Lupus Model Research Articles

Genetic Reduction of IRF5 Expression after Disease Initiation Reduces Disease in a Mouse Lupus Model by Impacting Systemic and End-Organ Pathogenic Pathways.

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. Global homozygous or heterozygous deficiency of IRF5 from birth confers protection in many lupus mouse models. However, less is known about the effects of IRF5 targeting after autoimmunity has already developed. This is an important point to clarify when considering IRF5 as a potential therapeutic target in lupus. In this study, we demonstrate that genetic reduction of IRF5 expression after disease initiation reduces disease severity in the FcγRIIB-/- Y-linked autoimmune accelerating mouse lupus model. Reduction of IRF5 expression resulted in a decrease in splenomegaly and lymphadenopathy and a reduction in splenic B cell activation and plasmablast numbers. Splenic T cell activation and differentiation were also impacted as demonstrated by an increase in the number of naive CD4+ and CD8+ T cells and a reduction in the number of memory/effector CD4+ and CD8+ T cells. Although serum antinuclear autoantibody levels were not altered, reduction in IRF5 expression led to decreased immune complex deposition and complement activation, diminished glomerular and interstitial disease, and a reduction in immune cell infiltrate in the kidney. Mechanistically, myeloid cells in the kidney produced less inflammatory cytokines after TLR7 and TLR9 activation. Overall, we demonstrate that genetic reduction of IRF5 expression during an active autoimmune process is sufficient to reduce disease severity. Our data support consideration of IRF5 as a therapeutic target and suggest that approaches targeting IRF5 in systemic lupus erythematosus may need to impact IRF5 activity both systemically and in target organs.

Preclinical Evidence for the Glucocorticoid-Sparing Potential of a Dual Toll-Like Receptor 7/8 Inhibitor in Autoimmune Diseases.

Toll-like receptor 7 (TLR7) and TLR8 are single-stranded RNA-sensing endosomal pattern recognition receptors that evolved to defend against viral infections. However, aberrant TLR7/8 activation by endogenous ligands has been implicated in the pathogenesis of autoimmune diseases including systemic lupus erythematosus. TLR activation and type I interferon (IFN) were shown recently to impart resistance to glucocorticoids (GC), which are part of the standard of care for multiple autoimmune diseases. While GCs are effective, a plethora of undesirable effects limit their use. New treatment approaches that allow for the use of lower and safer doses of GCs would be highly beneficial. Herein, we report that a dual TLR7/8 inhibitor (TLR7/8i) increases the effectiveness of GCs in inflammatory settings. Human peripheral blood mononuclear cell studies revealed increased GC sensitivity in the presence of TLR7/8i for reducing inflammatory cytokine production, a synergistic effect that was most pronounced in myeloid cells, particularly monocytes. Gene expression analysis by NanoString and single-cell RNA sequencing revealed that myeloid cells were substantially impacted by combining low-dose TLR7/8i and GC, as evidenced by the effects on nuclear factor-kappa B-regulated cytokines and GC-response genes, although IFNs were affected to a smaller degree. Low dose of TLR7/8i plus GC was more efficacious then either agent alone in the MRL/lpr mouse model of lupus, with improved proteinuria and survival. Overall, our findings indicate a GC-sparing potential for TLR7/8i compounds, suggesting TLR7/8i may offer a new strategy for the treatment of autoimmune diseases. SIGNIFICANCE STATEMENT: Some features of autoimmune diseases may be resistant to glucocorticoids, mediated at least in part by toll-like receptor (TLR) activation, necessitating higher doses that are associated with considerable toxicities. We demonstrate that TLR7/8 inhibition and glucocorticoids work synergistically to reduce inflammation in a cell-type specific manner and suppress disease in a mouse model of lupus. TLR7/8 inhibition is a promising strategy for the treatment of autoimmune diseases and has glucocorticoid-sparing potential.

Phenotypic and functional characteristics of murine CD11c+ B cells which is suppressed by metformin.

