex and the medulla. We will further analyze these populations via transcriptomics and functional assays after immune complex stimulation. This work identifies CX3CR1+ MNP’s as possible antigen-presenting cells in the kidney and a source of TL1A for costimulation of autoreactive T cells in nephritis.

Immunization of nonautoimmune BALB/c mice with multimeric DWEYSVWLSN, a peptide mimotope of DNA, induces anti-DNA and other lupus-associated Abs. To further investigate the pathogenesis of the autoantibody response induced by peptide immunization, we generated hybridomas from peptide-immunized mice that bound peptide, dsDNA, cardiolipin, Sm/ribonucleoprotein (RNP), or some combination of these Ags. Analysis of 24 IgM Abs led to the identification of three groups of Abs: 1) Abs reactive with peptide alone, 2) anti-peptide Abs cross-reactive with one or more autoantigens, and 3) autoantibodies that do not bind to peptide. The gene families and particular VH-VL combinations used in those hybridomas binding DNA were similar to those used in the anti-DNA response in spontaneous murine lupus. Another similarity to the spontaneous anti-DNA response was the generation of arginines in the complementarity-determining region-3 of DNA-binding hybridomas. Interestingly, one Ab had the VH-VL combination present in the original R4A anti-DNA Ab used to select the DWEYSVWLSN peptide from a phage display library. Many of the heavy and light chains displayed evidence of somatic mutation, suggesting that they were made by Ag-activated B cells. Analysis of the Ab repertoire in peptide-induced autoimmunity may provide insights into the generation of anti-DNA Abs following exposure to foreign Ag. Furthermore, the recovery of an Ab with the heavy and light chain combination of the Ab originally used to isolate the immunizing peptide confirms the utility of phage display peptide libraries in generating true molecular mimics.

Cutaneous lupus erythematosus (CLE) is a common disease manifestation in SLE patients. Ultraviolet-B irradiation (UVB) is well-known trigger of CLE; many lupus patients are photosensitive, and develop skin lesions following sun exposure. TWEAK, a TNF superfamily member, is a soluble cytokine that binds a sole signaling receptor, Fn14. TWEAK/Fn14 interactions are important in key biologic processes, including angiogenesis, cell death and inflammation, and are often upregulated during tissue injury. We found that MRL-lpr/lpr (MRL/lpr) lupus-prone mice deficient for the Fn14 receptor (Fn14KO) had markedly attenuated skin lesions following UVB irradiation, as compared to Fn14WT mice. Histologically, MRL/lpr Fn14WT mice had more keratinocyte apoptosis, degeneration of the basement membrane, and dermal inflammation. Further evaluation showed decreased infiltration of IBA-1+ macrophages, CD3+ T cells, and NGAL+ cells (neutrophils) in the skin of MRL/lpr Fn14KO mice. Depletion of macrophages in MRL/lpr mice using the CSF1R/fms kinase inhibitor GW2580 attenuated irradiation-induced tissue injury, confirming a pathogenic role for these Fn14-expressing cells in UVB-induced lesions. Several key chemokines (including MCP-1, MIP-1α, MIP-1β) were decreased following macrophage depletion and also differentially expressed between Fn14KO and WT mice, suggesting regulation through TWEAK/Fn14 signaling. Finally, both in vitro and in vivo, Fn14 was upregulated on dead and dying cells following irradiation, which can locally sensitize cells to the effects of TWEAK. Our data reveals important contributors to the pathogenesis of UVB-induced cutaneous lesions, and suggests a novel role for TWEAK/Fn14 signaling in skin disease.

