Carabin deficiency in B cells increases BCR-TLR9 costimulation-induced autoimmunity.
ABSTRACT: The mechanisms behind flares of human autoimmune diseases in general, and of systemic lupus in particular, are poorly understood. The present scenario proposes that predisposing gene defects favour clinical flares under the influence of external stimuli. Here, we show that Carabin is low in B cells of (NZB × NZW) F1 mice (murine SLE model) long before the disease onset, and is low in B cells of lupus patients during the inactive phases of the disease. Using knock-out and B-cell-conditional knock-out murine models, we identify Carabin as a new negative regulator of B-cell function, whose deficiency in B cells speeds up early B-cell responses and makes the mice more susceptible to anti-dsDNA production and renal lupus flare after stimulation with a Toll-like Receptor 9 agonist, CpG-DNA. Finally, in vitro analysis of NFκB activation and Erk phosphorylation in TLR9- and B-cell receptor (BCR)-stimulated Carabin-deficient B cells strongly suggests how the internal defect synergizes with the external stimulus and proposes Carabin as a natural inhibitor of the potentially dangerous crosstalk between BCR and TLR9 pathways in self-reactive B cells.
Project description:Plasmacytoid dendritic cells (pDCs) were considered to be the major IFN? source in systemic lupus erythematosus (SLE) but their phenotype and function in different disease status have not been well studied. To study the function and phenotype of pDCs in lupus-prone mice we used 7 strains of lupus-prone mice including NZB/W F1, NZB, NZW, NZM2410, B6.NZM(Sle1/2/3), MRL/lpr and BXSB/Mp mice and C57BL/6 as control mice. Increased spleen pDC numbers were found in most lupus mice compared to C57BL/6 mice. The IFN?-producing ability of BM pDCs was similar between lupus and C57BL/6 mice, whereas pDCs from the spleens of NZB/W F1 and NZB mice produced more IFN? than pDCs from the spleens of C57BL/6 mice. Furthermore, spleen pDCs from MRL-lpr and NZM2410 mice showed increased responses to Tlr7 and Tlr9, respectively. As the disease progressed, IFN signature were evaluated in both BM and spleen pDC from lupus prone mice and the number of BM pDCs and their ability to produce IFN? gradually decreased in lupus-prone mice. In conclusion, pDC are activated alone with disease development and its phenotype and function differ among lupus-prone strains, and these differences may contribute to the development of lupus in these mice.
Project description:Systemic lupus erythematosus is a debilitating autoimmune disease in which autoantibodies and autoreactive T cells destroy kidneys and other organs. Disease is clinically and genetically heterogeneous, suggesting that underlying mechanisms vary between patients. We previously used an autoantibody transgenic mouse reporter system to examine the effect of different autoimmune backgrounds on B-cell tolerance, failure of which is a fundamental defect in lupus. We identified a defect consistent with reversible anergy induced by endotoxin stimulation of B cells from Ig transgenic New Zealand Black (NZB) mice. Herein we report that the tolerance defect is revealed by TLR7 and TLR9 as well as TLR4 ligands, with additive effect, and is partially reversed by Mek inhibition. Gene expression analysis reveals significant differences in transcription of multiple TLR pathway genes and ptpn22 in stimulated NZB compared to B6 B cells. Additionally, the defect is detected in Ig transgenic NZB F1 hybrid strains (NZBxNZW)F1 and (B6xNZB)F1. These results implicate an inherited defect wherein NZB anergic B cells maintain coordinated TLR/BCR signaling that permits autoantibody production. Agents targeting these pathways may have therapeutic benefit in the subset of lupus patients that manifest similar defects in B-cell regulation.
Project description:The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.
Project description:Lupus is a systemic autoimmune disease characterized by the production of autoreactive antibodies against nuclear antigens. Women are disproportionately affected by lupus, and this sex bias is thought to be due, in large part, to the ability of estrogens to promote lupus pathogenesis. Previously, we have shown that global deletion of estrogen receptor alpha (ER?) significantly attenuated loss of tolerance, immune cell activation, autoantibody production, and the development of lupus nephritis. Here we show that targeted deletion of ER? specifically in B cells retards production of pathogenic autoantibodies and the development of nephritis in lupus-prone (NZB×NZW)F1 mice. Furthermore, we observed that ER? deletion in B cells was associated with decreased B cell activation in young, pre-autoimmune (NZB×NZW)F1 females. Altogether, these data suggest that ER? acts in a B cell-intrinsic manner to control B cell activation, autoantibody production, and lupus nephritis.