Since the description of age-associated or autoimmune-associated B cells (ABCs), there has been a growing interest in the role of these cells in autoimmunity. ABCs are differently defined depending on the research group and are heterogenous subsets. Here, we sought to characterize ABCs in Sle1/2/3 triple congenic (TC) mice, which is a well accepted mouse model of lupus. Compared to follicular (FO) B cells, ABCs have many distinct functional properties, including antigen presentation. They express key costimulatory molecules for T cell activation and a distinct profile of cytokines. Moreover, they exhibit an increased capacity for antigen uptake. ABCs were also compared with germinal center (GC) B cells, which are antigen activated B cell population. There are several phenotypic similarities between ABCs and GC B cells, but GC B cells do not produce proinflammatory cytokines or take up antigen. While T cell proliferation and activation is induced by both FO B and ABCs in an antigen-dependent manner, ABCs induce stronger T cell receptor signaling in naïve CD4+ T cells and preferentially induce differentiation of T follicular helper (Tfh) cells. We found that ABCs exhibit a distinct transcriptomic profile which is focused on metabolism, cytokine signaling and antigen uptake and processing. ABCs exhibit an increase in both glycolysis and oxidative phosphorylation compared to FO B cells. Treatment of ABCs with metformin suppresses antigen presentation by decreasing antigen uptake, resulting in decreased Tfh differentiation. Taken together, these findings define a fundamental connection between metabolism and function within ABCs.

Serum components influence antibody reactivity to glycan and DNA antigens

We previously generated three types of anti-glycan monoclonal IgM antibodies that react with certain structures on the glycans of glycosphingolipids and glycoproteins. As the nucleotide sequences for the variable regions of these IgM antibodies showed homology with those of anti-DNA antibodies deposited in public databases, we analyzed the reactivity of the anti-glycan IgM antibodies to DNA by ELISA. We found that anti-α2,6-sialyl LacNAc IgM in the supernatant of a hybridoma culture cross-reacted with DNA, and after purification of the IgM by zirconia column chromatography, the highly purified IgM showed increased cross-reactivity to DNA. As most of the contaminating bovine serum proteins in the culture supernatant were removed by the purification process, it is likely that a part of the removed components influences antibody reactivity to DNA. Purified anti-DNA antibodies prepared from lupus model NZB/W F1 and MRL/lpr mouse sera and normal human serum were then analyzed, and similar results showing increased reactivity to DNA were obtained. Furthermore, ELISA using these purified antibodies and various carbohydrate antigens showed that the antigen-binding specificity of these antibodies was altered by the purification process from serum-containing antibody preparations. Our results indicate that mammalian serum contains components that strongly influence antibody reactivity to carbohydrate antigens, including DNA.

HUC-MSC transplantation therapy effects on lupus-prone MRL/lpr mice at early disease stages

BackgroundThe efficacy of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation in treating systemic lupus erythematosus (SLE) has been confirmed by small-scale clinical trials. However, these trials focused on severe or refractory SLE, while few studies focused on mild SLE. Therefore, this study focused on the therapeutic effects of hUC-MSC transplantation in early-stage or mild MRL/lpr lupus model mice.MethodsCommercially available hUC-MSCs were transplanted into 8-week-old MRL/lpr mice by tail vein injection. Flow cytometry was used to analyze B cells and their subsets in the peripheral blood. Further, plasma inflammatory factors, autoantibodies, and plasma biochemical indices were detected using protein chip technology and ELISA kits. In addition, pathological staining and immunofluorescence were performed to detect kidney injury in mice.ResultshUC-MSC transplantation did not affect the mice’s body weight, and both middle and high dose hUC-MSC transplantation (MD and HD group) actually reduced spleen weight. hUC-MSC transplantation significantly decreased the proportion of plasmablasts (PB), IgG1− PB, IgG1+ PB, IgG1+ memory B (MB) cells, IgG1+ DN MB, and IgG1+ SP MB cells. The hUC-MSC transplantation had significantly reduced plasma levels of inflammatory factors, such as TNF-α, IFN-γ, IL-6, and IL-13. Pathological staining showed that the infiltration of glomerular inflammatory cells was significantly reduced and that the level of glomerular fibrosis was significantly alleviated in hUC-MSC-transplanted mice. Immunofluorescence assays showed that the deposition of IgG and IgM antibodies in the kidneys of hUC-MSC-transplanted mice was significantly lower than in the control.ConclusionhUC-MSC transplantation could inhibit the proliferation and differentiation of peripheral blood B cells in the early-stage of MRL/lpr mice, thereby alleviating the plasma inflammatory environment in mice, leading to kidney injury remission. The study provides a new and feasible strategy for SLE treatment.