In addition to an overt neuropsychiatric phenotype (depression and cognitive dysfunction), MRL/lpr mice display robust infiltration of lymphocytes through the brain choroid plexus (CP). However, the functional features of these cells, or their potential contribution to neuropsychiatric disease, have not been explored in detail. Localized tertiary lymphoid structures have been described in the kidney and peritoneum in spontaneous and inducible lupus models. Given the immunoprivileged nature of the brain and recent studies describing the critical role of the CP in immune surveillance, we evaluated 3D reconstructions of B-cell and T-cell infiltrates. We hypothesized that these CP infiltrates consist of tertiary lymphoid structures (ectopic germinal centers), induced in response to local and/or systemic inflammation. We found dramatically increased lymphotoxin-β and CXCL13, key molecules in lymphoid follicle organization, in the CP of MRL/lpr mice. Lymphotoxin-β and CXCL13 were upregulated by 8 weeks of age, well before any cellular infiltration was present. Organizational and phenotypic features of lymphoid follicles, including clusters of proliferating (PCNA+) T and B cells, organized according to local chemokine gradients, were present. Furthermore, we identified phenotypically distinct groups of B-cells (e.g. IgM+, GL7+, BCL6+) and T-cells (e.g. FoxP3+, BCL6+, GL7+), as well as follicular dendritic cells and extracellular reticular networks, consistent with lymphoid follicles. We therefore conclude that the CP of MRL/lpr mice contain germinal-center like structures. The contribution of these structures to the pathogenesis, including local production of neuropathic autoantibodies, remains to be determined.

Fn14, the sole receptor for the TNF family member TWEAK, is inducibly expressed in the brain in endothelial cells, astrocytes, microglia, and neurons. Previously, we had found that Fn14 knockout lupus MRL/lpr mice display significantly attenuated neuropsychiatric manifestations. To establish whether this improvement in disease is secondary to ablation of TWEAK-Fn14 signaling within the CNS or the periphery and determine whether TWEAK-mediated neuropsychiatric effects are strain dependent, we performed intracerebraventricular injection (ICV) of Fc-TWEAK or a control protein to B6 mice. Interestingly, we found that the Fc-TWEAK injected mice developed significant depressive-like behavior and abnormal cognitive dysfunction. Inflammatory mediators including MCP-1, C3, and iNOS were significantly elevated in the brains of Fc-TWEAK treated mice. Furthermore, Fc-TWEAK directly increased BBB disruption, as demonstrated by increased fibronectin deposition in the brain and reduced aquaporin-4 expression. Finally, Fc-TWEAK increased apoptotic cell death in the cortex, hippocampus, and brain stem. In conclusion, TWEAK can contribute to lupus-associated neurobehavioral deficits by acting within the CNS to enhance production of inflammatory mediators, promote permeability of the BBB, and induce apoptosis in resident brain cells. Our study provides further support that the TWEAK/Fn14 signaling pathway may be a potential therapeutic target for inflammatory diseases involving the CNS.

Neuropsychiatric disease is a common manifestation of systemic lupus erythematosus (SLE). Potential mechanisms underlying neuropsychiatric SLE (NPSLE) include thrombosis, complement deposition, brain-reactive autoantibodies, and cytokine mediated inflammation. However, it is not clear whether the neuropsychiatric manifestations of SLE are secondary to systemic disease, or a primary pathogenic process in the brain. In order to distinguish between central nervous system (CNS) and hematopoietic contributions, we generated bone marrow chimeras between lupus-prone MRL/lpr and congenic control MRL/+ mice. We monitored systemic disease progression, performed extensive behavioral testing, and analyzed brain tissue for evidence of inflammation. MRL/lpr (donor) → MRL/lpr (recipient) chimeric mice showed significantly elevated autoantibodies and marked proteinuria while MRL/+→MRL/+ and MRL/+→MRL/lpr did not, indicating abrogation of the MRL/lpr systemic disease phenotype. Surprisingly, MRL/+→MRL/lpr mice displayed a behavioral phenotype consistent with MRL/lpr→MRL/lpr mice, including depression-like behavior and increased spatial memory deficits. In contrast, MRL/+→MRL/+ mice behaved normally. Additionally, there was elevated RANTES expression, cellular infiltration, and neurodegeneration in MRL/lpr→MRL/lpr and MRL/+→MRL/lpr chimeric mice. The data presented herein indicate that the MRL/lpr CNS is sufficient to mediate NPSLE development absent significant hematopoietic contribution.

