Lupus-prone Mice Research Articles

Conversion of the Liver into a Biofactory for DNaseI Using Adeno-Associated Virus Vector Gene Transfer Reduces Neutrophil Extracellular Traps in a Model of Systemic Lupus Erythematosus.

Adeno-associated virus (AAV) vectors are proving to be clinically transformative tools in the treatment of monogenic genetic disease. Rapid ongoing development of this technology promises to not only increase the number of monogenic disorders amenable to this approach but also to bring diseases with complex multigenic and nongenetic etiologies within therapeutic reach. In this study, we explore the broader paradigm of converting the liver into a biofactory for systemic output of therapeutic molecules using AAV-mediated delivery of the endonuclease DNaseI as an exemplar. DNaseI can clear neutrophil extracellular traps (NETs), which are nuclear-protein structures possessing antimicrobial action, also involved in the pathophysiology of clinically troubling immune-mediated diseases. However, a translational challenge is short half-life of the enzyme in vivo (<5 h). This study demonstrates that AAV-mediated liver-targeted gene transfer stably induces serum DNaseI activity to >190-fold above physiological levels. In lupus-prone mice (NZBWF1), the activity was maintained for longer than 6 months, the latest time point tested, and resulted in a clear functional effect with reduced renal presence of neutrophils, NETs, IgG, and complement C3. However, treatment in this complex disease model did not extend lifespan, improve serological endpoints, or preserve renal function, indicating there are elements of pathophysiology not accessible to DNaseI in the NZBWF1 model. We conclude that a translational solution to the challenge of short half-life of DNaseI is AAV-mediated gene delivery and that this may be efficacious in treating disease where NETs are a dominant pathological mechanism.

Preclinical stage abundance and nuclear antigen reactivity of faecal Immunoglobulin A vary among males and females of lupus-prone mouse models.

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies with nuclear antigen (nAg) specificity. Using (SWRxNZB)F1 (SNF1) mice, we showed higher levels of Immunoglobulin A (IgA) production in the intestine and the nAg reactivity of faecal IgA under lupus susceptibility. Here, we determined whether the faecal IgA abundance and nAg reactivity are higher in, different among, various lupus-prone preclinical models (MRL/lpr, NZBxNZW-F1, SNF1, NZM2410 and NZM2328). We also determined whether the faecal IgA nAg reactivity at preseropositive ages correlates with the eventual serum autoantibody levels in males and females of these mouse models. We show that age-dependent increase in the abundance and nAg reactivity of faecal IgA can vary among different lupus-prone mouse models. Importantly, faecal IgA in these mice show significant levels of nAg reactivity, starting as early as at juvenile age. Furthermore, the pre-seropositive stage nAg reactivity of faecal IgA in most lupus-prone strains correlates well with that of eventual, seropositive stage systemic autoantibody levels. Gender differences in serum autoantibody levels were preceded by similar differences in the faecal IgA abundance and nAg reactivity. These observations suggest that faecal IgA features, nAg reactivity particularly, could serve as a biomarker for early prediction of the eventual systemic autoimmunity in lupus-prone subjects.

Phenotypic and functional alterations of peritoneal macrophages in lupus-prone mice

BackgroundSeveral studies have demonstrated the contribution of innate immune cells, including macrophages, in promoting systemic lupus erythematosus (SLE). Macrophages, one of the most abundant cell populations in the peritoneal cavity, are considered multifunctional cells with phenotypic plasticity. However, the functional properties of peritoneal macrophages in steady-state and during the progression of SLE remain poorly defined.Methods and resultsUsing the [NZB × NZW]F1 (BWF1) murine model of SLE, we analyzed the phenotype and function of peritoneal macrophages during the disease’s onset. We found a higher frequency of peritoneal macrophages and B1a cells in BWF1-diseased mice than age-matched controls. Additionally, macrophages from diseased animals expressed lower levels of CD206, MHC-II, and Sirpα. RNAseq analysis identified 286 differentially expressed genes in peritoneal macrophages from diseased-BWF1 mice compared to control mice. Functional experiments demonstrate that peritoneal macrophages from diseased-BWF1 mice secrete higher levels of pro-inflammatory cytokines when activated with TLR7 and TLR9 agonists, and they were less efficient in suppressing the activation and proliferation of peritoneal LPS-activated B cells. These data demonstrate that peritoneal macrophages from BWF1-diseased mice present phenotypic and functional alterations shifting to a more pro-inflammatory state.ConclusionsThe increase of macrophages with an altered phenotype and function together with the accumulation of B1a cells in the peritoneal cavity of diseased-BWF1 mice may promote the progression of the disease. Advancing awareness of the role and phenotype of peritoneal macrophages in SLE may contribute to a better understanding of these types of diseases and the development of novel therapies.

