Similarity and difference between systemic lupus erythematosus and NZB/W F1 mice by multi-omics analysis.

OBJECTIVES: Several animal disease models have been used to understand the mechanisms of systemic lupus erythematosus (SLE); however, the translation of findings from animals to humans has not been sufficiently examined in drug development. To confirm the validity of New Zealand black x New Zealand white (NZB/W) F1 mice as an SLE model, we extensively characterized SLE patients and NZB/W F1 mice by omics analysis. METHODS: Peripheral blood from patients and mice and spleen and lymph node tissue from mice were analysed using cell subset analysis, cytokine panel assays, and transcriptome analysis. RESULTS: CD4+ effector memory T cells, plasmablasts, and plasma cells were increased in both SLE patients and NZB/W F1 mice. Levels of tumor necrosis factor-α, interferon gamma induced protein-10, and B cell activating factor in plasma were significantly higher in SLE patients and NZB/W F1 mice than in their corresponding controls. Transcriptome analysis revealed an upregulation of genes involved in the interferon signalling pathway and T-cell exhaustion signalling pathway in both SLE patients and the mouse model. In contrast, death receptor signalling genes showed changes in the opposite direction between patients and mice. CONCLUSION: NZB/W F1 mice are a generally suitable model of SLE for analysing the pathophysiology and treatment response of T/B cells and monocytes/macrophages and their secreted cytokines.

Intestinal permeability and inflammatory features of juvenile age correlate with the eventual systemic autoimmunity in lupus-prone female SWR × NZB F1 (SNF1) mice.

The incidence of systemic lupus erythematosus (SLE) is about nine times higher in women than in men, and the underlying mechanisms that contribute to this gender bias are not fully understood. Previously, using lupus-prone (SWR × NZB)F1 (SNF1) mice, we have shown that the intestinal immune system could play a role in the initiation and progression of disease in SLE, and depletion of gut microbiota produces more pronounced disease protection in females than in males. Here, we show that the gut permeability features of lupus-prone female SNF1 mice at juvenile ages directly correlate with the expression levels of pro-inflammatory factors, faecal IgA abundance and nAg reactivity and the eventual systemic autoantibody levels and proteinuria onset. Furthermore, we observed that the disease protection achieved in female SNF1 mice upon depletion of gut microbiota correlates with the diminished gut inflammatory protein levels, intestinal permeability and circulating microbial DNA levels. However, faecal microbiota transplant from juvenile male and females did not result in modulation of gut inflammatory features or permeability. Overall, these observations suggest that the early onset of intestinal inflammation, systemic autoantibody production and clinical stage disease in lupus-prone females is linked to higher gut permeability in them starting at as early as juvenile age. While the higher gut permeability in juvenile lupus-prone females is dependent on the presence of gut microbes, it appears to be independent of the composition of gut microbiota.

Regulatory T cells inhibit autoantigen-specific CD4+ T cell responses in lupus-prone NZB/W F1 mice.

Introduction: Progressive loss of regulatory T cell (Treg)-mediated control over autoreactive effector T cells contributes to the development of systemic lupus erythematosus (SLE). Accordingly, we hypothesized that Treg may also have the capacity to suppress the activation of autoreactive CD4+ T cells that are considered to drive autoimmunity. Methods: To investigate whether Treg are involved in the control of autoreactive CD4+ T cells, we depleted CD25+ Treg cells either in vivo or in vitro, or combined both approaches before antigen-specific stimulation with the SLE-associated autoantigen SmD1(83-119) in the NZB/W F1 mouse model either after immunization against SmD1(83-119) or during spontaneous disease development. Frequencies of autoantigen-specific CD4+ T cells were determined by flow cytometry using the activation marker CD154. Results: Both in vitro and in vivo depletion of CD25+ Treg, respectively, increased the frequencies of detectable autoantigen-specific CD4+ T cells by approximately 50%. Notably, the combined in vivo and in vitro depletion of CD25+ Treg led almost to a doubling in their frequencies. Frequencies of autoantigen-specific CD4+ T cells were found to be lower in immunized haploidentical non-autoimmune strains and increased frequencies were detectable in unmanipulated NZB/W F1 mice with active disease. In vitro re-addition of CD25+ Treg after Treg depletion restored suppression of autoantigen-specific CD4+ T cell activation. Discussion: These results suggest that the activation and expansion of autoantigen-specific CD4+ T cells are partly controlled by Treg in murine lupus. Depletion of Treg therefore can be a useful approach to increase the detectability of autoantigen-specific CD4+ T cells allowing their detailed characterization including lineage determination and epitope mapping and their sufficient ex vivo isolation for cell culture.