A novel Toll-like receptor 7/8–specific antagonist E6742 ameliorates clinically relevant disease parameters in murine models of lupus

The sensing of self RNA by the endosomal Toll-like receptors (TLRs) 7 and 8 initiates pathogenic mechanisms underlying the autoimmune disease lupus. A blockade of the TLR7/8 signals may, therefore, be a novel therapeutic intervention for lupus. To test the hypothesis, a novel compound E6742 that blocks TLR7/8 activation was identified. The mode of action of E6742 was investigated by analysis of the tertiary structure of TLR7 and 8 in complex with E6742. The in vitro activities of the compound were examined in cellular systems and its therapeutic potential was evaluated in murine lupus models. Tertiary structures of the extracellular domain of TLR7 and 8 in complex with E6742 showed that E6742 binds specifically and non-covalently to the hydrophobic pocket located at the interface of TLR7 or TLR8 homodimers. E6742 potently and selectively inhibited several TLR7/8-mediated cytokine responses in human PBMC. In two mouse models of lupus, oral dosing of E6742 after the onset of disease suppressed increase in autoantibodies and blocked the advance of organ damage. Collectively, the data show that TLR7/8 activation contributes to disease progression and its blocking by E6742 has potential as a therapeutic intervention for lupus.

Disease in the Pld4thss/thss Model of Murine Lupus Requires TLR9.

Phospholipase D4 (PLD4) is an endolysosomal exonuclease of ssRNA and ssDNA, rather than a phospholipase as its name suggests. Human polymorphisms in the PLD4 gene have been linked by genome-wide association studies to systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. However, B6.129 Pld4-/- mice develop features of a distinct disease, macrophage activation syndrome, which is reversed in mice mutated in TLR9. In this article, we compare a Pld4 null mutant identified on the BALB/c background, Pld4thss/thss, which has distinct phenotypes: short stature, thin hair, and features of systemic lupus erythematosus. All phenotypes analyzed were largely normalized in Pld4thss/thssTlr9-/- mice. Thus, Pld4thss/thss represents a rare model in which mouse lupus etiology is TLR9 dependent. Compared with PLD4-deficient B6 mice, Pld4thss/thss mice had elevated levels of serum IgG, IgG anti-dsDNA autoantibodies, BAFF, and IFN-γ and elevated B cell numbers. Overall, the data suggest that PLD4 deficiency can lead to a diverse array of rheumatological abnormalities depending upon background-modifying genes, and that these diseases of PLD4 deficiency are largely driven by TLR9 recognition of ssDNA.