Rad52 mediates class-switch DNA recombination to IgD

In B cells, IgD is expressed together with IgM through alternative splicing of primary VHDJH-Cμ-s-m-Cδ-s-m RNAs, and also through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sμ with σδ. How such DSBs are resolved is still unknown, despite our previous report showing that Rad52 effects the ‘short-range’ microhomology-mediated synapsis of intra-Sμ region DSBs. Here we find that induction of IgD CSR downregulates Zfp318, and promotes Rad52 phosphorylation and recruitment to Sμ and σδ, thereby leading to alternative end-joining (A-EJ)-mediated Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52−/− B cells aborts IgD CSR in vitro and in vivo and dampens the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogates IgD CSR. Finally, Rad52 phosphorylation is associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in patients with systemic lupus erythematosus and in lupus-prone mice. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation.

Arctigenin inhibits abnormal germinal center reactions and attenuates murine lupus by inhibiting IFN-I pathway

Interferon-I (IFN-I) signaling pathway plays a vital role in the differentiation of germinal center B cells and the pathogenesis of systemic lupus erythematosus (SLE). Therefore, targeting the IFN-I signaling pathway could serve as an effective treatment strategy in SLE. Arctigenin is an active ingredient present in the seeds of Arctium lappa L. It has been reported to act as a negative regulator of inflammatory responses. However, the role of arctigenin remains unknown in the regulation process of the IFN-I-mediated differentiation of germinal center B cells and the pathogenesis of SLE. In the present study, we demonstrated that arctigenin alleviated the progression of spontaneous lupus in MRL/lpr mice and imiquimod-mediated lupus mice. Especially, arctigenin significantly reduced the proportions of germinal center B cells (7.1%, vs. 5.12%, p < 0.01), follicular helper T cells (11.49%, vs. 5.53%, p < 0.05), and plasma cells (2.44%, vs. 1.39%, p < 0.01) in the lupus-prone mice. In vitro studies have shown that arctigenin significantly inhibited the IFN-α-induced CD69 and interferon-stimulated gene (ISG) expressions along with the phosphorylation of JAK1 and STAT1 by nearly half in murine B cells via activating PP2A. Overall, these data highlighted the role of arctigenin in regulating the IFN-I-mediated differentiation of germinal center B cells and the pathogenesis of SLE. Thus, arctigenin may be used as a potentially effective therapeutic target for the treatment of SLE.

Host Genetics But Not Commensal Microbiota Determines the Initial Development of Systemic Autoimmune Disease in BXD2 Mice.

To determine the extent to which the gut microbiome influences systemic autoimmunity in a mouse model of lupus. We generated germ-free (GF) lupus-prone BXD2 mice, which under normal conditions develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. GF status was confirmed by gut bacterial culture. The autoimmune phenotypes of 6- and 12-month-old gnotobiotic GF BXD2 mice and specific pathogen-free (SPF) BXD2 mice were compared. Serum levels of autoantibodies were measured by enzyme-linked immunosorbent assay. Histologic sections of the mouse kidney and joints were evaluated. Flow cytometry was used to analyze GCs and age-associated B cells. CD4+ T cells were analyzed for PD-1+ICOS+ activated T cells, T follicular regulatory (Tfr) cells (Foxp3+CD25+ PD-1+CXCR5+), and PD-1+ICOS+ T cells expressing interleukin-17A (IL-17A) or interferon-γ (IFNγ) after stimulation with phorbol myristate acetate (PMA)/ionomycin. In 6-month-old mice, GF status did not affect splenomegaly, GC B cells, age-associated B cells, or serum autoantibody levels, except for IgG antihistone. GF BXD2 mice exhibited a significantly higher percentage of Tfr cells compared to their SPF counterparts (P < 0.05). At 12 months of age, however, GF BXD2 mice had significantly diminished IgG autoantibody levels and a lower percentage of GC B cells and age-associated B cells (P < 0.05). Following stimulation with PMA/ionomycin, PD-1+ICOS+ CD4+ T cells expressed significantly lower IL-17A, but not IFNγ, levels in GF BXD2 mice compared to SPF BXD2 mice (P < 0.01). SPF BXD2 mice and GF BXD2 mice developed equivalent renal and joint disease with no significant differences in severity. Our results suggest a model in which genetics plays a dominant role in determining the initial development of autoimmunity. In contrast, gut microbiomes may regulate the persistence of certain aspects of systemic autoimmunity.

