IL-6 produced by dendritic cells from lupus-prone mice inhibits CD4+CD25+ T cell regulatory functions

The B6.Sle1.Sle2.Sle3 triple congenic mouse (B6.TC) is a model of lupus coexpressing the three major NZM2410-derived susceptibility loci on a C57BL/6 background. B6.TC mice produce high titers of antinuclear nephrogenic autoantibodies and a highly penetrant glomerulonephritis. Previous studies have shown the Sle1 locus is associated with a reduced number of regulatory T cells (Treg) and that Sle3 results in intrinsic defects of myeloid cells that hyperactivate T cells. In this report, we show that B6.TC dendritic cells (DCs) accumulate in lymphoid organs and present a defective maturation process, in which bone marrow-derived, plasmacytoid, and myeloid DCs express a significantly lower level of CD80, CD86, and MHC class II. B6.TC DCs also induce a higher level of proliferation in CD4(+) T cells than B6 DCs, and B6.TC DCs block the suppressive activity of Treg. B6.TC DCs overproduce IL-6, which is necessary for the blockade of Treg activity, as shown by the effect of anti-IL-6 neutralizing Ab in the suppression assays. The overproduction of IL-6 by DCs and the blockade of Treg activity maps to Sle1, which therefore not only confers a reduced number of Treg but also blocks their ability to regulate autoreactive T cells. Taken together, these results provide a genetic and mechanistic evidence for systemic autoimmunity resulting from an impaired regulatory T cell compartment in both number and function and for Sle1-expressing DCs playing a major role in the latter defect though their production of IL-6.     

Atorvastatin inhibits autoreactive B cell activation and delays lupus development in New Zealand black/white F1 mice

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by a wide range of immunological abnormalities that underlie the loss of tolerance. In this study we show that administration of atorvastatin to lupus-prone NZB/W F(1) mice resulted in a significant reduction in serum IgG anti-dsDNA Abs and decreased proteinuria. Histologically, the treatment was associated with reduced glomerular Ig deposition and less glomerular injury. Disease improvement was paralleled by decreased expression of MHC class II on monocytes and B lymphocytes and reduced expression of CD80 and CD86 on B lymphocytes. Consequent upon this inhibition of Ag presentation, T cell proliferation was strongly impaired by atorvastatin in vitro and in vivo. A significant decrease in MHC class II expression was also observed in the target organ of lupus disease (i.e., the glomerulus). Serum cholesterol in atorvastatin-treated lupus mice fell to the level found in young NZB/W mice before disease onset. This is the first demonstration that atorvastatin can delay the progression of a spontaneous autoimmune disease and may specifically benefit patients with systemic lupus erythematosus.     

Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentiation, and cell death.

Polyclonal, generalized T cell defects, as well as Ag-specific Th clones, are likely to contribute to pathology in murine lupus, but the genetic bases for these mechanisms remain unknown. Mapping studies indicate that loci on chromosomes 1 (Sle1), 4 (Sle2), 7 (Sle3), and 17 (Sle4) confer disease susceptibility in the NZM2410 lupus strain. B6.NZMc7 mice are C57BL/6 (B6) mice congenic for the NZM2410-derived chromosome 7 susceptibility interval, bearing Sle3. Compared with B6 controls, B6.NZMc7 mice exhibit elevated CD4:CD8 ratios (2.0 vs 1.34 in 1- to 3-mo-old spleens); an age-dependent accumulation of activated CD4+ T cells (33.4% vs 21.9% in 9- to 12-mo-old spleens); a more diffuse splenic architecture; and a stronger immune response to T-dependent, but not T-independent, Ags. In vitro, Sle3-bearing T cells show stronger proliferation, increased expansion of CD4+ T cells, and reduced apoptosis (with or without anti-Fas) following stimulation with anti-CD3. With age, the B cells in this strain acquire an activated phenotype. Thus, the NZM2410 allele of Sle3 appears to impact generalized T cell activation, and this may be causally related to the low grade, polyclonal serum autoantibodies seen in this strain. Epistatic interactions with other loci may be required to transform this relatively benign phenotype into overt autoimmunity, as seen in the NZM2410 strain.     

