NADPH deficiency exacerbates lupus in murine models and in patients, but the mechanisms remain unknown. One hypothesis is that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, non-autoimmune mice. Prior work implicated cytochrome b-245, beta polypeptide (CYBB), a component of the NADPH oxidase complex, and RUN and cysteine-rich domain containing Beclin 1 interacting protein (RUBICON), as requisite for LAP.To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Fas lpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite-RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradicts the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found instead that LAP is not dependent on either of these two pathways. Our data thus reveal RUBICON as a novel regulator of SLE, possibly by a B cellintrinsic mechanism, but do not support a role for LAP in lupus.
TNF inhibitors are widely used to treat inflammatory diseases; however, 30-50% of treated patients develop new autoantibodies and 0.5-1% develop secondary autoimmune diseases, including lupus. TNF is required for formation of germinal centers (GCs), the site where high affinity autoantibodies are often made. We found that TNF deficiency in Sle1 mice induced TH17 T cells and enhanced the production of germline encoded, T-dependent IgG anticardiolipin antibodies but did not induce GC formation or precipitate clinical disease. We then asked whether a second hit could restore GC formation or induce pathogenic autoimmunity in TNF deficient mice. By using a range of immune stimuli, we found that somatically mutated autoantibodies and clinical disease can arise in the setting of TNF deficiency via extrafollicular pathways or via atypical GC-like pathways. This breach of tolerance may be due to defects in regulatory signals that modulate the negative selection of pathogenic autoreactive B cells.
The induction of autoantibodies and autoimmunity in patients treated with TNF inhibitors (TNFi) is well-known, but the mechanism by which TNFi induce breach of B cell tolerance is not known. TNF deficiency induces TFH and TH17 cell expansions and disrupts germinal centers (GC), that regulate high affinity antigen specific antibody production and autoreactive B cell selection. We found that TNF deficiency in Sle1 mice (Sle1.TNF−/−) induced high titers of germline encoded, T-dependent IgG anti-cardiolipin. Upon treated with TLR9 ligand (DNA/curli amyloid protein), Sle1.TNF−/− mice developed high IgG anti-dsDNA; although CD95+PNA+/GL7lowGC phenotype B cells were induced, they did not form GC nor acquire dark zone phenotype, and their Ig heavy chains accumulated low numbers of mutations. Similarly, without proper GC, GC phenotype B cells were found in the TNFR1/2−/−NZM2328 mice, who developed accelerated lupus, suggesting disturbed GC by TNF deficiency dysregulated GC phenotype B cell selection. Using an antibody to Neu5Gc (N-Glycolylneuraminic acid, a CD22 high affinity ligand which is the hydrolyzed product of GL7 ligand, N-acetylneuraminic acid, Neu5Ac), we found that GC phenotype B cells from TNF deficient mice failed to stop hydrolyzing Neu5Ac to Neu5Gc, suggesting a failure in recruitment of CD22 to the BCR complex upon activation. Furthermore, activated B cells from Sle1.TNF−/− mice constitutively expressed high level of pS6 and SHIP-1; whereas, additional stimulants, anti-Ig and/or anti-CD40, were required to induce pS6 expression in those of the Sle1 mice. Here, we report a possible mechanism by which TNF deficiency alters the homeostasis of regulatory molecules such as CD22 and FcgRIIB, and thus activated B cell selection.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease with significant morbidity and mortality - demanding further examination of disease reducing treatments. Previously, we showed that a 2-week course of combined CTLA4Ig and anti-CD40L costimulatory blockade prevented loss of tolerance to dsDNA in NZB/W mice while maintaining immune function against exogenous antigen, explained by an altered selection of autoreactive B cells in germinal centers (GC). Here, we examined both B and T cells to clarify if restoration of tolerance is due to a change in GC B cell profile or a decrease in T cell help. 3 months after treatment, large germinal centers persisted in the treated group, but due to smaller spleen size total GC B cells and TFH Cells were decreased compared with controls. The metabolic profile of splenic B and T cells showed a decreased spare respiratory capacity in both subsets and a decrease in B cell glycolysis among the treated group. RNA-seq analysis of GC dark zone and light zone B cells from treated mice showed decreased expression of markers of oxidative phosphorylation and cell proliferation and increased expression of signaling molecules downstream of BCR, including PLCγ, compared with untreated controls. GC B cell stimulation assays showed a delay in phosphorylation of PLCγ in response to anti-IgG in treated mice. These data show a long-lasting effect of short-term costimulatory blockade on both B and T cells with metabolic remodeling of GC B cells and an alteration of BCR signaling. Since both a profound decrease in BCR signaling and an increase in B cell glycolysis have been reported to enhance loss of tolerance in lupus prone mice, our findings help to explain the restoration of tolerance observed after treatment.
SLE is associated with adverse pregnancy outcomes, particularly in the presence of antiphospholipid antibodies (aPL). Pathogenic mechanisms damaging the fetal-maternal unit, however, remain poorly understood. A role for endosomal TLR8 was recently hypothesized in aPL induced placental damage. The role of TLR8 in systemic autoimmunity has remained elusive as TLR8 function differs from mouse to man. We investigated the effect of human (hu)TLR8 expression on pregnancy outcomes in Sle1 mice that develop aPL antibodies. huTLR8 transgenic(tg) Sle1 mice were generated and huTLR8 DNA copy number and mRNA expression was confirmed by qDigital- and qRT-PCR. Pregnancies were closely followed and terminal C-sections performed at signs of dystocia/prolonged labor. Pup and placental weight was assessed. Placental tissues were characterized by H&E and Myeloperoxidase (MPO) staining and inflammatory cytokine expression was assessed in placental tissue. Splenic and bone marrow immune populations were analyzed using flow cytometry. Female Sle1.huTLR8tg mice developed aPL and sm/RNP autoantibodies earlier than Sle1 controls and ±50% loss of litters was observed, compared to 0% in controls. IL-6, TNFa and MPO mRNA expression was significantly increased in placentas of affected litters and negatively correlated with placental weight. Abnormal placental morphology included placental infarcts, loss of placental lakes, arterial wall thickening and increased netting neutrophils. Here we identify a new model to study adverse pregnancy complications in SLE. In the presence of both Sle1 and huTLR8, we observed early onset of aPS autoantibodies and pregnancy loss attributed to placental developmental abnormalities, infarcts and inflammation.
Although SLE causal variants at the FAM167A-BLK locus are thought to reside in the BLK promoter region, our results reveal that genetic variants at distal regulatory elements modulate promoter activity, changing BLK and FAM167A gene expression and disease risk. Our results suggest that global haplotype-specific 3-dimensional chromatin looping architecture has a strong influence on local allelic BLK and FAM167A gene expression, providing mechanistic details for how regional variants controlling the BLK promoter may influence disease risk.
Anemia commonly occurs in systemic lupus erythematosus, a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death, while sparing other hematopoietic cell lineages. Whereas chronic inflammation is involved in this mechanism, less is known about the impact of systemic lupus erythematosus on the BM erythropoietic niche. We discovered that expression of the endosomal ssRNA sensor human TLR8 induces fatal anemia in Sle1.Yaa lupus mice. We observed that anemia was associated with a decrease in erythromyeloblastic islands and a block in differentiation at the CFU-E to proerythroblast transition in the BM. Single-cell RNAseq analyses of isolated BM erythromyeloblastic islands from human TLR8-expressing mice revealed that genes associated with essential central macrophage functions including adhesion and provision of nutrients were down-regulated. Although compensatory stress erythropoiesis occurred in the spleen, red blood cell half-life decreased because of hemophagocytosis. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation.
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.
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