TLR7 Activation Accelerates Cardiovascular Pathology in a Mouse Model of Lupus.
ABSTRACT: We report a novel model of lupus-associated cardiovascular pathology accelerated by the TLR7 agonist R848 in lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice. R848-treated TC mice but not non-autoimmune C57BL/6 (B6) controls developed microvascular inflammation and myocytolysis with intracellular vacuolization. This histopathology was similar to antibody-mediated rejection after heart transplant, although it did not involve complement. The TC or B6 recipients of serum or splenocytes from R848-treated TC mice developed a reactive cardiomyocyte hypertrophy, which also presents spontaneously in old TC mice as well as in TC.Rag-/- mice that lack B and T cells. Each of these cardiovascular lesions correspond to abnormalities that have been reported in lupus patients. Lymphoid and non-lymphoid immune cells as well as soluble factors contribute to lupus-associated cardiovascular lesions in TC mice, which can now be dissected using this model with and without R848 treatment.
Project description:The autoimmune disease systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies. It has been postulated that gut microbial dysbiosis may be one of the mechanisms involved in SLE pathogenesis. Here, we demonstrate that the dysbiotic gut microbiota of triple congenic (TC) lupus-prone mice (B6.<i>Sle1.Sle2.Sle3</i>) stimulated the production of autoantibodies and activated immune cells when transferred into germfree congenic C57BL/6 (B6) mice. Fecal transfer to B6 mice induced autoimmune phenotypes only when the TC donor mice exhibited autoimmunity. Autoimmune pathogenesis was mitigated by horizontal transfer of the gut microbiota between co-housed lupus-prone TC mice and control congenic B6 mice. Metabolomic screening identified an altered distribution of tryptophan metabolites in the feces of TC mice including an increase in kynurenine, which was alleviated after antibiotic treatment. Low dietary tryptophan prevented autoimmune pathology in TC mice, whereas high dietary tryptophan exacerbated disease. Reducing dietary tryptophan altered gut microbial taxa in both lupus-prone TC mice and control B6 mice. Consequently, fecal transfer from TC mice fed a high tryptophan diet, but not a low tryptophan diet, induced autoimmune phenotypes in germfree B6 mice. The interplay of gut microbial dysbiosis, tryptophan metabolism and host genetic susceptibility in lupus-prone mice suggest that aberrant tryptophan metabolism may contribute to autoimmune activation in this disease.
Project description:Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4+ T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4+ T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4+ T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4+ T cells were introduced into Rag1-/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4+ T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4+ T cells in a nonredundant manner with myeloid/stromal cells.
Project description:Lupus anti-nuclear Ab show the characteristics of Ag-driven T-cell-dependent (TD) humoral responses. If autoAg elicit the same response as exogenous Ag, lupus should enhance humoral responses to immunization. Blunted responses to various immunizations have, however, been reported in a significant portion of lupus patients. In this study, we show that lupus-prone C57BL/6.Sle1.Sle2.Sle3 (B6.TC) mice produce significantly less Ab in response to TD immunization than congenic controls, while producing significantly more total Ig. This blunted Ab response to TD Ag could be reconstituted with B6.TC B and CD4+ T cells. Multiple defects were found in the B6.TC response to 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin (NP-KLH) compared with total Ig, including a smaller percentage of B cells participating in the NP-response, a reduced entry into germinal centers, and highly defective production of NP-specific long-lived plasma cells (PC) in the bone marrow. B6.TC PC expressed reduced levels of FcgammaRIIb, which suggests that reduced apoptosis in resident PC prevents the establishment of newly formed NP-specific PC in bone marrow niches. Overall, these results show that lupus-prone mice responded differently to auto- and exogenous Ag and suggest that low FcgammaRIIb, hypergammaglobulinemia, and high autoAb production would be predictive of a poor response to immunization in lupus patients.
Project description:Marginal zone (MZ) B cells contain a large number of autoreactive clones and the expansion of this compartment has been associated with autoimmunity. MZ B cells also efficiently transport blood-borne antigen to the follicles where they activate T cells and differentiate into plasma cells. Using the B6.NZM2410.Sle1.Sle2.Sle3 (B6.TC) model of lupus, we show that the IgM+ CD1d(hi)/MZ B-cell compartment is expanded, and a large number of them reside inside the follicles. Contrary to the peripheral B-cell subset distribution and their activation status, the intrafollicular location of B6.TC IgM+ CD1d(hi)/MZ B cells depends on both bone marrow- and stromal-derived factors. Among the factors responsible for this intrafollicular location, we have identified an increased response to CXCL13 by B6.TC MZ B cells and a decreased expression of VCAM-1 on stromal cells in the B6.TC MZ. However, the reduced number of MZ macrophages observed in B6.TC MZs was independent of the IgM+ CD1d(hi)/B-cell location. B7-2 but not B7-1 deficiency restored IgM+ CD1d(hi)/MZ B-cell follicular exclusion in B6.TC mice, and it correlated with tolerance to dsDNA and a significant reduction of autoimmune pathology. These results suggest that follicular exclusion of IgM+ CD1d(hi)/MZ B cells is an important B-cell tolerance mechanism, and that B7-2 signaling is involved in breaching this tolerance checkpoint.