Male microbiota-associated metabolite restores macrophage efferocytosis in female lupus-prone mice via activation of PPARγ/LXR signaling pathways.

Systemic lupus erythematosus development is influenced by both sex and the gut microbiota. Metabolite production is a major mechanism by which the gut microbiota influences the immune system, and we have previously found differences in the fecal metabolomic profiles of lupus-prone female and lupus-resistant male BWF1 mice. Here we determine how sex and microbiota metabolite production may interact to affect lupus. Transcriptomic analysis of female and male splenocytes showed genes that promote phagocytosis were upregulated in BWF1 male mice. Because patients with systemic lupus erythematosus exhibit defects in macrophage-mediated phagocytosis of apoptotic cells (efferocytosis), we compared splenic macrophage efferocytosis in vitro between female and male BWF1 mice. Macrophage efferocytosis was deficient in female compared to male BWF1 mice but could be restored by feeding male microbiota. Further transcriptomic analysis of the genes upregulated in male BWF1 mice revealed enrichment of genes stimulated by PPARγ and LXR signaling. Our previous fecal metabolomics analyses identified metabolites in male BWF1 mice that can activate PPARγ and LXR signaling and identified one in particular, phytanic acid, that is a very potent agonist. We show here that treatment of female BWF1 splenic macrophages with phytanic acid restores efferocytic activity via activation of the PPARγ and LXR signaling pathways. Furthermore, we found phytanic acid may restore female BWF1 macrophage efferocytosis through upregulation of the proefferocytic gene CD36. Taken together, our data indicate that metabolites produced by BWF1 male microbiota can enhance macrophage efferocytosis and, through this mechanism, could potentially influence lupus progression.

Combination CTLA4Ig and Anti-CD40 Ligand Treatment Modifies T and B Cell Metabolic Profiles and Promotes B Cell Receptor Remodeling in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti-CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Development of a novel Poly (I:C)-induced murine model with accelerated lupus nephritis and examination of the therapeutic effects of mycophenolate mofetil and a cathepsin S inhibitor.

Systemic lupus erythematosus (SLE) is an autoimmune disease involving multi-organ systems with a widely heterogeneous clinical presentation. Renal involvement, observed mainly in lupus nephritis (LN), is the most common organ lesion associated with SLE and a determinant of prognosis. However, treatment of LN remains controversial and challenging, prompting the need for novel therapeutic approaches. In particular, development of a clinically relevant LN animal model would greatly facilitate the development of new treatments. Here, we report a novel murine model for LN established by administering polyinosinic-polycytidylic acid (Poly (I:C)) to NZB/W F1 mice. We investigated the effectiveness of administering Poly (I:C) to NZB/W F1 mice for accelerating nephritis onset and explored the optimal conditions under which to enroll mice with nephritis with similar pathology for studying treatment candidates. Gene-expression analysis revealed that activation of macrophages, which are reported to be involved in the progression of LN in patients, was a unique characteristic in this accelerated nephritis model. Evaluation of the therapeutic effect of mycophenolate mofetil (MMF), a recommended first-choice agent for LN, in this novel LN model showed that MMF significantly reduced proteinuria. The cathepsin S (CatS) inhibitor ASP1617, which has been reported to prevent development of lupus-like glomerulonephritis in the spontaneous NZB/W F1 mouse model, also showed marked therapeutic effect in this model. Our novel Poly (I:C) accelerated LN model would thus be very useful for screening clinical candidates for LN, and CatS may be an attractive therapeutic target for the treatment of LN.

The oxidative phosphorylation inhibitor IM156 suppresses B-cell activation by regulating mitochondrial membrane potential and contributes to the mitigation of systemic lupus erythematosus.

