Human Lupus Research Articles

Porcine ubiquitin-like protein MNSFβ promotes cell apoptosis and covalently binds to BCL-G to enhance staurosporine-induced apoptosis.

BackgroundMonoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family. It functions as a regulator of cell apoptosis and a potential tumor suppressor, playing a vital role in the processes of immune cell function and apoptosis.MethodsThe present study constructed GFP-pMNSFβ swine umbilical vein endothelial cell (SUVEC) lines and investigated the function of porcine MNSFβ (pMNSFβ) in apoptosis, as well as its interactions with pBCL-G. Results revealed that stably expressed pMNSFβ protein in SUVEC lines significantly enhanced staurosporine (STS)-induced apoptosis. pMNSFβ proteins interacted with pBCL-G proteins and the expression of these interacting proteins synergized to further enhance STS-induced apoptosis.ResultsGFP-pMNSFβ stably expressed SUVEC lines through transient transfection and neomycin screening methods. Over 90% of the SUVEC cultures expressed GFP signals, and 41.5 kDa GFP-pMNSFβ proteins were detected with western blotting methods. Annexin V-PE/PI staining and flow cytometry analyses showed that overexpression of pMNSFβ proteins significantly elevated STS-induced apoptosis rates. Co-immunoprecipitation methods revealed an interaction between pMNSFβ and pBCL-G proteins. BCL-G is a proapoptotic member of the BCL-2 family that has been shown to be misexpressed in human systemic lupus erythematosus, as well as mammary and prostate cancers. Here, we demonstrated that the co-expression and potential conjugation of pMNSFβ and pBCL-G proteins synergistically enhanced STS-induced apoptosis.ConclusionsThe present study was the first to characterize the function of MNSFβ in porcine cells, and to clarify the function of MNSFβ in apoptosis. These results reveal that pMNSFβ is a potential molecular model for future investigations of diseases related to human MNSFβ dysfunction.

Bcl-3 inhibits lupus-like phenotypes in BL6/lpr mice.

Bcl-3 is an atypical member of the IκB family that modulates NF-κB activity in nuclei. lpr mice carry the lpr mutation in Fas, resulting in functional loss of this death receptor; they serve as models for lupus erythematosus and autoimmune lymphoproliferation syndrome (ALPS). To explore the biologic roles of Bcl-3 in this disease model, we generated BL6/lpr mice lacking Bcl-3. Unlike lpr mice on an MRL background, BL6/lpr mice present with very mild lupus- or ALPS-like phenotypes. Bcl-3 KO BL6/lpr mice, however, developed severe splenomegaly, dramatically increased numbers of double negative T cells-a hallmark of human lupus, ALPS, and MRL/lpr mice-and exhibited inflammation in multiple organs, despite low levels of autoantibodies, similar to those in BL6/lpr mice. Loss of Bcl-3 specifically in T cells exacerbated select lupus-like phenotypes, specifically organ infiltration. Mechanistically, elevated levels of Tnfα in Bcl-3 KO BL6/lpr mice may promote lupus-like phenotypes, since loss of Tnfα in these mice reversed the pathology due to loss of Bcl-3. Contrary to the inhibitory functions of Bcl-3 revealed here, this regulator has also been shown to promote inflammation in different settings. Our findings highlight the profound, yet highly context-dependent roles of Bcl-3 in the development of inflammation-associated pathology.

In vitro regulation of IL-6 and TGF-ß by mesenchymal stem cells in systemic lupus erythematosus patients.