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4(+) T cells play an essential role. CD4(+) T cells rely on glycolysis for inflammatory effector functions, but recent studies have shown that mitochondrial metabolism supports their chronic activation. How these processes contribute to lupus is unclear. We show that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4(+) T cells from lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to non-autoimmune controls. In vitro, both the mitochondrial metabolism inhibitor metformin and the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) reduced interferon-? (IFN-?) production, although at different stages of activation. Metformin also restored the defective interleukin-2 (IL-2) production by TC CD4(+) T cells. In vivo, treatment of TC mice and other lupus models with a combination of metformin and 2DG normalized T cell metabolism and reversed disease biomarkers. Further, CD4(+) T cells from SLE patients also exhibited enhanced glycolysis and mitochondrial metabolism that correlated with their activation status, and their excessive IFN-? production was significantly reduced by metformin in vitro. These results suggest that normalization of T cell metabolism through the dual inhibition of glycolysis and mitochondrial metabolism is a promising therapeutic venue for SLE.
Project description:The breakdown in tolerance of autoreactive B cells in the lupus-prone NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) mice results in the secretion of autoantibodies. TC dendritic cells (DCs) enhance B cell proliferation and antibody secretion in a cytokine-dependent manner. However, the specific cytokine milieu by which TC DCs activate B cells was not known. In this study, we compared TC and C57BL/6 (B6) control for the distribution of DC subsets and for their production of cytokines affecting B cell responses. We show that TC DCs enhanced B cell proliferation through the production of IL-6 and IFN-?, while antibody secretion was only dependent on IL-6. Pre-disease TC mice showed an expanded PDCA1(+) cells prior to disease onset that was localized to the marginal zone and further expanded with age. The presence of PDCA1(+) cells in the marginal zone correlated with a Type I Interferon (IFN) signature in marginal zone B cells, and this response was higher in TC than B6 mice. In vivo administration of anti-chromatin immune complexes upregulated IL-6 and IFN-? production by splenic DCs from TC but not B6 mice. The production of BAFF and APRIL was decreased upon TC DC stimulation both in vitro and in vivo, indicating that these B cell survival factors do not play a role in B cell modulation by TC DCs. Finally, TC B cells were defective at downregulating IL-6 expression in response to anti-inflammatory apoptotic cell exposure. Overall, these results show that the TC autoimmune genetic background induces the production of B cell-modulating inflammatory cytokines by DCs, which are regulated by the microenvironment as well as the interplay between DC.
Project description:AM14 rheumatoid factor (RF) B cells in the MRL/lpr mice are activated by dual BCR and TLR7/9 ligation and differentiate into plasmablasts via an extrafollicular (EF) route. It was not known whether this mechanism of activation of RF B cells applied to other lupus-prone mouse models. We investigated the mechanisms by which RF B cells break tolerance in the NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) strain in comparison with C57BL/6 (B6) controls, each expressing the AM14 H chain transgene in the presence or absence of the IgG2a(a) autoantigen. The TC, but not B6, genetic background promotes the differentiation of RF B cells into Ab-forming cells (AFCs) in the presence of the autoantigen. Activated RF B cells preferentially differentiated into plasmablasts in EF zones. Contrary to the MRL/lpr strain, TC RF B cells were also located within germinal centers, but only the formation of EF foci was positively correlated with the production of RF AFCs. Immunization of young TC.AM14 H chain transgenic mice with IgG2a(a) anti-chromatin immune complexes (ICs) activated RF B cells in a BCR- and TLR9-dependent manner. However, these IC immunizations did not result in the production of RF AFCs. These results show that RF B cells break tolerance with the same general mechanisms in the TC and the MRL/lpr lupus-prone genetic backgrounds, namely the dual activation of the BCR and TLR9 pathways. There are also distinct differences, such as the presence of RF B cells in GCs and the requirement of chronic IgG2a(a) anti-chromatin ICs for full differentiation of RF AFCs.