Cutaneous lupus erythematosus (CLE) in one of its various forms is seen in the majority of patients with systemic lupus. Photosensitivity is common in lupus, and sunlight (via UVB irradiation) is a well-recognized trigger of CLE. A TNF superfamily member, TNF-like weak inducer of apoptosis (TWEAK), is a soluble cytokine that binds to its sole known signaling receptor, Fn14. TWEAK and Fn14 are upregulated with inflammation and/or injury, and this pathway is important in critical biologic processes including angiogenesis, cell survival, and local inflammation. Previously, we found that lupus-prone MRL/lpr mice deficient for Fn14 (Fn14 KO) had markedly attenuated skin disease (which develops spontaneously in this strain) as compared to Fn14 wild-type (WT) mice. Here, we demonstrate that absence of the Fn14 receptor is protective against the expected UVB-induced eruption. MRL/lpr Fn14 WT mice developed significantly more severe skin involvement following exposure to UVB when compared to strain, age, and gender matched Fn14 KO mice. Histologically, MRL/lpr Fn14 WT mice had significantly more apoptotic keratinocytes, basement membrane degeneration, and dermal inflammation comprised of macrophages following UVB. RT-PCR revealed higher levels of CXCL9, CXCL10, CXCL11 and RANTES in MRL/lpr Fn14 WT mice. Our data suggests a novel role for the TWEAK/Fn14 signaling pathway in the induction of cutaneous lupus lesions following UVB.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of anti-nuclear autoantibodies and inflammatory damage in target organs, commonly the kidney and skin. Macrophages are thought to be important in the pathogenesis of lupus end organ disease, although studies to date have not been sufficiently conclusive. In this study, we investigated the role of macrophages in a spontaneous mouse model of SLE, the MRL/lpr mouse. PLX3397 (Plexxikon), a small molecule inhibitor of CSF1R, c-Kit, and FMS-like tyrosine kinase 3a, was administered in chow form to MRL/lpr mice starting at 9 weeks of age. Compared to matched control treated mice, PLX3397-treated MRL/lpr mice exhibited significantly less proteinuria and improved renal function. Furthermore, anti-dsDNA antibodies were significantly reduced at several time points. IBA-1 staining confirmed macrophage depletion within the kidneys of the PLX3397 treated mice. Interestingly, PLX3397-treated mice were also protected from the development of spontaneous skin lesions common to MRL/lpr mice, with control treated mice displaying significantly worse cutaneous disease macroscopically at 16 and 22 weeks of age. The improvement in skin lesions in PLX3397-treated mice was confirmed histologically in skin obtained at sacrifice. Our results indicate an important role for macrophages in the pathogenesis of skin and kidney disease in MRL/lpr mice, and implicate macrophages as a potential therapeutic target in SLE.

The TNF superfamily member TWEAK acts through its receptor, Fn14, to mediate several key pathological processes known to be involved in lupus nephritis. To explore the potential for renal protection by targeting this pathway, we introduced the Fn14 null allele into the MRL/lpr lupus mouse strain. At 26 to 38 weeks of age, female MRL/lpr Fn14 knockout (KO) mice (backcross #8) had significantly lower levels of proteinuria, BUN, and creatinine as compared to Fn14 wild type (WT) mice. Furthermore, Fn14KO mice had significantly improved renal pathology, accompanied by attenuated glomerular and tubulointerstitial inflammation and less glomerular immune deposits. There were no detectable differences between the strains in either serum autoantibody levels or in splenic immune cell subsets, although less lymphadenopathy was observed in Fn14KO mice. Importantly, Fn14KO mice displayed substantial preservation of podocytes in kidney glomeruli. Moreover, we demonstrated that TWEAK signaling via Fn14 directly damaged the integrity of the glomerular filtration barrier through effects on both podocytes and glomerular endothelial cells. Inhibition of TWEAK signaling due to deficiency of the Fn14 receptor significantly improves renal disease in a spontaneous lupus nephritis model, likely through prevention of the direct injurious effects of TWEAK on the filtration barrier. Thus, blocking the TWEAK/Fn14 axis is a novel therapeutic target in immune-mediated proliferative glomerulonephritis.