Aim To analyse the ability of mesenchymal stem cells (MSCs) to regulate interleukin 6 (IL-6) and transforming growth factor (TGF-β) expression in vitro under co-culture conditions in human systemic lupus erythematosus (SLE). Method This study used a post-test group design that used peripheral blood mononuclear cells (PBMCs) from SLE patients at Kariadi Hospital, Semarang, Indonesia, and MSCs from a human umbilical cord. The cells were divided into two groups. The control group of PBMCs was treated with a standard medium, and the treatment group was co-cultured with the MSCs at a 1:40 ratio. Following 24 h incubation, the levels of IL-6 and TGF-β released in the culture medium were measured using a specific ELISA assay. Results This study showed a significant decrease in IL-6 level (p<0.05) and a significant increase in TGF-β level (p<0.001) following 24 h of co-culture incubation of human SLE PBMCs cells and MSCs. Conclusion The PBMCs-to-MSCs ratio of 1:40 can regulate the IL-6 and TGF-β levels in human SLE PBMCs.

EGR2 is elevated and positively regulates inflammatory IFN\u03b3 production in lupus CD4+ T cells

BackgroundRecent studies have shown that early growth response 2 (EGR2) is highly induced in activated T cells and regulates T cell functions. In normal C57BL/6 (B6) mice, deletion of EGR2 in lymphocytes results in the development of lupus-like systemic autoimmune disease, which implies indirectly an autoimmune protective role of EGR2. Conversely, increased EGR2 gene expression is suggested to link with high risk of human lupus. In the present studies we sought to clarify the expression and inflammation regulatory role of EGR2 in murine lupus T cells directly.ResultsWe performed RT-qPCR analysis and found a significant increase of EGR2 mRNA expression in human lupus PBMCs and in CD4+ T cells from three different murine lupus models including MRL-lpr, B6-lpr, and B6.sle123 mice at diseased stage when compared to age-matched control MRL or B6 mice. By performing intracellular flow cytometry analysis, we found that EGR2 protein expression was significantly increased in resting lupus (either MRL-lpr or B6.sle123) CD4+ T cells when compared to CD4+ T cells from their respective non-autoimmune controls. However, there was no difference of EGR2 protein expression in anti-CD3 and anti-CD28 stimulated control and lupus CD4+ T cells since there was a stronger induction of EGR2 in activated control CD4+ T cells. EGR2 expression was significantly increased in MRL-lpr mice at an age when lupus is manifested. To understand further the function of elevated EGR2 in lupus CD4+ T cells, we inhibited EGR2 with a specific siRNA in vitro in splenocytes from MRL-lpr and control MRL mice at 15 weeks-of-age. We found that EGR2 inhibition significantly reduced IFNγ production in PMA and ionomycin activated MRL-lpr lupus CD4+ T cells, but not control MRL CD4+ T cells. We also found that inhibition of EGR2 in vitro suppressed the Th1 differentiation in both MRL and MRL-lpr naïve CD4+ T cells.ConclusionsEGR2 is highly upregulated in human and murine lupus cells. Our in vitro data suggest a positive role of EGR2 in the regulation of Th1 differentiation and IFNγ production in lupus effector CD4+ T cells.

Correlation of the levels of DNA-binding inhibitor Id3 and regulatory T cells with SLE disease severity

E proteins, a subset of basic helix-loop-helix (bHLH) proteins, are transcription activators and their functions are inhibited by DNA-binding inhibitor (Id) 1–4. Studies have shown that Treg levels are decreased in Id3 knockout mice. Mice over-expressing Id1 in CD4 T cells possessed a greater number of regulatory T cells (Treg) and exhibited attenuated experimental autoimmune encephalomyelitis (EAE). The significance of Id proteins in human systemic lupus erythematosus (SLE) remains unclear. In this study, we systematically analyzed Id transcription in naïve, memory CD4 cells and regulatory T cells in peripheral blood mononuclear cells (PBMCs) in patients with active or inactive SLE. In parallel, Treg subsets in PBMCs were analyzed using different strategies. Id expression levels were correlated with Treg numbers as well as clinical indicators. We found that Id genes expressed in human peripheral CD4 cells were mainly Id2 and Id3. Id3 levels were significantly elevated in CD4+CD25hi T cells of patients with active SLE. Likewise, Id3 levels were positively correlated with increased CD4+FoxP3+ and CD4+Helios+FoxP3+ Treg cells in these patients. Id3 levels were found to be positively correlated with erythrocyte sedimentation rate (ESR), lupus anticoagulant (LAC), ribosomal antibody and SLE Disease Activity Index (SLEDAI) in patients with active SLE. Mice overexpressing Id1 in CD4+ T cells possessed significantly higher Treg levels in spleen and lower autoantibody concentrations in serum. Our results suggest that during the pathogenesis of SLE, up-regulation of Id3 can promote Treg differentiation to play an inhibitory effect on autoimmune responses.