Failure of CD25+ T cells from lupus-prone mice to suppress lupus glomerulonephritis and sialoadenitis

The development of organ-specific autoimmune diseases in mice thymectomized on day 3 of life (d3tx mice) can be prevented by transferring CD4(+)CD25(+) T cells from syngeneic, normal adult mice. Using a d3tx model, we asked whether CD4(+)CD25(+) T cell deficiency contributes to glomerulonephritis (GN) in lupus-prone mice. New Zealand Mixed 2328 (NZM2328) mice spontaneously develop autoantibodies to dsDNA and female-dominant, fatal GN. After d3tx, both male and female NZM2328 mice developed 1) accelerated dsDNA autoantibody response, 2) early onset and severe proliferative GN with massive mesangial immune complexes, and 3) autoimmune disease of the thyroid, lacrimal gland, and salivary gland. The d3tx male mice also developed autoimmune prostatitis. The transfer of CD25(+) cells from 6-wk-old asymptomatic NZM2328 donors effectively suppressed dsDNA autoantibody and the development of autoimmune diseases, with the exception of proliferative lupus GN and sialoadenitis. This finding indicates that NZM2328 lupus mice have a selective deficiency in T cells that regulates the development of lupus GN and sialoadenitis. After d3tx, the proliferative GN of female mice progressed to fatal GN, but largely regressed in the male, thereby revealing a checkpoint in lupus GN progression that depends on gender.     

Aberrant phenotype and function of myeloid dendritic cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by a systemic autoimmune response with profound and diverse T cell changes. Dendritic cells (DCs) are important orchestrators of immune responses and have an important role in the regulation of T cell function. The objective of this study was to determine whether myeloid DCs from individuals with SLE display abnormalities in phenotype and promote abnormal T cell function. Monocyte-derived DCs and freshly isolated peripheral blood myeloid DCs from lupus patients displayed an abnormal phenotype characterized by accelerated differentiation, maturation, and secretion of proinflammatory cytokines. These abnormalities were characterized by higher expression of the DC differentiation marker CD1a, the maturation markers CD86, CD80, and HLA-DR, and the proinflammatory cytokine IL-8. In addition, SLE patients displayed selective down-regulation of the maturation marker CD83 and had abnormal responses to maturation stimuli. These abnormalities have functional relevance, as SLE DCs were able to significantly increase proliferation and activation of allogeneic T cells when compared with control DCs. We conclude that myeloid DCs from SLE patients display significant changes in phenotype which promote aberrant T cell function and could contribute to the pathogenesis of SLE and organ damage.     

Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells

An increasing number of studies indicate that a subset of CD4(+) T cells with regulatory capacity (regulatory T cells; T(regs)) can function to control organ-specific autoimmune disease. To determine whether abnormalities of thymic-derived T(regs) play a role in systemic lupus erythematosus, we evaluated T(reg) prevalence and function in (New Zealand Black x New Zealand White)F(1) (B/W) lupus-prone mice. To explore the potential of T(regs) to suppress disease, we evaluated the effect of adoptive transfer of purified, ex vivo expanded thymic-derived T(regs) on the progression of renal disease. We found that although the prevalence of T(regs) is reduced in regional lymph nodes and spleen of prediseased B/W mice compared with age-matched non-autoimmune mice, these cells increase in number in older diseased mice. In addition, the ability of these cells to proliferate in vitro was comparable to those purified from non-autoimmune control animals. Purified CD4(+)CD25(+)CD62L(high) B/W T(regs) were expanded ex vivo 80-fold, resulting in cells with a stable suppressor phenotype. Adoptive transfer of these exogenously expanded cells reduced the rate at which mice developed renal disease; a second transfer after treated animals had developed proteinuria further slowed the progression of renal disease and significantly improved survival. These studies indicate that thymic-derived T(regs) may have a significant role in the control of autoimmunity in lupus-prone B/W mice, and augmentation of these cells may constitute a novel therapeutic approach for systemic lupus erythematosus.     