TIGIT-Fc fusion protein alleviates murine lupus nephritis through the regulation of SPI-B-PAX5-XBP1 axis-mediated B-cell differentiation

ObjectivesT cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is a newly discovered immune checkpoint (IC) that exhibits immunosuppressive function in the regulation of immune system. Activation of TIGIT signaling has emerged as a promising approach for autoimmune disease immunotherapy, such as systemic lupus erythematosus (SLE). MethodsWe generated a chimeric protein, TIGIT-immunoglobulin (Ig), by fusing the extracellular domain of murine TIGIT to the Fc region of mouse IgG2a, which was used to investigated the effect of activating the TIGIT signaling in murine lupus models (MRL/lpr and chronic graft-versus-host disease mice). Treated mice were harvested, and samples of serum, kidney, and spleen were collected for outcome evaluation. In vitro treatment of TIGIT-Ig in B cells was used for exploring the roles of TIGIT in toll-like receptor 7 (TLR7)-mediated B cell differentiation and antibody production. ResultsTIGIT-Ig treatment delayed disease progression in both lupus models, accompanied by a decrease in the production of anti-double stranded DNA antibodies (anti-dsDNA), proteinuria, proteinuria/creatinine, and Ig kidney deposition. Additionally, the group treated with TIGIT-Ig displayed a decreased proportion of T helper cell (Th)1 cells, T follicular helper (Tfh) cells, and B-cell subsets, including germinal center B cells (GC B), plasmablasts, and plasma cells, compared to the group treated with control IgG. Interestingly, we also observed an increased proportion of Tregs in the spleen of the TIGIT-Ig group. We have discovered a new way in which activating the TIGIT pathway can regulate B-cell differentiation through the SPI-B-PAX5-XBP1 pathway, resulting in a reduction in autoantibodies. ConclusionTogether, TIGIT may be a promising IC target for SLE treatment.

Endogenous erythropoietin has immunoregulatory functions that limit the expression of autoimmune kidney disease in mice.

Administration of recombinant erythropoietin (EPO), a kidney-produced hormone with erythropoietic functions, has been shown to have multiple immunoregulatory effects in mice and humans, but whether physiological levels of EPO regulate immune function in vivo has not been previously evaluated. We generated mice in which we could downregulate EPO production using a doxycycline (DOX)-inducible, EPO-specific silencing RNA (shEPOrtTAPOS), and we crossed them with B6.MRL-Faslpr/J mice that develop spontaneous lupus. We treated these B6.MRL/lpr shEPOrtTAPOS with DOX and serially measured anti-dsDNA antibodies, analyzed immune subsets by flow cytometry, and evaluated clinical signs of disease activity over 6 months of age in B6.MRL/lpr shEPOrtTAPOS and in congenic shEPOrtTANEG controls. In B6.MRL/lpr mice, Epo downregulation augmented anti-dsDNA autoantibody levels and increased disease severity and percentages of germinal center B cells compared with controls. It also increased intracellular levels of IL-6 and MCP-1 in macrophages. Our data in a murine model of lupus document that endogenous EPO reduces T- and B-cell activation and autoantibody production, supporting the conclusion that EPO physiologically acts as a counterregulatory mechanism to control immune homeostasis.

TLR7/TLR8 activation and susceptibility genes synergize to breach gut barrier in a mouse model of lupus.

Mounting evidence suggests that increased gut permeability, or leaky gut, and the resulting translocation of pathobionts or their metabolites contributes to the pathogenesis of Systemic Lupus Erythematosus. However, the mechanisms underlying the induction of gut leakage remain unclear. In this study, we examined the effect of a treatment with a TLR7/8 agonist in the B6.Sle1.Sle2.Sle3 triple congenic (TC) mouse, a spontaneous mouse model of lupus without gut leakage. Lupus-prone mice (TC), TC.Rag1-/- mice that lack B and T cells, and congenic B6 healthy controls were treated with R848. Gut barrier integrity was assessed by measuring FITC-dextran in the serum following oral gavage. Claudin-1 and PECAM1 expression as well as the extent of CD45+ immune cells, B220+ B cells, CD3+ T cells and CD11b+ myeloid cells were measured in the ileum by immunofluorescence. NKp46+ cells were measured in the ileum and colon by immunofluorescence. Immune cells in the ileum were also analyzed by flow cytometry. R848 decreased gut barrier integrity in TC but not in congenic control B6 mice. Immunofluorescence staining of the ileum showed a reduced expression of the tight junction protein Claudin-1, endothelial cell tight junction PECAM1, as well as an increased infiltration of immune cells, including B cells and CD11b+ cells, in R848-treated TC as compared to untreated control mice. However, NKp46+ cells which play critical role in maintaining gut barrier integrity, had a lower frequency in treated TC mice. Flow cytometry showed an increased frequency of plasma cells, dendritic cells and macrophages along with a decreased frequency of NK cells in R848 treated TC mice lamina propria. In addition, we showed that the R848 treatment did not induce gut leakage in TC.Rag1-/- mice that lack mature T and B cells. These results demonstrate that TLR7/8 activation induces a leaky gut in lupus-prone mice, which is mediated by adaptive immune responses. TLR7/8 activation is however not sufficient to breach gut barrier integrity in non-autoimmune mice.