Project description:Previous studies have suggested that MHC and non-MHC genes contribute to the development of autoimmune disease in F1 hybrids of New Zealand black (NZB) and white (NZW) mice. We conducted a genome-wide screen of 148 female (NZB x NZW)F1 x NZB backcross mice to map dominant NZW genetic loci linked with lupus disease traits. In this backcross analysis, inheritance of the NZW MHC (H2(d/z) vs. H2(d/d)) was strongly linked with the development of lupus nephritis (P approximately 1 x 10(-16)), increasing the risk of disease by over 30-fold. H2(d/z) was also linked with elevated serum levels of IgG autoantibodies to single-stranded DNA, double-stranded DNA, histones, and chromatin but not with anti-gp70 autoantibodies, measured as circulating gp70-anti-gp70 immune complexes. Non-MHC contributions from NZW seemed weak in comparison to MHC, although NZW loci on chromosomes 7 and 16 were noted to be suggestively linked with autoantibody production. Strikingly, H2(d/z) (compared with H2(d/d)) enhanced antinuclear antibodies in a coordinate fashion but did not affect anti-gp70 production in the current backcross. However, the opposite influence was noted for H2(d/z) (compared with H2(z/z)) when (NZB x NZW)F1 x NZW backcross mice were analyzed. These results suggest that H2(z) and H2(d) haplotypes differentially regulate two different sets of nephritogenic autoantibody responses. This study confirms a critical role for H2(z) compared with other dominant NZW loci in (NZB x NZW)F1 mice and provides an explanation as to why H2(d/z) heterozygosity is required for full expression of disease in this model.
Project description:To investigate the mechanism by which interferon-? (IFN?) accelerates systemic lupus erythematosus (SLE) in (NZB×NZW)F1 (NZB/NZW) mice.NZB/NZW mice were treated with an adenovirus expressing IFN?. In some mice, T cells were depleted with an anti-CD4 antibody. The production of anti-double-stranded DNA (anti-dsDNA) antibodies was measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. Germinal centers and antibody-secreting cells (ASCs) in spleens and IgG deposition and leukocyte infiltrates in kidneys were visualized by immunofluorescence staining. The phenotype of splenic cells was determined by flow cytometry. Finally, somatic hypermutation and gene usage in VH regions of IgG2a and IgG3 were studied by single-cell polymerase chain reaction.IFN?-accelerated lupus in NZB/NZW mice was associated with elevated serum levels of IgG2 and IgG3 anti-dsDNA antibodies and accumulation of many IgG ASCs in the spleen, which did not develop into long-lived plasma cells. Furthermore, IgG2a and IgG3 antibodies in the mice were highly somatically mutated and used distinct repertoires of VH genes. The induction of SLE in the mice was associated with an increase in B cell Toll-like receptor 7 expression, increased serum levels of BAFF, interleukin-6 (IL-6), and tumor necrosis factor ?, and induction of T cells expressing IL-21. Although IFN? drove a T cell-independent increase in serum levels of IgG, autoantibody induction and the development of nephritis were both completely dependent on CD4+ T cell help.These findings demonstrate that, although IFN? activates both innate and adaptive immune responses in NZB/NZW mice, CD4+ T cells are necessary for IFN?-driven induction of anti-dsDNA antibodies and clinical SLE.