The small heat shock protein HSPB5 attenuates the severity of lupus nephritis in lupus-prone mice

Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus. The current treatments for LN are accompanied with severe immunotoxicity and have limits of effectiveness. Since our in vitro experiments demonstrated that a small heat shock protein (HSP), alpha-B crystallin (HSPB5; CRYAB), selectively modulates myeloid cells towards anti-inflammatory and tolerogenic phenotypes, the aim of this study was to investigate whether HSPB5 can attenuate the severity of LN. MRL/lpr mice were treated intravenously with HSPB5 at 2.5 or 10 μg/dose twice per week after disease onset, from 11 to 21 weeks of age. Disease progression was monitored by weekly measurements of proteinuria, and sera, spleens, and kidneys were collected for assessment at the terminal time point. Treatment with 10 μg HSPB5 substantially reduced endocapillary proliferation and tubular atrophy, which significantly reduced proteinuria and blood urea nitrogen (BUN). Compared to vehicle, 10 μg HSPB5 treatment substantially decreased activation/proliferation of splenocytes, increased IL-10+ macrophages, T and B regulatory cells (Treg, Breg), increased serum IL-10, and lowered expression of IL-6 in kidneys, which correlated with improved kidney function and pathology. This study demonstrated the utility of exogenous human HSPB5 to attenuate severe nephropathy in MRL/lpr mice and provides evidence in favour of a novel therapeutic approach for lupus nephritis.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1β, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA’s effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE‐mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

Adenosine receptor 2a agonists target mouse CD11c+T-bet+ B cells in infection and autoimmunity

CD11c+T-bet+ B cells are recognized as an important component of humoral immunity and autoimmunity. These cells can be distinguished from other B cells by their higher expression of the adenosine receptor 2a. Here we address whether A2A receptor activation can affect CD11c+T-bet+ B cells. We show that administration of the A2A receptor agonist CGS-21680 depletes established CD11c+T-bet+ B cells in ehrlichial-infected mice, in a B cell-intrinsic manner. Agonist treatment similarly depletes CD11c+T-bet+ B cells and CD138+ B cells and reduces anti-nuclear antibodies in lupus-prone mice. Agonist treatment is also associated with reduced kidney pathology and lymphadenopathy. Moreover, A2A receptor stimulation depletes pathogenic lymphocytes and ameliorates disease even after disease onset, highlighting the therapeutic potential of this treatment. This study suggests that targeting the adenosine signaling pathway may provide a method for the treatment of lupus and other autoimmune diseases mediated by T-bet+ B cells.

GDF15 Suppresses Lymphoproliferation and Humoral Autoimmunity in a Murine Model of Systemic Lupus Erythematosus

Growth and differentiation factor 15 (GDF15), a divergent member of the transforming growth factor-β superfamily, has been associated with acute and chronic inflammatory conditions including autoimmune disease, i.e., type I diabetes and rheumatoid arthritis. Still, its role in systemic autoimmune disease remains elusive. Thus, we studied GDF15-deficient animals in Fas-receptor intact (C57BL/6) or deficient (C57BL/6lpr/lpr) backgrounds. Further, lupus nephritis (LN) microdissected kidney biopsy specimens were analyzed to assess the involvement of GDF15 in human disease. GDF15-deficiency in lupus-prone mice promoted lymphoproliferation, T-, B- and plasma cell-expansion, a type I interferon signature, and increased serum levels of anti-DNA autoantibodies. Accelerated systemic inflammation was found in association with a relatively mild renal phenotype. Splenocytes of phenotypically overall-normal Gdf15−/− C57BL/6 and lupus-prone C57BL/6lpr/lpr mice displayed increased in vitro lymphoproliferative responses or interferon-dependent transcription factor induction in response to the toll-like-receptor (TLR)-9 ligand CpG, or the TLR-7 ligand Imiquimod, respectively. In human LN, GDF15 expression was downregulated whereas type I interferon expression was upregulated in glomerular- and tubular-compartments versus living donor controls. These findings demonstrate that GDF15 regulates lupus-like autoimmunity by suppressing lymphocyte-proliferation and -activation. Further, the data indicate a negative regulatory role for GDF15 on TLR-7 and -9 driven type I interferon signaling in effector cells of the innate immune system.