Current treatment strategies for autoimmune diseases may not sufficiently control aberrant metabolism in B-cells. To address this concern, we investigated a biguanide derivative, IM156, as a potential regulator for B-cell metabolism in vitro and in vivo on overactive B-cells stimulated by the pro-inflammatory receptor TLR-9 agonist CpG oligodeoxynucleotide, a mimic of viral/bacterial DNA. Using RNA sequencing, we analyzed the B-cell transcriptome expression, identifying the major molecular pathways affected by IM156 in vivo. We also evaluated the anti-inflammatory effects of IM156 in lupus-prone NZB/W F1 mice. CD19+B-cells exhibited higher mitochondrial mass and mitochondrial membrane potential compared to T-cells and were more susceptible to IM156-mediated oxidative phosphorylation inhibition. In vivo, IM156 inhibited mitochondrial oxidative phosphorylation, cell cycle progression, plasmablast differentiation, and activation marker levels in CpG oligodeoxynucleotide-stimulated mouse spleen B-cells. Interestingly, IM156 treatment significantly increased overall survival, reduced glomerulonephritis and inhibited B-cell activation in the NZB/W F1 mice. Thus, our data indicated that IM156 suppressed the mitochondrial membrane potentials of activated B-cells in mice, contributing to the mitigation of lupus activity. Hence, IM156 may represent a therapeutic alternative for autoimmune disease mediated by B-cell hyperactivity.

HSPB5 suppresses renal inflammation and protects lupus-prone NZB/W F1 mice from severe renal damage.

BACKGROUND: Lupus nephritis (LN) is an inflammatory disease of the kidneys affecting patients with systemic lupus erythematosus. Current immunosuppressive and cytotoxic therapies are associated with serious side effects and fail to protect 20-40% of LN patients from end-stage renal disease. In this study, we investigated whether a small heat shock protein, HSPB5, can reduce kidney inflammation and the clinical manifestations of the disease in NZB/W F1 mice. Furthermore, we investigated whether HSPB5 can enhance the effects of methylprednisolone, a standard-of-care drug in LN, in an endotoxemia mouse model. METHODS: NZB/W F1 mice were treated with HSPB5, methylprednisolone, or vehicle from 23 to 38 weeks of age. Disease progression was evaluated by weekly proteinuria scores. At the end of the study, the blood, urine, spleens, and kidneys were collected for the assessment of proteinuria, blood urea nitrogen, kidney histology, serum IL-6 and anti-dsDNA levels, immune cell populations, and their phenotypes, as well as the transcript levels of proinflammatory chemokine/cytokines in the kidneys. HSPB5 was also evaluated in combination with methylprednisolone in a lipopolysaccharide-induced endotoxemia mouse model; serum IL-6 levels were measured at 24 h post-endotoxemia induction. RESULTS: HSPB5 significantly reduced terminal proteinuria and BUN and substantially improved kidney pathology. Similar trends, although to a lower extent, were observed with methylprednisolone treatment. Serum IL-6 levels and kidney expression of BAFF, IL-6, IFNγ, MCP-1 (CCL2), and KIM-1 were reduced, whereas nephrin expression was significantly preserved compared to vehicle-treated mice. Lastly, splenic Tregs and Bregs were significantly induced with HSPB5 treatment. HSPB5 in combination with methylprednisolone also significantly reduced serum IL-6 levels in endotoxemia mice. CONCLUSIONS: HSPB5 treatment reduces kidney inflammation and injury, providing therapeutic benefits in NZB/W F1 mice. Given that HSPB5 enhances the anti-inflammatory effects of methylprednisolone, there is a strong interest to develop HSBP5 as a therapeutic for the treatment of LN.

Impact of dietary vitamin D on immunoregulation and disease pathology in lupus-prone NZB/W F1 mice.