Cutaneous manifestations are very common in the systemic autoimmune disease systemic lupus erythematosus (SLE). TWEAK is a soluble cytokine member of the TNF superfamily that binds to a sole receptor, Fn14. TWEAK/Fn14 signaling is involved in cell survival, apoptosis, cytokine production, and angiogenesis, and as such has been found to be important in both tissue repair and inflammatory diseases. Whether TWEAK is involved in autoimmune skin disease is not known. We found that lupus-prone MRL/lpr mice deficient for Fn14 (Fn14KO) had attenuated cutaneous disease as compared to Fn14WT mice. MRL/lpr Fn14KO mice had significantly less epidermal acanthosis and follicular plugging, and there was decreased infiltration of T cells and macrophages as compared to Fn14WT mice. In vitro, TWEAK treatment of murine keratinocytes stimulated the secretion of RANTES (via Fn14), and promoted apoptosis. Parthenolide decreased production of RANTES, indicating that this effect of TWEAK is mediated via NF-KB. Ultraviolet light, specifically UVB, is recognized as a potent trigger of cutaneous lupus. We found that TWEAK co-treatment exacerbates UVB light- induced keratinocyte apoptosis and increases the amount of RANTES secreted, when compared to cells treated with UVB alone. These synergistic effects of TWEAK+UVB on keratinocytes were likely due to upregulation of Fn14 expression by UVB. Our data strongly implicate TWEAK/Fn14 signaling in the pathogenesis of the cutaneous manifestations of lupus.

Neuropsychiatric disease (NPSLE) is one of the most common manifestations of human lupus, but remains poorly understood. TWEAK is a cytokine member of the TNF superfamily; its sole receptor, Fn14, is expressed in brain endothelial cells, astrocytes, microglia and neurons. In vitro, TWEAK signaling promotes expression of MCP-1, IL-6 and IL-8, decreases ZO-1 expression, and increases the permeability of human brain microvascular endothelial cells. Our previous study showed that Fn14 knockout (KO) in the MRL/lpr lupus strain led to markedly attenuated neuropsychiatric disease, as revealed by significant reduction in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of NPSLE. We found that Fn14KO mice had improved BBB integrity as shown by decreased fibronectin and IgG deposition in the brain. Further, there were fewer cellular infiltrates in Fn14KO mice. Additionally, Fn14KO mice displayed reduced neuron apoptosis and hippocampal gliosis. Interestingly, there were no differences in neurogenesis and microglia activation, or in circulating autoantibody titers, between Fn14KO and Fn14WT mice. Our studies indicate that TWEAK/Fn14 interactions play a central role in the pathogenesis of NPSLE by improving BBB integrity and reducing neuronal damage, suggesting a novel target for therapeutic intervention for this major disease manifestation.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies and inflammatory damage in target organs. Kidney involvement affects up to 60% of lupus patients, and is responsible for significant morbidity and mortality. Previous studies using methods to reduce macrophage number have reached conflicting conclusions regarding the role of macrophages in lupus nephritis (LN). Moreover, “off target” effects occur in mice congenitally deficient in macrophages. In this study, we investigated the role of macrophages in mice receiving anti-glomerular antibodies, or nephrotoxic serum (NTS), an experimental model which closely mimics the immune complex mediated disease seen in LN. GW2580, an oral kinase inhibitor monospecific for the CSF-1 receptor, was used as a novel and highly selective method for macrophage depletion. We found that GW2580-treated NTS challenged mice did not develop the significant increases in levels of proteinuria, serum creatinine or serum urea, seen in control treated mice. Further, GW2580-treated mice were protected from the robust kidney expression of inflammatory cytokines associated with LN seen in control treated mice, including RANTES, IP-10, VCAM1, MCP-1 and IL-6. IBA-1 staining confirmed profound depletion of macrophages in glomeruli of GW2580-treated mice. Our results support an important role of macrophages in LN, and suggest targeting this cell type as a promising approach to the treatment of LN.