IFNβ signaling inhibits osteogenesis in human SLE bone marrow.

Bone marrow mesenchymal stem cells are multipotent adult stem cells that can differentiate into osteoblasts, adipocytes, and chondrocytes. Our recently published data demonstrate that systemic lupus erythematous bone marrow mesenchymal stem cells produce increased quantities of interferon β based on a positive feedback loop involving the innate signaling molecule mitochondrial antiviral signaling protein. Moreover, this pathway contributes to human systemic lupus erythematous bone marrow mesenchymal stem cell senescence-like features. Here we investigate the differentiation defects of systemic lupus erythematous bone marrow mesenchymal stem cells and the potential for therapeutic interventions. The six systemic lupus erythematous patients recruited in this study satisfy the American College of Rheumatology 1997 classification criteria for systemic lupus erythematous. Systemic Lupus Erythematous Disease Activity Index-2K was used to determine disease activity. Systemic lupus erythematous bone marrow mesenchymal stem cells were isolated with Ficoll centrifugation and phenotyped using flow cytometry. In vitro studies included real-time polymerase chain reaction and western blotting. We compared six age-paired bone marrow aspirates from healthy controls and systemic lupus erythematous patients. Systemic lupus erythematous bone marrow mesenchymal stem cells display significantly reduced alkaline phosphatase staining, as well as reduced expression of osteogenic markers alkaline phosphatase, Runt-related transcription factor 2, and bone sialoprotein. When healthy bone marrow mesenchymal stem cells were treated with interferon β for 6 hours, expression of these same osteogenic markers was markedly reduced. Conversely the application of interferon β neutralizing antibody enhanced the expression of osteoblastogenesis markers. When the underlying mechanisms for interferon β inhibition of osteoblastogenesis were investigated, we found that IFNβ pre-treatment activates the inhibitory Smad6 and Smad7 expression through JAK1/STAT1, leading to reduced Smad1 phosphorylation and nuclear translocation. Our present work suggests that interferon β affects osteogenesis. By revealing the essential role of interferon β on systemic lupus erythematous bone marrow mesenchymal stem cell differentiation, our study sheds light on systemic lupus erythematous pathogenesis and provides a new potential therapeutic target for the bone complications found in systemic lupus erythematous.

Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury.

Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge8-/- , leading to defective clearance of apoptotic cells; and either C1q-/- or C3-/- , leading to low complement levels. We report that proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge8 -/- C1q -/- and Sle1.Mfge8 -/- C3 -/- triple-mutant [TM] strains [C1q -/-TM and C3-/- TM, respectively]), with structural kidney changes resembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q-/- TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this strain. Interestingly, enhanced thrombin activation in C3-/- TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention.

B cell depletion in murine lupus using cytotoxic T lymphocytes in vivo: Feasibility and benefit

Given the promising results in human lupus with B cell depletion, we tested whether in vivo cytotoxic T lymphocyte (CTL) could eliminate autoreactive B cells in the setting of murine lupus. Using the parent-into-F1 (P → F1) model to generate CTL that eliminate B cells, we found that transfer ofNZB parental splenocytes into lupus-prone female NZB/W F1 mice resulted in profound B cell reduction whereas NZW → F1 mice exhibited defective B cell elimination. Using pre-disease or early disease B/W mice as hosts, NZB → F1 mice exhibited B cell depletion and improved proteinuria but no improvement in survival whereas NZW → F1 mice had significantly reduced proteinuria and prolonged survival. Thus, despite the defective IL-2 environment in B/W F1 mice, generation of CTL and B cell depletion is feasible in NZB → F1 mice. The surprising increase in survival for NZW → F1 mice despite defective B cell elimination suggests that NZW splenocytes may contain a beneficial down regulatory cell.