Project description:CD4+ T cells have numerous features of over-activated cellular metabolism in lupus patients and mouse models of the disease. This includes a higher glycolysis than in healthy controls. Glucose transporters play an essential role in glucose metabolism by controlling glucose import into the cell from the extracellular environment. We have previously shown that treatment of lupus-prone mice with 2-deoxy-D-glucose, which inhibits the first step of glycolysis was sufficient to prevent autoimmune activation. However, direct targeting of glucose transporters has never been tested in a mouse model of lupus. Here, we show that CG-5, a novel glucose transporter inhibitor, ameliorated autoimmune phenotypes in a spontaneous lupus-prone mouse model, B6.NZM2410.Sle1.Sle2.Sle3 (Triple-congenic, TC), and in a chronic graft- vs. host-disease (cGVHD) model of induced lupus. In vitro, CG-5 blocked glycolysis in CD4+ T cells, and limited the expansion of CD4+ T cells induced by alloreactive stimulation. CG-5 also modulated CD4+ T cell polarization by inhibiting Th1 and Th17 differentiation and promoting regulatory T (Treg) induction. Moreover, CG-5 treatment reduced lupus phenotypes including the expansion of germinal center B (GC B) cells, as well as the production of autoantibodies in both TC mice and cGVHD models. Finally, CG-5 blocked glycolysis in human T cells. Overall, our data suggest that blocking glucose uptake with a small molecule inhibitor ameliorates autoimmune activation, at least partially due to its inhibition of glycolysis in CD4+ T cells.
Project description:Patients with systemic lupus erythematosus show an overexpression of type I IFN-responsive genes that is referred to as "IFN signature." We found that B6.NZMSle1/Sle2/Sle3 (Sle1,2,3) lupus-prone mice also express an IFN signature compared with non-autoimmune C57BL/6 mice. In vitro, myeloid dendritic cells (mDCs) (GM-CSF bone marrow-derived dendritic cells; BMDCs) from Sle1,2,3 mice constitutively overexpressed IFN-responsive genes such as IFN-?, Oas-3, Mx-1, ISG-15, and CXCL10 and members of the IFN signaling pathway STAT1, STAT2, and IRF7. The IFN signature was similar in Sle1,2,3 BMDCs from young, pre-autoimmune mice and from mice with high titers of autoantibodies, suggesting that the IFN signature in mDCs precedes disease onset and is independent from the autoantibodies. Sle1,2,3 BMDCs hyperresponded to stimulation with IFN-? and the TLR7 and TLR9 agonists R848 and CpGs. We propose that this hyperresponse is induced by the IFN signature and only partially contributes to the signature, as oligonucleotides inhibitory for TLR7 and TLR9 only partially suppressed the constitutive IFN signature, and pre-exposure to IFN-? induced the same hyperresponse in wild-type BMDCs as in Sle1,2,3 BMDCs. In vivo, mDCs and to a lesser extent T and B cells from young prediseased Sle1,2,3 mice also expressed the IFN signature, although they lacked the strength that BMDCs showed in vitro. Sle1,2,3 plasmacytoid DCs expressed the IFN signature in vitro but not in vivo, suggesting that mDCs may be more relevant before disease onset. We propose that Sle1,2,3 mice are useful tools to study the role of the IFN signature in lupus pathogenesis.
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive follicular helper T (Tfh) cells license high-affinity autoantibody production. Strikingly, the frequency of circulating Tfh is correlated with disease activity in SLE patients. As such, understanding the molecular mechanisms responsible for the aberrant Tfh cell generation and activation in lupus is of fundamental significance. We previously demonstrated that expanded Tfh cells in the B6.Sle1.Sle2.Sle3 (TC for triple congenic) lupus model exhibit high glycolysis and oxidative metabolism, which can be constrained by inhibiting glycolysis with 2-deoxyglucose (2DG). We performed RNA-seq analyses of splenic Tfh and naïve CD4+ T cells (Tn) comparing between TC and B6 mice. First, data revealed a large number of shared gene signatures in Tfh and Tn comparing between TC and B6 group, implicating that the aberrant development of Tfh initiates as early as Tn state. Further alignment of the Tfh transcriptome obtained from RNA-seq and earlier microarray assays demonstrated concerted alterations in numerous gene signatures of overactivation of T cells including dysregulated tyrosine kinase signaling and MAPK signaling pathways. Gene set enrichment analyses (GSEA) further revealed altered metabolic pathways (e.g., oxidative phosphorylation and pyruvate metabolism) among splenic TC Tfh cells. Overall design: Splenic CD4+CD44+CD162lo/-PD-1+ Tfh cells and CD4+CD44- Tn cells from B6.NZM-Sle1NZM2410/AegSle2NZM2410/AegSle3NZM2410/Aeg/LmoJ (TC) congenic mice and B6 control at the matched ages of 8 months old were sorted for RNA-seq analysis.