Cytoarchitectureal changes in hippocampal subregions of the NZB/W F1 mouse model of lupus

Over 50% of clinical patients affected by the systemic lupus erythematosus disease display impaired neurological cognitive functions and psychiatric disorders, a form called neuropsychiatric systemic lupus erythematosus. Hippocampus is one of the brain structures most sensitive to the cognitive deficits and psychiatric disorders related to neuropsychiatric lupus. The purpose of this study was to compare, layer by layer, neuron morphology in lupus mice model NZB/W F1 versus Wild Type mice.By a morphometric of cells identified on Nissl-stained sections, we evaluated structural alterations between NZB/W F1 and Wild Type mice in seven hippocampal subregions: Molecular dentate gyrus, Granular dentate gyrus, Polymorph dentate gyrus, Oriens layer, Pyramidal layer, Radiatum layer and Lacunosum molecular layer. By principal component analysis we distinguished healthy Wild Type from NZB/W F1 mice. In NZB/W F1 mice hippocampal cytoarchitecture, the neuronal cells resulted larger in size and more regular than those of Wild Type. In NZB/W F1, neurons were usually denser than in WT. The Pyramidal layer neurons were much denser in Wild Type than in NZB/W F1. Application of principal component analysis, allowed to distinguish NZB/W F1 lupus mice from healthy, showing as NZBW subjects presented a scattered distribution and intrasubject variability.Our results show a hypertrophy of the NZB/W F1 hippocampal neurons associated with an increase in perikaryal size within the CA1, CA2, CA3 region and the DG. These results help advance our understanding on hippocampal organization and structure in the NZB/W F1 lupus model, suggesting the hypothesis that the different subregions could be differentially affected in neuropsychiatric systemic lupus erythematosus disease. Leveraging an in-depth analysis of the morphology of neural cells in the hippocampal subregions and applying dimensionality reduction using PCA, we propose an efficient methodology to distinguish pathological NZBW mice from WT mice.”

Impaired regulatory function of granzyme B-producing B cells against T cell inflammatory responses in lupus mice

ObjectiveRecently, a new subtype of granzyme B (GrB)-producing Breg cells has been identified, which was proven to be involved in autoimmune disease. Our recent report demonstrated that GrB-producing Breg cells...

Impaired dynamic X-chromosome inactivation maintenance in T cells is a feature of spontaneous murine SLE that is exacerbated in female-biased models