Lactobacillus acidophilus Supplementation Exerts a Synergistic Effect on Tacrolimus Efficacy by Modulating Th17/Treg Balance in Lupus-Prone Mice via the SIGNR3 Pathway.

ObjectiveTacrolimus (Tac) is an immunosuppressant used in the treatment of systemic lupus erythematosus (SLE); however, it induces T cell subset imbalances by reducing regulatory T (Treg) cells. Lactobacillus acidophilus (LA) is reported to have therapeutic efficacy in immune-mediated diseases via T cell regulation.MethodsThis study investigated whether a combination therapy of LA and Tac improves the therapeutic efficacy of Tac by modulating T cell subset populations in an animal model of SLE. Eight-week-old MRL/lpr mice were orally administered with 5 mg/kg of Tac and/or 50 mg/kg of LA daily for 8 weeks. Cecal microbiota compositions, serum autoantibodies levels, the degree of proteinuria, histological changes in the kidney, and populations of various T cell subsets in the spleen were analyzed.ResultsMice presented with significant gut dysbiosis, which were subsequently recovered by the combination treatment of Tac and LA. Double negative T cells in the peripheral blood and spleens of MRL/lpr mice were significantly decreased by the combination therapy. The combination treatment reduced serum levels of anti-dsDNA antibodies and Immunoglobulin G2a, and renal pathology scores were also markedly alleviated. The combination therapy induced Treg cells and decreased T helper 17 (Th17) cells both in vitro and in vivo. In vitro treatment with LA induced the production of indoleamine-2,3-dioxygenase, programmed death-ligand 1, and interleukin-10 via the specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 receptor signals.ConclusionThe present findings indicate that LA augments the therapeutic effect of Tac and modulates Th17/Treg balance in a murine model of SLE.

Anti-TLR7 Antibody Protects Against Lupus Nephritis in NZBWF1 Mice by Targeting B Cells and Patrolling Monocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple organ damage. Toll-like receptor 7 (TLR7), an innate immune RNA sensor expressed in monocytes/macrophages, dendritic cells (DCs), and B cells, promotes disease progression. However, little is known about the cellular mechanisms through which TLR7 drives lupus nephritis. Here, we show that the anti-mouse TLR7 mAb, but not anti-TLR9 mAb, protected lupus-prone NZBWF1 mice from nephritis. The anti-TLR7 mAb reduced IgG deposition in glomeruli by inhibiting the production of autoantibodies to the RNA-associated antigens. We found a disease-associated increase in Ly6Clow patrolling monocytes that expressed high levels of TLR7 and had upregulated expression of lupus-associated IL-10, CD115, CD31, and TNFSF15 in NZBWF1 mice. Anti-TLR7 mAb abolished this lupus-associated increase in patrolling monocytes in the circulation, spleen, and glomeruli. These results suggested that TLR7 drives autoantibody production and lupus-associated monocytosis in NZBWF1 mice and, that anti-TLR7 mAb is a promising therapeutic tool targeting B cells and monocytes/macrophages.

CD38 Correlates with an Immunosuppressive Treg Phenotype in Lupus-Prone Mice.

CD38 is a transmembrane glycoprotein expressed by T-cells. It has been reported that patients with systemic lupus erythematosus (SLE) showed increased CD38+CD25+ T-cells correlating with immune activation and clinical signs. Contrariwise, CD38 deficiency in murine models has shown enhanced autoimmunity development. Recent studies have suggested that CD38+ regulatory T-cells are more suppressive than CD38− regulatory T-cells. Thus, we have suggested that CD38 overexpression in SLE patients could play a role in regulating immune activation cells instead of enhancing it. This study found a correlation between CD38 with FoxP3 expression and immunosuppressive molecules (CD69, IL-10, CTLA-4, and PD-1) in T-cells from lupus-prone mice (B6.MRL-Faslpr/J). Additionally, B6.MRL-Faslpr/J mice showed a decreased proportion of CD38+ Treg cells regarding wild-type mice (WT). Furthermore, Regulatory T-Cells (Treg cells) from CD38-/- mice showed impairment in expressing immunosuppressive molecules and proliferation after stimulation through the T-cell receptor (TCR). Finally, we demonstrated an increased ratio of IFN-γ/IL-10 secretion in CD38-/- splenocytes stimulated with anti-CD3 compared with the WT. Altogether, our data suggest that CD38 represents an element in maintaining activated and proliferative Treg cells. Consequently, CD38 could have a crucial role in immune tolerance, preventing SLE development through Treg cells.