Verdinexor, a selective inhibitor of nuclear export, decreases plasma cell survival in human lupus

Abstract Systemic lupus erythematous (SLE) is a complex autoimmune disease affecting multiple organs. Anti-nuclear antibody (Ab) producing plasma cells (PCs) play a critical role in the disease pathogenesis and are an important target for developing therapies against SLE. Karyopharm Therapeutics (KPTI) is an oncology-focused pharmaceutical company dedicated to the discovery, development, and commercialization of novel first-in-class drugs for the treatment of cancer and other major diseases. KPTI’s lead compound, XPOVIO™ (selinexor), received accelerated approval from the FDA in combination with dexamethasone as a treatment for patients with heavily pretreated multiple myeloma. KPTI’s selective inhibitors of nuclear export (SINE, verdinexor) are effective in reducing nephritis in the NZBW/F1 mouse model, and dramatically reduced autoreactive PCs. To further define whether SINEs directly decrease PC survival and/or generation, we studied PCs in human lupus ex vivo. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from healthy (n=3) and SLE donors (n=3) were treated with verdinexor and PC survival was determined by IgG ELISPOT and apoptosis assay. Verdinexor treatment significantly reduced the number of Ab secreting cells from healthy and SLE donor PBMCs as well as BMMCs (IC50 =0.1uM). Upon ex vivo verdinexor treatment (0.5uM), the levels of live blood plasmablasts and bone marrow CD19+ PCs were reduced by 30% with increased level of apoptotic cells. In contrast, ex vivo verdinexor treatment had no effect on naïve B cells and T cells from healthy and SLE PBMCs and BMMCs. These results support the hypothesis that SINEs have a direct effect on PC survival and represent a novel treatment approach for SLE.

Plasmacytoid dendritic cells from parent strains of the NZB/W F1 lupus mouse contribute different characteristics to autoimmune propensity.

The NZB/W F1 (F1) mice develop severe disease that is similar to human systemic lupus erythematosus. By contrast, each parent strain, NZB or NZW, has limited autoimmunity, suggesting traits of both strains contribute to pathogenesis. Although many of the contributing genes have been identified, the contributing cellular abnormality associated with each parent strain remains unresolved. Given that plasmacytoid dendritic cells (pDCs) are key to the pathogenesis of lupus, we investigated the properties of pDCs from NZB and NZW mice. We found that NZB mouse had higher numbers of pDCs, with much of the increase being contributed by a more abundant CD8+ pDC subset. This was associated with prolonged survival and stronger proliferation of CD4+ T cells. By contrast, NZW pDCs had heightened capacity to produce interferon-α (IFNα) and IFNλ, and promoted stronger B-cell proliferation upon CpG stimulation. Thus, our data reveal the different functional and numerical characteristics of pDCs from NZW and NZB mouse.

A Missense Variant Affecting the C-Terminal Tail of UNC93B1 in Dogs with Exfoliative Cutaneous Lupus Erythematosus (ECLE).