There is strong experimental evidence that anti DNA antibodies are instrumental in LN pathogenesis. We found that exposure of murine mesangial cells to pathogenic anti DNA mAbs induces overexpression of Lipocalin 2, a protein known to be upregulated following kidney insult. In this study we explored the upregulation of Lipocalin 2 in the context of murine lupus nephritis. Kidney mesangial and tubular cells from MRL/lpr lupus prone mice were treated with the pathogenic anti DNA mAb 1A3F; consistent upregulation of Lipocalin 2 was detected. Similarly, IL1β, an inflammatory cytokine secreted by epithelial cells and macrophages, and agonists of TLR2 and TLR4 significantly stimulated the expression of Lipocalin 2. Interestingly, we found that 1A3F binds to HMGB1, an endogenous ligand for TLR 2/4. Old MRL/lpr mice displayed higher kidney RNA expression of Lipocalin 2 than BALB/c mice (p We conclude that Lipocalin 2 is upregulated in lupus kidneys, although whether this is only due to injury or plays a role in pathogenicity is yet unknown. Nevertheless, antibody deposition mediates the upregulation of Lipocalin 2 in intrinsic kidney cells with possible involvement of TLR 2/4 signaling.

SLE is a chronic, recurrent, potentially fatal multisystem inflammatory disorder mainly affecting women. SLE patients produce antibodies to many different self-antigens. Yet, currently used serological markers for SLE are lacking in specificity and/or sensitivity. The aim of this study was to develop an improved diagnostic test by measuring and multiplexing specific autoantibody reactivities in SLE patients. Autoantibody reactivity profiles in serum samples collected from 97 SLE patients within three years of the diagnosis were compared with those of 56 healthy controls. Autoantibody profiles were determined using the ImmunArray iCHIP™ - a proprietary protein microarray technology that allows the display of antigens representing a wide range of SLE-associated biochemical pathways on a single chip. Using this novel platform, SLE patients could be differentiated from healthy subjects by a relatively small subset of auto-antigens and Epstein Barr Virus (EBV) antigens. The autoantibody reactivity profile that allowed SLE diagnosis with high sensitivity and specificity displayed differential response to known SLE-specific antigens, such as single-stranded DNA and EBV, and to several novel ones. Conclusion: An antibody profile for SLE diagnosis using a single multiplexed chip was successfully developed. This profile may differentiate between SLE patients and healthy individuals. Validation of this profile in additional patient samples is ongoing.

We previously demonstrated an important role of the constant (C) region in the pathogenicity of anti-DNA antibodies (Ab) through modulation of immunoglobulin affinity and specificity. However, the mechanisms by which the C region affects the binding of autoantibodies to nuclear antigens (Ag) have yet to be elucidated. From a parent IgG3 anti-nuclear Ab, PL9-11, we generated IgG1, IgG2b, and IgG2a variants that share identical variable regions but that differ in their C region. The affinity of these Abs for histone antigens was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine the wavelength shift of the Ab panel upon the addition of DNA and histone Ags. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure of the PL9-11 panel corresponding to Ab-Ag interactions. SPR analysis revealed significant differences of histone 2A/2B binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the Ab isotype, while increasing salt concentrations lowered KD values. Circular dichroism analysis determined that changes in Ab secondary structure content differed between the IgG isotypes upon Ag binding. In conclusion, the Ag-binding affinity of anti-nuclear Abs is dependent on the particular C region. Ag binding effects on Trp fluorescence, as well as secondary structure shifts, were also C region dependent. Alteration of secondary structures influenced by C regions may explain differences in fine specificity of anti-DNA Abs between Abs with similar variable regions, as well as cross-reactivity of anti-DNA Abs with non-DNA Ags.

Neuropsychiatric disease in systemic lupus erythematosus (NPSLE) is a common and dangerous disease manifestation. However, the mechanisms underlying NPSLE are not fully understood, and the optimal treatment is not known. One postulated mechanism involves lupus autoantibodies with affinity for brain tissue, which due to a compromised blood brain barrier that allows for the passage from the serum into the brain can deposit in the CNS to induce cell damage and neuropsychiatric abnormalities. Therefore, B-cell depletion would be expected to be an effective intervention for NPSLE. To address this question experimentally, we studied B-cell depletion in a murine lupus model with prominent neuropsychiatric manifestations, the MRL-lpr/lpr strain (JHD-MRL/lpr). Comprehensive neurobehavioral tests including forced swim, anhedonia, open field, object recognition, object placement, and social preference were employed to evaluate the neuropsychiatric manifestations in B cell depleted JHD-MRL/lpr as compared to B cell sufficient MRL/lpr mice. Unexpectedly, we found that complete B cell depletion did not ameliorate major NPSLE features, including depression-like behavior and cognitive dysfunction. However, improved motor activity was observed in JHD-MRL/lpr mice. Thus, the absence of B cells and autoantibodies is not sufficient to ameliorate the expression of NPSLE in MRL/lpr mice. Whether therapeutic depletion or conditional B cell knockout would display similar effects is not yet known.