Overexpression of Human TLR8 Causes Fatal Anemia in SLE-Prone Mice By Altering the Bone Marrow Erythropoietic Niche

Anemia is a common hematologic abnormality in patients with active systemic lupus erythematosus (SLE), a disease characterized by innate immune system activation by nucleic acid containing immune complexes and cell debris. Work in mouse models has shown that overactivation of several cytoplasmic innate immune sensors by either self-DNA or self-RNA specifically leads to erythroid cell death in the fetal liver while sparing other hematopoietic cell lineages.The endosomal receptors TLR7 and TLR8 both recognize ssRNA in humans. TLR7 overexpression in mice causes a lupus syndrome that includes the development of mild to moderate anemia (Hb >10) and thrombocytopenia and is associated with spleen histiocytosis, autoimmune hemolysis, erythrophagocytosis and compensatory stress erythropoiesis in the spleen. A recent study demonstrated that patients with TLR8 gain of function mutations present with immunodeficiency, inflammation and bone marrow failure. However the role of TLR8 in SLE has been difficult to study in mice because it has a 5 amino acid deletion that attenuates its RNA binding capacity.To address the role of TLR8 in SLE, we overexpressed human TLR8 in a lupus mouse model (huTLR8tg.Sle1.Yaa) using a BAC transgene. 50% of homozygous huTLR8tg.Sle1.Yaa mice developed severe anemia (Hb<9) resulting in early mortality starting at 3-4.5 months of age. This phenotype required both the Sle1 and Yaa loci that promote the formation of high titer anti-chromatin and anti-RNA antibodies and onset of nephritis at >6 months of age. There was no difference in autoantibody titers between Sle1.Yaa wt and huTLR8tg mice and early death was not due to premature onset of renal disease. All mice had normal RBC indices prior to 10 weeks of age. Anemia was associated with an increase in bone marrow (BM) trabecular bone and a decrease in erythroblastic islands (EBI) in the BM with compensatory stress erythropoiesis leading to reticulocytosis and vast splenomegaly. RBC half-life was normal even after the development of reticulocytosis, but decreased in severely anemic mice as a pre-terminal event. Using CFSE labeling of RBCs we showed that hemophagocytosis occurred in vivo as a result of expansion of phagocytic red pulp macrophages.Flow cytometry of BMs from transgenic mice showed normal erythroid progenitors but a block at the late CFU-E/early proerythroblast stage leading to overall decreased terminal erythroid differentiation. Single cell RNAseq of EBIs isolated from transgenic BMs confirmed the block in erythropoiesis. The erythroblast cluster proximal to the block had a signature of mitochondrial stress and decreased proliferation. Spleen EBIs from transgenic mice were characterized by a low frequency of early erythroblasts compared with BM EBIs. We found 6 related clusters of EBI central macrophages in the BMs of both wt and transgenic mice. In transgenic mice, most of these displayed an inflammatory and Type 1 IFN signature. One cluster (M2) expressed all the classical central macrophage phenotypic markers in wt mice but was characterized in transgenic mice by downregulation of multiple phagocytic receptors and a 5-fold decrease of VCAM1 expression. Loss of VCAM1 and downregulation of CD169 in central macrophages of transgenic BMs was confirmed by flow cytometry. By contrast spleen central macrophages from transgenic mice retained VCAM and CD169 expression.Together, these results suggest that failure of BM erythropoiesis in huTLR8 transgenic SLE.Yaa lupus-prone mice, in which the acquisition of anti-nucleic acid autoantibodies drives excess innate stimulus through TLR7 and huTLR8, is due to a block in differentiation from CFU-E to the early proerythroblast stage; this is associated with an inflammatory phenotype specifically in BM erythroblastic island central macrophages and down regulation of adhesion and phagocytic receptors. Stress erythropoiesis in the spleens is associated with vast expansion of red pulp macrophages with phagocytic properties and fatal anemia is associated with a decrease in red blood cell half-life, suggesting that excessive RBC phagocytosis, coupled with insufficient erythroblast progenitors, eventually exceeds the capability of stress erythropoiesis to replace the RBC mass. DisclosuresKalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Paulson: Forma Therapeutics: Consultancy. Blanc: Keros Therapeutics, Inc.: Consultancy.