ObjectiveSystemic lupus erythematosus (SLE) is a highly female-biased systemic autoimmune disease, but the molecular basis for this female bias remains incompletely elucidated. B and T lymphocytes from patients with SLE and female-biased mouse models of SLE exhibit features of epigenetic dysregulation on the X chromosome which may contribute to this strong female bias. We therefore examined the fidelity of dynamic X-chromosome inactivation maintenance (dXCIm) in the pathogenesis of two murine models of spontaneous lupus—NZM2328 and MRL/lpr—with disparate levels of female-bias to determine whether impaired dXCIm contributes to the female bias of disease. MethodsCD23+ B cells and CD3+ T cells were purified from age-matched C57BL/6 (B6), MRL/lpr, and NZM2328 male and female mice, activated in vitro, and processed for Xist RNA fluorescence in situ hybridization, H3K27me3 immunofluorescence imaging, qPCR, and RNA sequencing analyses. ResultsThe dynamic relocalization of Xist RNA and the canonical heterochromatin mark, H3K27me3, to the inactive X chromosome was preserved in CD23+ B cells, but impaired in activated CD3+ T cells from the MRL/lpr model (p < 0.01 vs. B6), and even more impaired in the heavily female-biased NZM2328 model (p < 0.001 vs. B6; p < 0.05 vs. MRL/lpr). RNAseq of activated T cells from NZM2328 mice revealed the female-biased upregulation of 32 X-linked genes distributed broadly across the X chromosome, many of which have roles in immune function. Many genes encoding Xist RNA-interacting proteins were also differentially expressed and predominantly downregulated, which may account for the observed mislocalization of Xist RNA to the inactive X chromosome. ConclusionsAlthough evident in T cells from both the MRL/lpr and NZM2328 models of spontaneous SLE, impaired dXCIm is more severe in the heavily female-biased NZM2328 model. The aberrant X-linked gene dosage in female NZM2328 mice may contribute towards the development of female-biased immune responses in SLE-prone hosts. These findings provide important insights into the epigenetic mechanisms contributing to female-biased autoimmunity.

S1P/S1PR1 axis promotes macrophage M1 polarization through NLRP3 inflammasome activation in Lupus nephritis

Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) as well as the leading cause of mortality in patients. Previous studies revealed that S1P level is elevated in plasma samples of SLE patients and murine lupus models. FTY720, targeting S1P receptors, exhibited therapeutic effects in improving the nephritis symptoms of lupus mouse models. However, few studies have discussed the potential relevance of S1P/S1PR to the pathogenesis of LN. Macrophages have been shown to be an important causative agent of renal inflammation, while the pro-inflammatory M1-type promotes kidney injury and inflammation during LN. Importantly, macrophages express various S1P receptors, and how they respond to S1P in the setting of LN remains unclear. Therefore, we examined the level of S1P in the lupus MRL/lpr mice and explored the ensuing interaction of macrophages and S1P. We found that S1P level was elevated in the MRL/lpr mice with a subsequent enhancement of the S1PR1 expression, and blocking S1PR1 by FTY720, the nephritis symptoms of MRL/lpr mice were improved. Mechanistically, we demonstrated that elevated S1P level increase the M1-type macrophage accumulation. And the in-vitro studies proved that S1P/S1PR1 was involved in the promotion of macrophage polarization towards M1 type through activation of NLRP3 inflammasome. These findings confer a novel role to macrophage S1PR1 and provide a new perspective for targeting S1P during LN.

Pharmacologic inhibition of glycolysis prevents the development of lupus by altering the gut microbiome in mice

Gut dysbiosis has been associated with lupus pathogenesis, and fecal microbiota transfers (FMT) from lupus-prone mice shown to induce autoimmune activation into healthy mice. The immune cells of lupus patients exhibit an increased glucose metabolism and treatments with 2-deoxy-D-glucose (2DG), a glycolysis inhibitor, are therapeutic in lupus-prone mice. Here, we showed in two models of lupus with different etiologies that 2DG altered the composition of the fecal microbiome and associated metabolites. In both models, FMT from 2DG-treated mice protected lupus-prone mice of the same strain from the development of glomerulonephritis, reduced autoantibody production as well as the activation of CD4+ Tcells and myeloid cells as compared to FMT from control mice. Thus, we demonstrated that the protective effect of glucose inhibition in lupus is transferable through the gut microbiota, directly linking alterations in immunometabolism to gut dysbiosis in the hosts.

Involvement of Transcriptional Factor Pbx1 in Peripheral B Cell Homeostasis to Constrain Lupus Autoimmunity.