Cutaneous lupus erythematosus (CLE) in humans encompasses multiple subtypes that exhibit a wide array of skin lesions and, in some cases, are associated with the development of systemic lupus erythematosus (SLE). We investigated dogs with exfoliative cutaneous lupus erythematosus (ECLE), a dog-specific form of chronic CLE that is inherited as a monogenic autosomal recessive trait. A genome-wide association study (GWAS) with 14 cases and 29 controls confirmed a previously published result that the causative variant maps to chromosome 18. Autozygosity mapping refined the ECLE locus to a 493 kb critical interval. Filtering of whole genome sequence data from two cases against 654 controls revealed a single private protein-changing variant in this critical interval, UNC93B1:c.1438C>A or p.Pro480Thr. The homozygous mutant genotype was exclusively observed in 23 ECLE affected German Shorthaired Pointers and an ECLE affected Vizsla, but absent from 845 controls. UNC93B1 is a transmembrane protein located in the endoplasmic reticulum and endolysosomes, which is required for correct trafficking of several Toll-like receptors (TLRs). The p.Pro480Thr variant is predicted to affect the C-terminal tail of the UNC93B1 that has recently been shown to restrict TLR7 mediated autoimmunity via an interaction with syndecan binding protein (SDCBP). The functional knowledge on UNC93B1 strongly suggests that p.Pro480Thr is causing ECLE in dogs. These dogs therefore represent an interesting spontaneous model for human lupus erythematosus. Our results warrant further investigations of whether genetic variants affecting the C-terminus of UNC93B1 might be involved in specific subsets of CLE or SLE cases in humans and other species.

TLR7 and TLR8 Differentially Activate the IRF and NF-κB Pathways in Specific Cell Types to Promote Inflammation.

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-κB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-κB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation.

Regulatory role of SphK1 in TLR7/9-dependent type I interferon response and autoimmunity.

Plasmacytoid dendritic cells (pDCs) express Toll like receptors (TLRs) that modulate the immune response by production of type I interferons. Here, we report that sphingosine kinase 1 (SphK1) which produces the bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P), plays a critical role in the pDC functions and interferon production. Although dispensable for the pDC development, SphK1 is essential for the pDC activation and production of type I IFN and pro-inflammatory cytokines stimulated by TLR7/9 ligands. SphK1 interacts with TLRs and specific inhibition or deletion of SphK1 in pDCs mitigates uptake of CpG oligonucleotide ligands by TLR9 ligand. In the pristane-induced murine lupus model, pharmacological inhibition of SphK1 or its genetic deletion markedly decreased the IFN signature, pDC activation, and glomerulonephritis. Moreover, increases in the SphK1 expression and S1P levels were observed in human lupus patients. Taken together, our results indicate a pivotal regulatory role for the SphK1/S1P axis in maintaining the balance between immunosurveillance and immunopathology and suggest that specific SphK1 inhibitors might be a new therapeutic avenue for the treatment of type I IFN-linked autoimmune disorders.

Bioengineered Immunotherapy Techniques for the Treatment of Select Secondary Immunodeficiency Disorders

The inner workings of the immune system are quite complex. Each component works diligently in order to protect us from invaders such as pathogens: viruses, microbes, and fungi. Unfortunately, the immune system may become dysfunctional, which can result in secondary immunodeficiency diseases. The treatments available for these disorders and diseases of the immune system do not typically serve to cure, and much too often the side effects associated can exacerbate the conditions. As an alternative, the area of immunotherapy seeks to remedy the limitations of today’s medicine. Through this review, insight into the current conventional treatments available for select secondary immunodeficiency diseases will be provided, particularly in Human Immunodeficiency Virus disease and Systemic Lupus Erythematosus. The modern immunotherapy approaches that are underway will then be analyzed in their capacity and potential to treat these defects of the immune system.

Oxidative stress and immune complexes: Pathogenic mechanisms in pristane induced murine model of lupus.