B7x (B7-H4, B7S1), a member of the B7/CD28 co-stimulatory superfamily, is an inhibitor of T cell activation and proliferation. B7x is highly expressed in peripheral, non-lymphoid tissues. However, the in vivo function of B7x is largely unknown. We hypothesized that B7x may modulate the pathogenesis of antibody mediated nephritis through its effects on T cells. We induced nephritis in B7x-deficient (B7x-/-) (n=10) and B7x-wild type (WT) B6 mice (n=10) pre-immunized with rabbit IgG by intravenous injection of nephrotoxic sera (rabbit anti-mouse glomerular antibodies). Following nephritis induction we observed a significant increase in the levels of serum IgG, particularly of the IgG2b and IgG1 isotype, in B7x-/- mice as compared to B7x WT mice. At the mRNA level, IL-23, CCL2 and CCR5 were significantly upregulated in B7x-/- kidney tissue. Immunohistochemistry revealed significantly increased glomerular infiltration of CD3+ T cells and CD68+ macrophages in B7x-/- kidneys. Furthermore, kidney histopathology was more severe in B7x-/- mice, in terms of infiltrating polymorphonuclear cells in both glomerular and interstitial compartments, crescent formation, and tubular atrophy/dilatation, as compared to B7x WT mice. In conclusion, B7x plays a protective role in immune mediated nephritis, likely through exerting an inhibitory effect on T cell activation and restricting the migration of immune cells. Our results indicate a potential role for modulation of the B7x pathway for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease (Goodpasture’s syndrome), lupus nephritis, and related conditions.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the earliest manifestations in human SLE, and a major cause of morbidity and mortality in this disease. While the pathways underlying NPSLE are not fully understood, blood brain barrier (BBB) disruption and altered brain perfusion have been postulated to contribute to the pathogenesis. The purpose of this study was to evaluate BBB integrity, macromolecule content, and other indicators of neurological damage in MRL-lpr/lpr mice which spontaneously develop NPSLE, compared to the background control strain MRL/+. Female MRL-lpr/lpr and MRL/+ mice were imaged by MRI at 9-10 weeks of age, at which time major neuropsychiatric symptoms including depression and cognitive dysfunction were already present. Dynamic contrast enhancement MRI indicated increased gadolinium uptake in MRL/lpr mice in all regions of the brain, with the largest differences visualized in the hypothalamus where these changes were significant. Furthemore, MRL/lpr mice displayed significantly higher concentrations of albumin, total immunoglobulin, and antibodies to double stranded DNA in their cerebrospinal fluid. In contrast, whole brain analysis of magnetic transfer contrast and quantitative T2 imaging indicated no significant differences between the MRL-lpr/lpr and MRL/+ mice. Thus, a compromised blood brain barrier (particularly in specific regions) in lupus prone MRL/lpr mice may allow for the passage of circulating autoantibodies from the serum into the brain, and induction of some of the neuropsychiatric abnormalities observed in this mouse strain. Preventing the regional breakdown in barrier integrity may be a novel approach to treatment of neuropsychiatric involvement in SLE.

Lupus nephritis (LN) is associated with significant morbidity and mortality. microRNAs (miRs) are small, non-coding RNAs that regulate translation. Previous, studies report changes in miR expression in kidney tissue, urine and PBMC that correlate with LN disease activity. However, LN WHO class-specific miRs have not been described. Using deep-sequencing, we aimed to identify WHO class-specific miRs in urine from adult and pediatric patients with biopsy-proven LN. Cell-free urine from adult (n=25) and pediatric (n=8) female patients with WHO class IV and V LN were obtained at time of active disease and during remission. Total RNA was used to prepare small RNA cDNA libraries for sequencing. Multiplexing through sample-specific 3' adapters was applied. Sequence reads were mapped to the human genome and small RNA databases. miRs were quantified by relative read abundance. We obtained reproducible profiles of miRs. In a paired-sample analysis comparing miR abundance in urine of adult and pediatric patients with active class IV versus class V LN, we found significant changes in 6 miRs, including up-regulation of miR-193a-5p, -423, 501-3p, and -874 by 400-1,000x in WHO class IV LN. In conclusion, we detected higher counts of specific miRs in urine of adult and pediatric patients with LN class IV, versus class V. Given the worse prognosis of class IV LN identifying miRs associated with this LN class is an important step in biomarker discovery of this particulary aggressive disease.