Disruption of B cell homeostasis and subsequent dominance of effector B cell subsets are critical for the development of systemic lupus erythematosus (SLE). Revealing the key intrinsic regulators involved in the homeostatic control of B cells has important therapeutic value for SLE. This study was undertaken to determine the regulatory role of the transcription factor Pbx1 in B cell homeostasis and lupus pathogenesis. We constructed mice with B cell-specific deletion of Pbx1. T cell-dependent and T cell-independent humoral responses were induced by intraperitoneal injection of nitrophenyl-containing hapten (NP) conjugated to keyhole limpet hemocyanin or NP-Ficoll. The regulatory effects of Pbx1 on autoimmunity were observed in a Bm12-induced lupus murine model. We investigated mechanisms of Pbx1 using RNA sequencing, the cleavage under targets and tagmentation assay, and chromatin immunoprecipitation-quantitative polymerase chain reaction assay. We transduced B cells from SLE patients with plasmids that overexpressed PBX1 to explore the in vitro therapeutic efficacy of PBX1. Pbx1 was specifically down-regulated in autoimmune B cells and negatively correlated with disease activity. The deficiency of Pbx1 in B cells resulted in excessive humoral responses following immunization. In the Bm12-induced lupus model, mice with B cell-specific Pbx1 deficiency displayed enhancements in germinal center responses, plasma cell differentiation, and autoantibody production. Pbx1-deficient B cells had increased survival and proliferative advantages after activation. Pbx1 regulated genetic programs by directly targeting critical components of the proliferation and apoptosis pathways. In SLE patients, PBX1 expression was negatively correlated with effector B cell expansion; when PBX1 expression was enforced, the survival and proliferative capacity of SLE B cells were attenuated. Our study reveals the regulatory function and mechanism of Pbx1 in adjusting B cell homeostasis and highlights Pbx1 as a therapeutic target in SLE.

Gut epithelial barrier dysfunction in lupus triggers a differential humoral response against gut commensals.

Systemic lupus erythematosus is an autoimmune disease with multisystemic involvement including intestinal inflammation. Lupus-associated intestinal inflammation may alter the mucosal barrier where millions of commensals have a dynamic and selective interaction with the host immune system. Here, we investigated the consequences of the intestinal inflammation in a TLR7-mediated lupus model. IgA humoral and cellular response in the gut was measured. The barrier function of the gut epithelial layer was characterised. Also, microbiota composition in the fecal matter was analysed as well as the systemic humoral response to differential commensals. The lupus-associated intestinal inflammation modifies the IgA+ B cell response in the gut-associated lymphoid tissue in association with dysbiosis. Intestinal inflammation alters the tight junction protein distribution in the epithelial barrier, which correlated with increased permeability of the intestinal barrier and changes in the microbiota composition. This permeability resulted in a differential humoral response against intestinal commensals. Lupus development can cause alterations in microbiota composition, allowing specific species to colonize only the lupus gut. Eventually, these alterations and the changes in gut permeability induced by intestinal inflammation could lead to bacterial translocation.

Loss of interleukin-1 beta is not protective in the lupus-prone NZM2328 mouse model.

Aberrant activation of the innate immune system is a known driver of lupus pathogenesis. Inhibition of the inflammasome and its downstream signaling components in murine models of lupus has been shown to reduce the severity of disease. Interleukin-1 beta (IL-1β) is a proinflammatory cytokine released from cells following inflammasome activation. Here, we examine how loss of IL-1β affects disease severity in the lupus-prone NZM2328 mouse model. We observed a sex-biased increase in immune complex deposition in the kidneys of female mice in the absence of IL-1β that corresponds to worsened proteinuria. Loss of IL-1β did not result in changes in overall survival, anti-dsDNA autoantibody production, or renal immune cell infiltration. RNA-sequencing analysis identified upregulation of TNF and IL-17 signaling pathways specifically in females lacking IL-1β. Increases in these signaling pathways were also found in female patients with lupus nephritis, suggesting clinical relevance for upregulation of these pathways. Together, these data suggest that inhibition of the inflammasome or its downstream elements that block IL-1β signaling may need to be approached with caution in SLE, especially in patients with renal involvement to prevent potential disease exacerbation.