Systemic lupus erythematosus is a systemic autoimmune inflammatory disease with a broad spectrum of clinical presentations. Mouse models play an indispensible role in understanding the disease pathology and for developing new therapeutics. This study investigated the effect of pristane administration on female BALB/c mice. The various manifestations of lupus replicated in this mouse model were analysed and their relevance to human disease was assessed. Pristane injection replicates the two prime manifestations of lupus i.e. oxidative stress and inflammation. An increase in oxidative stress was determined by the decreasing anti-oxidants, increasing ROS and lipid peroxidation. Inflammatory cytokines (IL-6 and TNF-α) also increased in the plasma. The deteriorating organ functions after pristane administration were evident histopathologically. An increase in inflammatory cells, necrosis, oil vacuoles and pigment cells were marked in kidney, liver, lungs and spleen, whereas skin did not manifest any alteration. Liver function (bilirubin, SGPT) and kidney function (creatinine and proteinuria) tests were also altered. Pristane injection caused the generation of anti-nuclear antibodies, which were apparent from the different immunofluorescence patterns observed. Immune deposits were evident in all the vital organs stating the similarity this model holds with lupus patients. Replicating various manifestations of human lupus disorder, pristane induced mouse model of lupus serves as an appropriate model to study lupus complications and in the development of novel therapeutics for disease management.

Selective Histone Deacetylase 6 Inhibition Normalizes B Cell Activation and Germinal Center Formation in a Model of Systemic Lupus Erythematosus.

Autoantibody production by plasma cells (PCs) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. Histone deactylase 6 (HDAC6) is a unique cytoplasmic HDAC that modifies the interaction of a number of tubulin- associated proteins; inhibition of HDAC6 has been shown to be beneficial in murine models of SLE, but the downstream pathways accounting for the therapeutic benefit have not been clearly delineated. In the current study, we sought to determine whether selective HDAC6 inhibition would abrogate abnormal B cell activation in SLE. We treated NZB/W lupus mice with the selective HDAC6 inhibitor, ACY-738, for 4 weeks beginning at 20 weeks-of age. After only 4 weeks of treatment, manifestation of lupus nephritis (LN) were greatly reduced in these animals. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune, and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggest that a critical event might be modulation of cellular metabolism.

BAFF inhibition in SLE-Is tolerance restored?

The B cell activating factor (BAFF) inhibitor, belimumab, is the first biologic drug approved for the treatment of SLE, and exhibits modest, but durable, efficacy in decreasing disease flares and organ damage. BAFF and its homolog APRIL are TNF-like cytokines that support the survival and differentiation of B cells at distinct developmental stages. BAFF is a crucial survival factor for transitional and mature B cells that acts as rheostat for the maturation of low-affinity autoreactive cells. In addition, BAFF augments innate B cell responses via complex interactions with the B cell receptor (BCR) and Toll like receptor (TLR) pathways. In this manner, BAFF impacts autoreactive B cell activation via extrafollicular pathways and fine tunes affinity selection within germinal centers (GC). Finally, BAFF and APRIL support plasma cell survival, with differential impacts on IgM- and IgG-producing populations. Therapeutically, BAFF and combined BAFF/APRIL inhibition delays disease onset in diverse murine lupus strains, although responsiveness to BAFF inhibition is model dependent, in keeping with heterogeneity in clinical responses to belimumab treatment in humans. In this review, we discuss the mechanisms whereby BAFF/APRIL signals promote autoreactive B cell activation, discuss whether altered selection accounts for therapeutic benefits of BAFF inhibition, and address whether new insights into BAFF/APRIL family complexity can be exploited to improve human lupus treatments.

Human Lupus Plasma Pro-Atherogenic Effects on Cultured Macrophages Are Not Mitigated by Statin Therapy: A Mechanistic LAPS Substudy.