Vitamin D (VD) deficiency is a highly prevalent worldwide phenomenon and is extensively discussed as a risk factor for the development of systemic lupus erythematosus (SLE) and other immune-mediated diseases. In addition, it is now appreciated that VD possesses multiple immunomodulatory effects. This study aims to explore the impact of dietary VD intake on lupus manifestation and pathology in lupus-prone NZB/W F1 mice and identify the underlying immunological mechanisms modulated by VD. Here, we show that low VD intake accelerates lupus progression, reflected in reduced overall survival and an earlier onset of proteinuria, as well higher concentrations of anti-double-stranded DNA autoantibodies. This unfavorable effect gained statistical significance with additional low maternal VD intake during the prenatal period. Among examined immunological effects, we found that low VD intake consistently hampered the adoption of a regulatory phenotype in lymphocytes, significantly reducing both IL-10-expressing and regulatory CD4+ T cells. This goes along with a mildly decreased frequency of IL-10-expressing B cells. We did not observe consistent effects on the phenotype and function of innate immune cells, including cytokine production, costimulatory molecule expression, and phagocytic capacity. Hence, our study reveals that low VD intake promotes lupus pathology, likely via the deviation of adaptive immunity, and suggests that the correction of VD deficiency might not only exert beneficial functions by preventing osteoporosis but also serve as an important module in prophylaxis and as an add-on in the treatment of lupus and possibly other immune-mediated diseases. Further research is required to determine the most appropriate dosage, as too-high VD serum levels may also induce adverse effects, possibly also on lupus pathology.

SB431542 alleviates lupus nephritis by regulating B cells and inhibiting the TLR9/TGFß1/PDGFB signaling.

Lupus nephritis (LN) is the most common cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Currently, immunosuppressive treatments for LN are suboptimal and can induce significant side effects. SB431542 is a selective and potent inhibitor of the TGFß/Activin/NODAL pathway. Here, we study the effects of SB431542 treatment on LN and discuss the potential mechanisms. SB431542 ameliorated clinical outcomes with a consequent histological improvement in NZB/W mice. A comparative transcriptional profiling analysis revealed 586 differentially expressed genes (247 downregulated genes) in the SB431542 group compared to the control group. We found that the downregulated genes were mainly enriched in the biological processes of B cell activation, B cell proliferation, B cell differentiation, and B cell receptor signaling. Kyoto encyclopedia of genes and genomes pathway analysis revealed that the hematopoietic cell linage pathway was significantly downregulated in the SB431542 group. In addition, we observed that SB431542 reduced the splenic or renal levels of CD20 and the serum levels of anti-dsDNA antibody (IgG) in NZB/W mice. Furthermore, qRT-PCR and immunohistochemistry confirmed that SB431542 inhibits the production of TLR9, TGFß1, and PDGFB. Thus, due to its immunomodulatory activities, SB431542 could be considered for clinical therapy development for LN.

Modulation of the Itaconate Pathway Attenuates Murine Lupus.

OBJECTIVE: Itaconic acid, a Krebs cycle-derived immunometabolite, is synthesized by myeloid cells in response to danger signals to control inflammasome activation, type I interferon (IFN) responses, and oxidative stress. As these pathways are dysregulated in systemic lupus erythematosus (SLE), we investigated the role of an itaconic acid derivative in the treatment of established murine lupus. METHODS: Female (NZW × NZB)F1 lupus-prone mice were administered 4-octyl itaconate (4-OI) or vehicle starting after clinical onset of disease (30 weeks of age) for 4 weeks (n = 10 mice /group). At 34 weeks of age (peak disease activity), animals were euthanized, organs and serum were collected, and clinical, metabolic, and immunologic parameters were evaluated. RESULTS: Proteinuria, kidney immune complex deposition, renal scores of severity and inflammation, and anti-RNP autoantibodies were significantly reduced in the 4-OI treatment group compared to the vehicle group. Splenomegaly decreased in the 4-OI group compared to vehicle, with decreases in activation markers in innate and adaptive immune cells, increases in CD8+ T cell numbers, and inhibition of JAK1 activation. Gene expression analysis in splenocytes showed significant decreases in type I IFN and proinflammatory cytokine genes and increased Treg cell-associated markers in the 4-OI group compared to the vehicle group. In human control and lupus myeloid cells, 4-OI in vitro treatment decreased proinflammatory responses and B cell responses. CONCLUSIONS: These results support targeting immunometabolism as a potentially viable approach in autoimmune disease treatment, with 4-OI displaying beneficial roles attenuating immune dysregulation and organ damage in lupus.

Polymeric dexamethasone prodrugs attenuate lupus nephritis in MRL/lpr mice with reduced glucocorticoid toxicity.