The effect of amaranth oil on autoantibodies in lupus prone mice

Background: Autoimmune diseases, such as systemic lupus erythematosus, can have severe impacts on quality of life. They are one of the leading causes of death for women in the United States. Distinguished by the body damaging its own tissues and organs, they are often classified and diagnosed based on autoantibody levels. Treatments often include immunosuppressant drugs, which can have adverse effects. Aim of Study: Amaranth is a good functional food candidate, possessing antioxidants, bioactive compounds, and a variety of health benefits, such as lowering cholesterol, and aiding diabetes and hypertension. Previous studies have largely focused on the grain or seed, but amaranth oil is less explored. This study examines whether orally administered amaranth oil had any effects on autoantibodies and splenic immune cell populations in murine subjects.Methods: Mice in the experimental group (n = 3) were given 4μl of amaranth oil per gram of mouse weight for 5 days a week over 84 days. Control mice (n = 2) were sham treated on the same schedule with no oil. To determine autoantibody levels, enzyme-linked immunosorbent assays (ELISAs) were first conducted on wells pre-coated with double stranded DNA, single stranded DNA, histones, or double stranded DNA and then histones (nucleosomes). Autoantibody presence was quantified by measuring absorbance at 405nm. Splenic cell populations were examined with flow cytometry and compared using a student’s t-test. Results: Compared to the control group, the mice receiving amaranth oil showed decreased IgG and IgM histone autoantibody absorbance levels throughout the whole study. IgG dsDNA, ssDNA, and nucleosome autoantibody absorbances were lower than that of the control group for the first 42 days. IgM dsDNA, ssDNA, and nucleosome autoantibody absorbances were lower only for the first 14 days. There were no significant differences found amongst splenic immune cell populations between the control and experimental groups.Conclusion: These preliminary data show that amaranth oil may help decrease autoantibody levels in lupus prone murine subjects. However, given the small number of subjects in this study, further research is needed to confirm observed effects and determine the most effective dose and administration schedule. Keywords: autoantibody, IgG, IgM, histone, dsDNA, ssDNA, nucleosome, amaranth oil, immunoglobulin, lupus

The effect of amaranth oil on proteinuria in lupus prone mice

Background: Kidney disease is a leading cause of death in the United States and the world. Proteinuria signifies kidney damage and can exacerbate kidney disease. It has become an important indicator of kidney disease; reducing proteinuria results in renoprotective effects and slows renal disease. Existing treatments do not work for every patient.Aim of Study: Amaranth is a tropical plant that was regularly consumed in the ancient Central American diet. It has a plethora of health effects and is a strong functional food candidate. This study examines whether a regular oral dose of amaranth oil decreased proteinuria concentration in murine subjects. Methods: Mice in the experimental group (n = 3) were given 4μl of amaranth oil per gram of mouse weight for 5 days a week over 84 days. Control mice (n = 2) were sham treated on the same schedule with no oil. Urine protein concentration was determined by Bradford assay, measuring absorbance at 595nm, then comparing with a BSA standard curve. Results: The experimental group showed decreased proteinuria levels throughout the entire 84 days of studyConclusion: Results show amaranth oil may help decrease proteinuria levels in lupus prone murine subjects. Given the small sample size, the data are preliminary. More research is needed to validate the results and determine the optimal dose and treatment schedule.Key words: proteinuria, amaranth oil, renoprotective, kidney disease, renal disease, lupus, functional food

A Selective Neutraligand for CXCL12/SDF-1α With Beneficial Regulatory Functions in MRL/Lpr Lupus Prone Mice.