Background and Objectives: Atherosclerotic cardiovascular disease (CVD) remains a major cause of morbidity and mortality in persons with systemic lupus erythematosus (SLE, lupus). Atherosclerosis, which involves interplay between cholesterol metabolism and cellular inflammatory pathways, is primarily treated with statins since statins have lipid-lowering and anti-inflammatory properties. The Lupus Atherosclerosis Prevention Study (LAPS) was designed to investigate the efficacy of statins against CVD in SLE patients. LAPS demonstrated that 2 years of atorvastatin administration did not reduce atherosclerosis progression in lupus patients. In this LAPs substudy, we use cultured macrophages to explore the atherogenic properties of plasma from LAPS subjects to explain the mechanistic rationale for the inability of statins to reduce CVD in lupus. Materials and Methods: THP-1 differentiated macrophages were treated for 18 h with 10% SLE patient plasma obtained pre- and post-atorvastatin therapy or placebo. Gene expression of the following cholesterol transport genes was measured by qRT-PCR. For efflux—ATP binding cassette transporter (ABC)A1 and ABCG1, 27-hydroxylase, peroxisome proliferator-activated receptor (PPAR)γ, and liver X receptor (LXR)α; and for influx—cluster of differentiation 36 (CD36) and scavenger receptor (ScR)A1. Results: Macrophages exposed to plasma from both statin-treated and placebo-treated groups showed a significant decrease in cholesterol efflux proteins ATP binding cassette (ABC) transporters A1 and ABCG1, an increase in 27-hydroxylase, an increase in the LDL receptor and a decrease in intracellular free cholesterol. No change in influx receptors ScRA1 and CD36, nor nuclear proteins LXRα and PPARγ was observed. Conclusions: Statins do not normalize pro-atherogenic changes induced by lupus and these changes continue to worsen over time. This study provides mechanistic insight into LAPS findings by demonstrating that statins are overall ineffective in altering the balance of cholesterol transport gene expression in human macrophages. Furthermore, our study suggests that statins as a CVD treatment may not be useful in attenuating lipid overload in the SLE environment.

Vasculitis and crescentic glomerulonephritis in a newly established congenic mouse strain derived from ANCA-associated vasculitis-prone SCG/Kj mice

Lupus nephritis (LN) is the secondary glomerulonephritis (GN) involved in systemic lupus erythematosus (SLE) and a typical immune complex-type GN. Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease characterized by systemic vasculitis and pauci-immune-type crescentic glomerulonephritis (CrGN) with ANCA production. Human AAV causes death due to lung haemorrhage and end-stage renal disease, for which renal replacement therapies are necessary. The SLE/AAV overlap syndrome was recently reported in humans. The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a unique model of human AAV showing production of myeloperoxidase (MPO)-ANCA. We previously discovered seven disease susceptibility quantitative trait loci (QTL) derived from SCG/Kj mice by linkage analysis. To investigate the individual functions of each QTL, and to identify AAV susceptibility genes, we introduced them into a B6/lpr background to establish SCG/Kj interval congenic mice (SICM). B6/lpr.C1scg mice, a type of SICM, exhibited the production of autoantibodies, including MPO-ANCA. The GN in B6/lpr.C1scg mice was not pauci-immune type: deposition of immunoglobulins and complement components was observed in nephritic glomeruli, similar to that in LN. The incidence of GN in female B6/lpr.C1scg mice was 100%. Granulocyte infiltration was also observed in the glomerular tuft and crescents. B6/lpr.C1scg mice also displayed vasculitis in multiple organs, most frequently the lung and kidney. Vasculitis was characterized by the infiltration of mononuclear cells to vascular walls followed by granulocyte infiltration, resembling human lupus vasculitis. The incidence of lung vasculitis was over 90% in male and female B6/lpr.C1scg mice. Blood MPO-ANCA levels were significantly associated with histopathological disease phenotypes. MPO deposition was observed in nephritic glomeruli, and granulocytes infiltrated into inflamed vessels and glomeruli. These observations suggest that the activation of granulocytes and local MPO release contribute to the pathogenesis of GN and vasculitis. As a monocongenic mouse, B6/lpr.C1scg mice show the association between murine chromosome 1 segment and autoimmunity. This strain can be used as a model of the SLE/AAV overlap syndrome, and will be useful for elucidating the mechanism of ANCA generation and the pathogenesis of CrGN and vasculitis, as well as in the search for genetic factors related to AAV.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

BackgroundAdaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model.MethodsFor lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant.ResultsCompared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038).ConclusionIncreased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.