Due to their potent immunosuppressive and anti-inflammatory effects, glucocorticoids (GCs) are the most widely used medications in treating lupus nephritis (LN). Long-term use of GCs, however, is associated with numerous off-target adverse effects. To reduce GCs' adverse effects, we previously developed two polymeric dexamethasone prodrug nanomedicines: N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based dexamethasone prodrug (P-Dex), and micelle-forming polyethylene glycol (PEG)-based dexamethasone prodrug (ZSJ-0228). Both P-Dex and ZSJ-0228 provided sustained amelioration of LN in lupus-prone NZB/W F1 mice with reduced GC-associated adverse effects. Here, we have extended our investigation to the MRL/lpr mouse model of LN. Compared to dose equivalent daily dexamethasone sodium phosphate (Dex) treatment, monthly P-Dex or ZSJ-0228 treatments were more effective in reducing proteinuria and extending the lifespan of MRL/lpr mice. Unlike the daily Dex treatment, ZSJ-0228 was not associated with measurable GC-associated adverse effects. In contrast, adrenal gland atrophy was observed in P-Dex treated mice.

Different Spatial and Temporal Roles of Monocytes and Monocyte-Derived Cells in the Pathogenesis of an Imiquimod Induced Lupus Model.

Mounting evidence indicates the importance of aberrant Toll-like receptor 7 (TLR7) signaling in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanism of disease progression remains unclear. An imiquimod (IMQ)-induced lupus model was used to analyze the lupus mechanism related to the aberrant TLR7 signals. C57BL/6 mice and NZB/NZW mice were treated with topical IMQ, and peripheral blood, draining lymph nodes, and kidneys were analyzed focusing on monocytes and monocyte-related cells. Monocytes expressed intermediate to high levels of TLR7, and the long-term application of IMQ increased Ly6Clo monocytes in the peripheral blood and Ly6Clo monocyte-like cells in the lymph nodes and kidneys, whereas Ly6Chi monocyte-like cell numbers were increased in lymph nodes. Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice were supplied by bone marrow-derived cells as demonstrated using a bone marrow chimera. Ly6Clo monocytes obtained from IMQ-induced lupus mice had upregulated adhesion molecule-related genes, and after adoptive transfer, they showed greater infiltration into the kidneys compared with controls. RNA-seq and post hoc PCR analyses revealed Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice had upregulated macrophage-related genes compared with peripheral blood Ly6Clo monocytes and downregulated genes compared with kidney macrophages (MF). Ly6Clo monocyte-like cells in the kidneys upregulated Il6 and chemoattracting genes including Ccl5 and Cxcl13. The higher expression of Il6 in Ly6Clo monocyte-like cells compared with MF suggested these cells were more inflammatory than MF. However, MF in IMQ-induced lupus mice were characterized by their high expression of Cxcl13. Genes of proinflammatory cytokines in Ly6Chi and Ly6Clo monocytes were upregulated by stimulation with IMQ but only Ly6Chi monocytes upregulated IFN-α genes upon stimulation with 2'3'-cyclic-GMP-AMP, an agonist of stimulator of interferon genes. Ly6Chi and Ly6Clo monocytes in IMQ-induced lupus mice had different features. Ly6Chi monocytes responded in the lymph nodes of locally stimulated sites and had a higher expression of IFN-α upon stimulation, whereas Ly6Clo monocytes were induced slowly and tended to infiltrate into the kidneys. Infiltrated monocytes in the kidneys likely followed a trajectory through inflammatory monocyte-like cells to MF, which were then involved in the development of nephritis.

Reduction of Cell Surface T-Cell Receptor by Non-Mitogenic CD3 Antibody to Mitigate Murine Lupus.