Sle1ab mediates the aberrant activation of STAT3 and Ras-ERK signaling pathways in B lymphocytes

The Sle1ab genomic interval on murine chromosome 1 mediates the loss of immune tolerance to chromatin resulting in antinuclear Abs (ANA) production in the lupus-prone NZM2410 mouse. Global gene expression analysis was used to identify the molecular pathways that are dysregulated at the initiation of B lymphocyte autoimmunity in B6.Sle1ab mice. This analysis identified that STAT3 and ras-ERK signaling pathways are aberrantly activated in Sle1ab B lymphocytes, consistent with increased production of IL-6 by splenic B lymphocytes and monocytes in B6.Sle1ab mice. In vitro treatment of splenic mononuclear cells isolated from ANA-positive Sle1ab mice with anti-IL-6 Ab or AG490, an inhibitor of STAT3 signaling pathway, suppressed ANA production in short-term culture, indicating that this pathway was essential to the production of autoantibodies. In vivo treatment of ANA-positive B6.Sle1ab mice with the ras pathway inhibitor, perillyl alcohol, suppressed the increase of ANA. These findings identify IL-6 as a early key cytokine in Sle1ab-mediated disease development and indicate that the STAT3 and ras-ERK signaling pathways are potential therapeutic targets for treating systemic lupus erythematosus.     

Toxoplasma gondii-derived heat shock protein 70 stimulates maturation of murine bone marrow-derived dendritic cells via Toll-like receptor 4

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) induced maturation of bone marrow-derived dendritic cells (DCs) of wild-type (WT) C57BL/6 mice as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. These phenotypic and functional changes of T.g.HSP70-stimulated DCs were demonstrated in Toll-like receptor (TLR) 2- and myeloid differentiation factor 88 (MyD88)-deficient but not TLR4-deficient C57BL/6 mice. DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70 stimulation. T.g.HSP70-stimulated DCs from WT, TLR2-deficient, and MyD88-deficient, but not TLR4-deficient mice expressed IFN-beta mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.     

A New Zealand Black-derived locus suppresses chronic graft-versus-host disease and autoantibody production through nonlymphoid bone marrow-derived cells

The development of lupus pathogenesis results from the integration of susceptibility and resistance genes. We have used a chronic graft-versus-host disease (cGVHD) model to characterize a suppressive locus at the telomeric end of the NZM2410-derived Sle2 susceptibility locus, which we named Sle2c2. cGVHD is induced normally in Sle2c2-expressing mice, but it is not sustained. The analysis of mixed bone marrow chimeras revealed that cGVHD resistance was eliminated by non-B non-T hematopoietic cells expressing the B6 allele, suggesting that resistance is mediated by this same cell type. Furthermore, Sle2c2 expression was associated with an increased number and activation of the CD11b(+) GR-1(+) subset of granulocytes before and in the early stage of cGVHD induction. We have mapped the Sle2c2 critical interval to a 6-Mb region that contains the Cfs3r gene, which encodes for the G-CSFR, and its NZM2410 allele carries a nonsynonymous mutation. The G-CSFR-G-CSF pathway has been previously implicated in the regulation of GVHD, and our functional data on Sle2c2 suppression suggest a novel regulation of T cell-induced systemic autoimmunity through myeloid-derived suppressor cells. The validation of Csf3r as the causative gene for Sle2c2 and the further characterization of the Sle2c2 MDSCs promise to unveil new mechanisms by which lupus pathogenesis is regulated.     

CCR2-dependent trafficking of F4/80dim macrophages and CD11cdim/intermediate dendritic cells is crucial for T cell recruitment to lungs infected with Mycobacterium tuberculosis

We previously reported that CCR2(-/-) mice are susceptible to Mycobacterium tuberculosis infection. Susceptibility was associated with an early and sustained macrophage trafficking defect, followed by delayed recruitment of dendritic cells (DCs) and T cells to the lungs. However, the relative importance of the lack of CCR2 expression by macrophages and DCs vs T cells in susceptibility to infection was unclear. In this study, we used mixed bone marrow transplantation to create mice in which the genotype of the T cells was either CCR2(+/+) or CCR2(-/-) while maintaining the genotype of the myeloid cells as CCR2(+/+). After infection with M. tuberculosis, we found that the genotype of the macrophages and/or DCs, but not that of the T cells, was critical for both T cell and myeloid cell migration to the lungs. Further investigation revealed a critical role for CCR2 in the recruitment of F4/80(dim) macrophages and CD11c(dim/intermediate) DCs to the infected lung.     