Project description:Proteins involved in iron homeostasis have been identified as biomarkers for lupus nephritis, a serious complication of systemic lupus erythematosus (SLE). We tested the hypothesis that renal iron accumulation occurs and contributes to renal injury in SLE. Renal non-heme iron levels were increased in the (New Zealand Black x New Zealand White) F1 (NZB/W) mouse model of lupus nephritis compared with healthy New Zealand White (NZW) mice in an age- and strain-dependent manner. Biodistribution studies revealed increased transferrin-bound iron accumulation in the kidneys of albuminuric NZB/W mice, but no difference in the accumulation of non-transferrin bound iron or ferritin. Transferrin excretion was significantly increased in albuminuric NZB/W mice, indicating enhanced tubular exposure and potential for enhanced tubular uptake following filtration. Expression of transferrin receptor and 24p3R were reduced in tubules from NZB/W compared to NZW mice, while ferroportin expression was unchanged and ferritin expression increased, consistent with increased iron accumulation and compensatory downregulation of uptake pathways. Treatment of NZB/W mice with the iron chelator deferiprone significantly delayed the onset of albuminuria and reduced blood urea nitrogen concentrations. Together, these findings suggest that pathological changes in renal iron homeostasis occurs in lupus nephritis, contributing to the development of kidney injury.
Project description:Fc?RIIB-/-yaa mice develop severe lupus glomerulonephritis due to lack of an inhibitory immune cell receptor combined with a Y-chromosome linked autoimmune accelerator mutation. In the present study, we have investigated nephritis development and progression in Fc?RIIB-/-yaa mice to find shared features with NZB/NZW F1 lupus prone mice and human disease. We sacrificed 25 male Fc?RIIB-/-yaa mice at various disease stages, and grouped them according to activity and chronicity indices for lupus nephritis. Glomerular morphology and localization of electron dense deposits containing IgG were further determined by immune electron microscopy. Renal DNase I and pro-inflammatory cytokine mRNA levels were measured by real-time quantitative PCR. DNase I protein levels was assessed by immunohistochemistry and zymography. Our results demonstrate early development of electron dense deposits containing IgG in Fc?RIIB-/-yaa mice, before detectable levels of serum anti-dsDNA antibodies. Similar to NZB/NZW F1, electron dense deposits in Fc?RIIB-/-yaa progressed from being confined to the mesangium in the early stage of lupus nephritis to be present also in capillary glomerular basement membranes. In the advanced stage of lupus nephritis, renal DNase I was lost on both transcriptional and protein levels, which has previously been shown in NZB/NZW F1 mice and in human disease. Although lupus nephritis appears on different genetic backgrounds, our findings suggest similar processes when comparing different murine models and human lupus nephritis.
Project description:We have previously shown that immune tolerance induced by the anti-DNA Ig peptide pCons in (NZB×NZW)F(1) (NZB/W) lupus mice prolonged survival of treated animals and delayed the appearance of autoantibodies and glomerulonephritis. Part of the protection conferred by pCons could be ascribed to the induction of regulatory T cells (T(Reg)) that suppressed the production of anti-DNA antibodies in a p38 MAPK-dependent fashion. Here we show that another effect of pCons in the induction of immune tolerance in NZB/W lupus mice is the facilitation of effector T cell suppression by T(Reg). These new findings indicate that pCons exerts protective effects in NZB/W lupus mice by differentially modulating the activity of different T cell subsets, implying new considerations in the design of T(Reg)-based approaches to modulate T cell autoreactivity in SLE.
Project description:IFN-? is known to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanisms remain unclear. We previously showed that within weeks, exposure to IFN-? in vivo induces lupus in pre-autoimmune lupus-prone NZB×NZW F1 (NZB/W) but not in BALB/c mice. In the current study, we show that in vivo expression of IFN-? induces sustained B-cell proliferation in both BALB/c and NZB/W mice. In NZB/W but not BALB/c mice, B-cell proliferation was accompanied by a rapid and unabated production of autoantibody-secreting cells (ASCs) in secondary lymphoid organs, suggesting that a B-cell checkpoint is altered in the autoimmune background. The majority (>95%) of ASCs elicited in IFN-?-treated NZB/W mice were short-lived and occurred without the induction of long-lived plasma cells. A short course of cyclophosphamide caused a sharp drop in IFN-?-elicited short-lived plasma cells, but the levels recovered within days following termination of treatment. Thus, our work provides new insights into effectiveness and limitations of the current SLE therapies.