T-cells are critically involved in the pathogenesis of systemic lupus erythematosus. Although treatment with the anti-CD3 antibody has been reported to be effective in several autoimmune disease animal models including lupus, the immunosuppressive mechanisms remain obscure because of its pleiotropic in vivo kinetics. In this study, a conventional anti-CD3 (2C11C) and a non-mitogenic anti-CD3 with a manipulated Fc region (2C11S) were compared to elucidate the underlying mechanism of action. The efficacy and safety of 2C11S in vivo were demonstrated by sustained TCR reduction for a longer period as compared to 2C11C and no induction of cytokine release or T-cell depletion. Anti-CD3s were administered to NZB/W F1 (BWF1) mice at different time points for individual periods. The short-term treatment with 2C11S in the early phase of lupus suppressed the autoantibody associated with the reduction of germinal center B-cells. Treatment in the late phase attenuated lupus nephritis without affecting autoantibodies or differentiation of effector T-cells. The effect of reduced TCR in the development of autoimmunity was examined by CD3ζ heterozygous-deficient mice, in which T-cells had reduced TCR intensity but showed normal TCR signaling response. Autoantibody and lupus nephritis were attenuated significantly in CD3ζ heterozygous-deficient lupus-prone mice. Collectively, the reduction of surface TCR by non-mitogenic anti-CD3 could sufficiently suppress the development of lupus.

Small Molecule Inhibitors of Nuclear Export and the Amelioration of Lupus by Modulation of Plasma Cell Generation and Survival.

OBJECTIVE: To investigate the hypothesis that selective inhibitors of nuclear export (SINE compounds), recently approved for treatment of refractory plasma cell (PC) malignancy, may have potential in the treatment of lupus. METHODS: Female NZB/NZW mice were treated with the SINE compound KPT-350 or vehicle control. Tissue specimens were harvested and analyzed by flow cytometry, using standard markers. Nephritis was monitored by determining the proteinuria score and by histologic analysis of kidney specimens. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay, and total numbers of IgG-secreting and dsDNA-specific antibody-secreting cells were assessed by enzyme-linked immunospot assay. RESULTS: KPT-350 abrogated murine lupus nephritis at both early and late stages of the disease and rapidly impaired generation of autoreactive PCs in germinal centers (GCs). SINE compounds inhibited the production of NF-κB-driven homeostatic chemokines by stromal cells, altering splenic B and T cell strategic positioning and significantly reducing follicular helper T cell, GC B cell, and autoreactive PC counts. KPT-350 also decreased levels of cytokines and chemokines involved in PC survival and recruitment in the kidney of lupus-prone mice. Exportin 1, the target of SINE compounds, was detected in GCs of human tonsils, splenic B cells of lupus patients, and multiple B cell subsets in the kidneys of patients with lupus nephritis. CONCLUSION: Collectively, our results provide support for the therapeutic potential of SINE compounds, via their targeting of several molecular and cellular pathways critical in lupus pathogenesis, including autoantibody production by plasma cells.

Potential benefit of the cathepsin S inhibitor, ASP1617, as a treatment for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the dysregulation of various cell types and immunological pathways. Autoantibodies play an important role in its pathogenesis. The presence of autoantibodies suggests that self-antigen presentation through major histocompatibility complex (MHC) class II on antigen presenting cells is involved in the pathogenesis of autoimmune diseases, including SLE. Cathepsin S (CatS) is a key protease for antigen peptide loading onto lysosomal/endosomal MHC class II molecules through invariant chain degradation to promote antigen presentation. Inhibition of CatS is therefore expected to suppress antigen presentation via MHC class II, T and B cell activation, and antibody production from B cells. Here, we report the pharmacological profile of ASP1617, a novel CatS inhibitor. ASP1617 induced invariant chain accumulation and decreased the expression level of MHC class ΙΙ on the cell surface in both mouse and human B cells. Further, ASP1617 prevented DO11.10 mice T cell proliferation to ovalbumin antigen. We investigated the effects of ASP1617 and mycophenolate mofetil (MMF) on the development of lupus-like nephritis in NZB/W F1 mice, a widely used SLE mouse model. Oral administration of ASP1617 suppressed anti-dsDNA IgG, prevented progression of lupus-like glomerulonephritis, and significantly prevented proteinuria excretion. In contrast, MMF did not suppress anti-dsDNA IgG. Further, we found that plasma and/or urine CatS levels were increased in specimens from NZB/W F1 mice and several SLE patients. These results indicate that CatS may be an attractive therapeutic target for the treatment of SLE.

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

BACKGROUND: Several 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 RESULTS: Using 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. CONCLUSIONS: The 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.