Dysregulation of CXCL12/SDF-1-CXCR4/CD184 signaling is associated with inflammatory diseases and notably with systemic lupus erythematosus. Issued from the lead molecule chalcone-4, the first neutraligand of the CXCL12 chemokine, LIT-927 was recently described as a potent analogue with improved solubility and stability. We aimed to investigate the capacity of LIT-927 to correct immune alterations in lupus-prone MRL/lpr mice and to explore the mechanism of action implemented by this small molecule in this model. We found that in contrast to AMD3100, an antagonist of CXCR4 and agonist of CXCR7, LIT-927 reduces the excessive number of several B/T lymphocyte subsets occurring in the blood of sick MRL/lpr mice (including CD3+/CD4-/CD8-/B220+ double negative T cells). In vitro, LIT-927 downregulated the overexpression of several activation markers on splenic MRL/lpr lymphocytes. It exerted effects on the CXCR4 pathway in MRL/lpr CD4+ T spleen cells. The results underline the importance of the CXCL12/CXCR4 axis in lupus pathophysiology. They indicate that neutralizing CXCL12 by the neutraligand LIT-927 can attenuate hyperactive lymphocytes in lupus. This mode of intervention might represent a novel strategy to control a common pathophysiological mechanism occurring in inflammatory diseases.

Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice.

Autoimmune disease has presented an insurmountable barrier to restoration of durable immune tolerance. Previous studies indicate that chronic therapy with metabolic inhibitors can reduce autoimmune inflammation, but it remains unknown whether acute metabolic modulation enables permanent immune tolerance to be established. In an animal model of lupus, we determined that targeting glucose metabolism with 2-deoxyglucose (2DG) and mitochondrial metabolism with metformin enables endogenous immune tolerance mechanisms to respond to tolerance induction. A 2-week course of 2DG and metformin, when combined with tolerance-inducing therapy anti-CD45RB, prevented renal deposition of autoantibodies for 6 months after initial treatment and restored tolerance induction to allografts in lupus-prone mice. The restoration of durable immune tolerance was linked to changes in T cell surface glycosylation patterns, illustrating a role for glycoregulation in immune tolerance. These findings indicate that metabolic therapy may be applied as a powerful preconditioning to reinvigorate tolerance mechanisms in autoimmune and transplant settings that resist current immune therapies.

Haemoglobin drives inflammation and initiates antigen spread and nephritis in lupus.

Haemoglobin (Hb) has well-documented inflammatory effects and is normally efficiently scavenged; clearance mechanisms can be overwhelmed during erythrocyte lysis. Whether Hb is preferentially inflammatory in lupus and triggers broad anti-self responses was assessed. Peripheral blood mononuclear cells (PBMCs) derived from SLE patients secreted higher levels of lupus-associated inflammatory cytokines when incubated with human Hb than did PBMCs derived from healthy donors, an effect negated by haptoglobin. Ferric murine Hb triggered the preferential release of lupus-associated cytokines from splenocytes, B cells, CD4T cells, CD8T cells and plasmacytoid dendritic cells isolated from ageing, lupus-prone NZM2410mice, and also had mitogenic effects on B cells. Pull-downs, followed by mass spectrometry, revealed interactions of Hb with several lupus-associated autoantigens; co-incubation of ferric Hb with apoptotic blebs (structures that contain packaged autoantigens) revealed synergies-in terms of cytokine release and autoantibody production in vitro-that were also restricted to the lupus genotype. Murine ferric Hb activated multiple signalling pathways and, in combination with apoptotic blebs, preferentially triggered MAP kinase signalling specifically in splenocytes isolated from lupus-prone mice. Infusion of murine ferric Hb into lupus-prone mice led to enhanced release of lupus-associated cytokines, the generation of a spectrum of autoantibodies and enhanced-onset glomerulosclerosis. Given that the biased recognition of ferric Hb in a lupus milieu, possibly in concert with lupus-associated autoantigens, triggers inflammatory responses and the generation of lupus-associated cytokines, and also stimulates the generation of potentially pathogenic lupus-associated autoantibodies, neutralization of Hb could have beneficial effects.

Anti-Monomeric C-Reactive Protein Antibody Ameliorates Arthritis and Nephritis in Mice.

Conformation-specific Ags are ideal targets for mAb-based immunotherapy. Here, we demonstrate that the monomeric form of C-reactive protein (mCRP) is a specific therapeutic target for arthritis and nephritis in a murine model. Screening of >1800 anti-mCRP mAb clones identified 3C as a clone recognizing the monomeric, but not polymeric, form of CRP. The anti-mCRP mAb suppressed leukocyte infiltration in thioglycollate-induced peritonitis, attenuated rheumatoid arthritis symptoms in collagen Ab-induced arthritis model mice, and attenuated lupus nephritis symptoms in MRL/Mp-lpr/lpr lupus-prone model mice. These data suggest that the anti-mCRP mAb 3C has therapeutic potential against rheumatoid arthritis and lupus nephritis.