Decrease of Functional Activated T and B Cells and Treatment of Glomerulonephitis in Lupus-Prone Mice Using a Natural Flavonoid Astilbin

Treatment of systemic lupus erythematosus (SLE), a chronic inflammatory disease, involves the long-term use of immunosuppressive agents with significant side effects. New therapeutic approaches are being explored to find better treatment possibilities. In this study, age-matched female MRL/lpr mice were treated orally with a natural flavonoid astilbin. Astilbin administration started either at week 8 or week 12 of age though week 20. In the early treatment regimen, the treatment with astilbin reduced splenomegaly / lymphomegaly, autoantibody production and ameliorated lupus nephitis. Several serum cytokines were significantly decreased upon treatment including IFN-g, IL-17A, IL-1b, TNF-a and IL-6. Both spleen CD44^hiCD62L^lo activated T cells and CD138⁺B220^- plasma cells greatly declined. Furthermore, astilbin treatment resulted in decreased mitochondrial membrane potential in activated T cells and downregulated expression of the co-stimulatory molecules CD80 and CD86 on LPS stimulated B cells. Similar but less profound effectiveness was observed in the mice with established disease in the late treatment regimen. These results indicate that the natural product astilbin can mitigate disease development in lupus-prone mice by decreasing functional activated T and B cells.     

Regulation of dendritic cell function and T cell priming by the fatty acid-binding protein AP2

The fatty acid-binding protein (FABP) family consists of a number of conserved cytoplasmic proteins with roles in intracellular lipid transport, storage, and metabolism. Examination of a comprehensive leukocyte gene expression database revealed strong expression of the adipocyte FABP aP2 in human monocyte-derived dendritic cells (DCs). We isolated bone marrow-derived DC from aP2-deficient mice, and showed that expression of DC cytokines including IL-12 and TNF was significantly impaired in these cells. Degradation of IkappaBalpha was also impaired in aP2-deficient DCs, indicative of reduced signaling through the IkappaB kinase-NF-kappaB pathway. The cytokine defect was selective because there was no effect on Ag uptake or expression of MHC class II, CD40, CD80, or CD86. In an MLR, aP2-deficient DCs stimulated markedly lower T cell proliferation and cytokine production than did wild-type DCs. Moreover, aP2-deficient mice immunized with keyhole limpet hemocyanin/CFA showed reduced production of IFN-gamma by restimulated draining lymph node cells, suggesting a similar defect in DC function in vivo. Similarly, infection of aP2-deficient mice with the natural mouse pathogen ectromelia virus resulted in substantially lower production of IFN-gamma by CD8+ T cells. Thus, FABP aP2 plays an important role in DC function and T cell priming, and provides an additional link between metabolic processes and the regulation of immune responses.     

Splenic phagocytes promote responses to nucleosomes in (NZB x NZW) F1 mice

Autoantigen presentation to T cells is crucial for the development of autoimmune disease. However, the mechanisms of autoantigen presentation are poorly understood. In this study, we show that splenic phagocytes play an important role in autoantigen presentation in murine lupus. Nucleosomes are major autoantigens in systemic lupus erythematosus. We found that nucleosome-specific T cells were stimulated dominantly in the spleen, compared with lymph nodes, lung, and thymus. Among splenic APCs, F4/80(+) macrophages and CD11b(+)CD11c(+) dendritic cells were strong stimulators for nucleosome-specific T cells. When splenic phagocytes were depleted in (NZB x NZW) F(1) (NZB/W F(1)) mice, nucleosome presentation in the spleen was dramatically suppressed. Moreover, depletion of splenic phagocytes significantly suppressed anti-nucleosome Ab and anti-dsDNA Ab production. Proteinuria progression was delayed and survival was prolonged in phagocyte-depleted mice. The numbers of autoantibody- secreting cells were decreased in the spleen from phagocyte-depleted mice. Multiple injections of splenic F4/80(+) macrophages, not those of splenic CD11c(+) dendritic cells, induced autoantibody production and proteinuria progression in NZB/W F(1) mice. These results indicate that autoantigen presentation by splenic phagocytes including macrophages significantly contributes to autoantibody production and disease progression in lupus-prone mice.     

Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state

Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.     

Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis

In an effort to identify potential biomarkers in lupus nephritis, urine from mice with spontaneous lupus nephritis was screened for the presence of VCAM-1, P-selectin, TNFR-1, and CXCL16, four molecules that had previously been shown to be elevated in experimental immune nephritis, particularly at the peak of disease. Interestingly, all four molecules were elevated approximately 2- to 4-fold in the urine of several strains of mice with spontaneous lupus nephritis, including the MRL/lpr, NZM2410, and B6.Sle1.lpr strains, correlating well with proteinuria. VCAM-1, P-selectin, TNFR-1, and CXCL16 were enriched in the urine compared with the serum particularly in active disease, and were shown to be expressed within the diseased kidneys. Finally, all four molecules were also elevated in the urine of patients with lupus nephritis, correlating well with urine protein levels and systemic lupus erythematosus disease activity index scores. In particular, urinary VCAM-1 and CXCL16 showed superior specificity and sensitivity in distinguishing subjects with active renal disease from the other systemic lupus erythematosus patients. These studies uncover VCAM-1, P-selectin, TNFR-1, and CXCL16 as a quartet of molecules that may have potential diagnostic significance in lupus nephritis. Longitudinal studies are warranted to establish the clinical use of these potential biomarkers.     

Cutting Edge: Ectopic expression of CD40 ligand on B cells induces lupus-like autoimmune disease.

CD40 ligand (CD40L) is ectopically expressed on B cells in patients with systemic lupus erythematosus (SLE) and lupus-prone BXSB mice. To assess the role of the ectopic CD40L expression in development of SLE, we have established transgenic mice expressing CD40L on B cells. Some of the 12- to 14-mo-old CD40L-transgenic mice spontaneously produced autoantibodies such as antinuclear Abs, anti-DNA Abs, and antihistone Abs. Moreover, approximately half of the transgenic mice developed glomerulonephritis with immune-complex deposition, whereas the kidneys of the normal littermates showed either no pathological findings or only mild histological changes. These results indicate that CD40L on B cells causes lupus-like disease in the presence of yet unknown environmental factors that by themselves do not induce the disease. Thus, ectopic CD40L expression on B cells may play a crucial role in development of SLE.     

Dysregulated Cytokine Production by Dendritic Cells Modulates B Cell Responses in the NZM2410 Mouse Model of Lupus

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-γ, 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-γ 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.     

Flt3 ligand preferentially increases the number of functionally active myeloid dendritic cells in the lungs of mice

In the present study, we investigated the effects of in vivo Flt3L administration on the generation, phenotype, and function of lung dendritic cells (DCs) to evaluate whether Flt3L favors the expansion and maturation of a particular DC subset. Injection of Flt3L into mice resulted in an increased number of CD11c-expressing lung DCs, preferentially in the alveolar septa. FACS analysis allowed us to quantify a 19-fold increase in the absolute numbers of CD11c-positive, CD45R/B220 negative DCs in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DCs (CD11c positive, CD45R/B220 negative, and CD11b positive) and only a 3-fold increase of lymphoid DCs (CD11c positive, CD45R/B220 negative, and CD11b negative) from the lungs of Flt3L-treated mice over vehicle-treated mice. Flt3L-treated lung DCs were more mature than vehicle-treated lung DCs as demonstrated by a significantly higher percentage of cells expressing MHC class II, CD86, and CD40. Freshly isolated Flt3L lung DCs were not fully mature, because after an overnight culture they continued to increase accessory molecule expression. Functionally, Flt3L-treated lung DCs were more efficient than vehicle-treated DCs at stimulating naive T cell proliferation. Our data show that administration of Flt3L favors the expansion of myeloid lung DCs over lymphoid DCs and enhanced their ability to stimulate naive lymphocytes.