Cellular and Molecular Phenotypes of pConsensus Peptide (pCons) Induced CD8+ and CD4+ Regulatory T Cells in Lupus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with widespread inflammation, immune dysregulation, and is associated with the generation of destructive anti-DNA autoantibodies. We have shown previously the immune modulatory properties of pCons peptide in the induction of both CD4+ and CD8+ regulatory T cells which can in turn suppress development of the autoimmune disease in (NZB/NZW) F1 (BWF1) mice, an established model of lupus. In the present study, we add novel protein information and further demonstrate the molecular and cellular phenotypes of pCons-induced CD4+ and CD8+ Treg subsets. Flow cytometry analyses revealed that pCons induced CD8+ Treg cells with the following cell surface molecules: CD25highCD28high and low subsets (shown earlier), CD62Lhigh, CD122low, PD1low, CTLA4low, CCR7low and 41BBhigh. Quantitative real-time PCR (qRT-PCR) gene expression analyses revealed that pCons-induced CD8+ Treg cells downregulated the following several genes: Regulator of G protein signaling (RGS2), RGS16, RGS17, BAX, GPT2, PDE3b, GADD45ß and programmed cell death 1 (PD1). Further, we confirmed the down regulation of these genes by Western blot analyses at the protein level. To our translational significance, we showed herein that pCons significantly increased the percentage of CD8+FoxP3+ T cells and further increased the mean fluorescence intensity (MFI) of FoxP3 when healthy peripheral blood mononuclear cells (PBMCs) are treated with pCons (10 µg/ml, for 24-48 hours). In addition, we found that pCons reduced apoptosis in CD4+ and CD8+ T cells and B220+ B cells of BWF1 lupus mice. These data suggest that pCons stimulates cellular, immunological, and molecular changes in regulatory T cells which in turn protect against SLE autoimmunity.

Fecal microbiota changes in NZB/W F1 mice after induction of lupus disease.

The association between the gut microbiota and the development of lupus is unclear. We investigated alterations in the gut microbiota after induction of lupus in a murine model using viral peptide of human cytomegalovirus (HCMV). Three treatment arms for the animals were prepared: intraperitoneal injection of HCMVpp65 peptide, adjuvant alone, and PBS injection. Feces were collected before and after lupus induction biweekly for 16S rRNA sequencing. HCMVpp65 peptide immunization induced lupus-like effects, with higher levels of anti-dsDNA antibodies, creatinine, proteinuria, and glomerular damage, compared with mice treated with nothing or adjuvant only. The Simpson diversity value was higher in mice injected with HCMVpp65 peptide, but there was no difference in ACE or Chao1 among the three groups. Statistical analysis of metagenomic profiles showed a higher abundance of various families (Saccharimonadaceae, Marinifiaceae, and Desulfovibrionaceae) and genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) in HCMVpp65 peptide-treated mice. Significant correlations between increased abundances of related genera (Candidatus Saccharimonas, Roseburia, Odoribacter, and Desulfovibrio) and HCMVpp65 peptide immunization-induced lupus-like effects were observed. This study provides insight into the changes in the gut microbiota after lupus onset in a murine model.

SMS2 deficiency impairs PKCδ-regulated B cell tolerance in the germinal center.

B cell tolerance prevents autoimmunity by deleting or deactivating autoreactive B cells that otherwise may cause autoantibody-driven disorders, including systemic lupus erythematosus (lupus). Lupus is characterized by immunoglobulin Gs carrying a double-stranded (ds)-DNA autospecificity derived mainly from somatic hypermutation in the germinal center (GC), pointing to a checkpoint breach of GC B cell tolerance that leads to lupus. However, tolerance mechanisms in the GC remain poorly understood. Here, we show that upregulated sphingomyelin synthase 2 (SMS2) in anti-dsDNA GC B cells induces apoptosis by directly activating protein kinase C δ (PKCδ)'s pro-apoptotic activity. This tolerance mechanism prevents lupus autoimmunity in C57/BL6 mice and can be stimulated pharmacologically to inhibit lupus pathogenesis in lupus-prone NZBWF1 mice. Patients with lupus consistently have substantially reduced SMS2 expression in B cells and to an even greater extent in autoimmune-prone, age-associated B cells, suggesting that patients with lupus have insufficient SMS2-regulated B cell tolerance.