Dual inhibition of phosphoinositide 3-kinases delta and gamma reduces chronic B cell activation and autoantibody production in a mouse model of lupus.

Phosphoinositide 3-kinase delta (PI3Kδ) plays key roles in normal B cell activation and is chronically activated in malignant B cells. Targeting of PI3Kδ using FDA-approved drugs Idelalisib or Umbralisib has shown efficacy in treatment of multiple B cell malignancies. Duvelisib, an inhibitor targeting both PI3Kδ and PI3Kγ (PI3Kδγi) has also been used for treatment of several leukemias and lymphomas and was suggested to offer potential additional benefits in supressing T cell and inflammatory responses. Transcriptomics analyses indicated that while most B cell subsets predominantly express PI3Kδ, plasma cells upregulate PI3Kγ. We thus assessed whether PI3Kδγi treatment can impact chronic B cell activation in the context of an autoantibody-mediated disease. Using the TAPP1R218LxTAPP2R211L (TAPP KI) mouse model of lupus-like disease driven by dysregulated PI3K pathway activity, we performed 4 week PI3Kδγi treatments and found significant reduction in CD86+ B cells, germinal center B cells, follicular helper T cells and plasma cells in multiple tissues. This treatment also significantly attenuated the abnormally elevated serum levels of IgG isotypes observed in this model. The profile of autoantibodies generated was markedly altered by PI3Kδγi treatment, with significant reductions in IgM and IgG targeting nuclear antigens, matrix proteins and other autoantigens. Kidney pathology was also impacted, with reduced IgG deposition and glomerulonephritis. These results indicate that dual inhibition of PI3Kδ and PI3Kγ can target autoreactive B cells and may have therapeutic benefits in autoantibody-mediated disease.

Type 1 interferons promote anti-RBC antibody responses in interferon-dependent lupus mice

Abstract Red blood cell (RBC) transfusion can induce anti-RBC antibodies that promote hemolytic events. Patients with systemic lupus erythematosus (SLE) have an increased frequency of RBC alloimmunization, and two-thirds of patients express a type 1 interferon (IFNα/β) gene signature. We previously reported that IFNα/β induces RBC alloimmunization in the IFNα/β-dependent pristane lupus mouse model. However, it is unclear whether the lupus-like phenotype or IFNα/β directly promoted alloimmunization. Thus, we examined alloantibody responses in MRL/lpr mice, which have an IFNα/β-independent lupus-like phenotype. MRL/lpr mice produced anti-dsDNA lupus antibodies. However, unlike pristane-treated WT mice, IFNα/β-stimulated genes (ISGs) were not elevated in MRL/lpr mice. After transfusion with allogeneic RBCs, untreated WT and MRL/lpr mice produced minimal levels of anti-RBC antibodies, compared to elevated levels produced by pristane-treated WT mice. To determine whether IFNα/β can induce alloimmunization in MRL/lpr mice, WT and MRL/lpr mice were treated with recombinant IFNα (rIFNα) or the IFNα/β stimulus, poly(I:C), prior to transfusion. While rIFNa and poly(I:C) induced RBC alloimmunization in WT mice, they failed to induce alloimmunization in MRL/lpr mice. Thus, MRL/lpr mice are resistant to IFNα/β-induced alloimmunization due to a currently explored mechanism. These findings also illustrate that allogeneic transfusion induces alloimmunization in an IFNα/β-dependent lupus model, but not in an IFNα/β-independent model. Thus, the lupus-like phenotype is not sufficient to induce alloimmunization. Collectively, these findings warrant investigation of the role of IFNα/β in RBC alloimmunization in patients with SLE. NIH (R01 HL165169-01); NIH (R03 HL158637-01); NIH (K08 HL141446-03)