B7x (B7-H4, B7S1) is an important member of the B7/CD28 superfamily of T cell costimulatory proteins. B7x is a negative regulator of CD4+ and CD8+ T cell activation and proliferation, and is expressed in both hematopoietic and non-hematopoietic tissues. We hypothesized that B7x is instrumental in the pathogenesis of antibody mediated nephritis. To examine this question we induced nephritis in B7x-deficient (B7x-/-) (n=10) and WT B6 mice (n=10) by passive transfer (i.v.) of nephritogenic antibodies. Following induction of nephritis, we found a marked increase in the levels of serum IgG (p=0.023), IgG2b (p=0.055), and IgG1 (p=0.064) in B7x-/- mice compared to WT mice. Moreover, renal damage was more severe in B7x deficient mice. Serum creatinine and BUN were elevated in the B7x-/- group compared to the WT mice. Furthermore, B7x-/- mice demonstrated worse kidney histopathology, with an increase in infiltrating PMN cells in glomeruli (p

NGAL (lipocalin-2), a member of the lipocalin superfamily, is expressed by neutrophils, hepatocytes, and renal resident cells following ischemia or inflammatory conditions & is involved in innate immune responses during infection. However, its role in adaptive immunity has not been described. Previously, we had found that NGAL is instrumental in the pathogenesis of antibody mediated nephritis. To investigate a possible role for NGAL in the generation of autoreactive antibody responses, we examined its effect in an experimental lupus model. We injected pristane (0.5 ml per mouse i.p.) into wild type B6 (n=10) and NGAL-deficient (NGAL-/-) (n=10) mice and followed the course of disease. Analyzing the levels of serum autoantibodies at 3 months post injection, NGAL-/- mice had significant increases in IgG2a (p=0.007) and IgG2b (p=0.019) anti-double stranded DNA antibodies, and IgG2a (p=0.002) & IgG2b (p=0.010) anti-single stranded DNA antibodies, and IgG anti-histone antibodies (p=0.025) as compared to pristane challenged, NGAL sufficient mice. Immunoprecipitation studies showed elevated serum anti-Sm/RNP antibodies in NGAL-/- mice compared to the WT mice at 3 months post injection, with an increase in titer and epitope diversity of this response at 6 months post injection. In conclusion, NGAL delays the onset of anti-nuclear antibodies in pristane induced lupus. The mechanisms by which NGAL plays a protective role in humoral autoimmunity are under investigation.

Lupus nephritis (LN), which manifests histologically by glomerulonephritis, tubulointerstitial inflammation and mesangial cell proliferation, is a major cause of mortality and morbidity in systemic lupus (SLE). TNF ligands and receptors are important in the pathogenesis of LN; furthermore, we recently found that TWEAK, a member of the TNF ligand superfamily, was significantly increased in urine from patients with active LN as compared to patients with inactive renal disease. We studied the effects of TWEAK in human kidney cells including mesangial cells (MC), podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. TWEAK induces kidney cells to secret multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1a, GM-CSF, ICAM-1, and VCAM-1. Cytokine production is primarily mediated through NF-kB activation, and could be inhibited by novel anti-TWEAK mAb. TWEAK-stimulated chemokines induced PBMC migration, especially monocyte/macrophages, which may then infiltrate into lupus kidneys in vivo. Furthermore, we found that TWEAK promotes MC proliferation. In conclusion, TWEAK may play a pathogenic role in the development of LN by enhancing renal inflammation and MC proliferation. Furthermore, blocking TWEAK/Fn14 interactions may be a promising therapeutic target in LN and perhaps other immune-mediated renal diseases.