BWF1 Mice Research Articles

The lymphotoxin β receptor is a potential therapeutic target in renal inflammation.

Accumulation of inflammatory cells in different renal compartments is a hallmark of progressive kidney diseases including glomerulonephritis (GN). Lymphotoxin β receptor (LTβR) signaling is crucial for the formation of lymphoid tissue, and inhibition of LTβR signaling has ameliorated several non-renal inflammatory models. Therefore, we tested whether LTβR signaling could also have a role in renal injury. Renal biopsies from patients with GN were found to express both LTα and LTβ ligands, as well as LTβR. The LTβR protein and mRNA were localized to tubular epithelial cells, parietal epithelial cells, crescents, and cells of the glomerular tuft, whereas LTβ was found on lymphocytes and tubular epithelial cells. Human tubular epithelial cells, mesangial cells, and mouse parietal epithelial cells expressed both LTα and LTβ mRNA upon stimulation with TNF in vitro. Several chemokine mRNAs and proteins were expressed in response to LTβR signaling. Importantly, in a murine lupus model, LTβR blockade improved renal function without the reduction of serum autoantibody titers or glomerular immune complex deposition. Thus, a preclinical mouse model and human studies strongly suggest that LTβR signaling is involved in renal injury and may be a suitable therapeutic target in renal diseases.

A CD153+CD4+ T follicular cell population with cell-senescence features plays a crucial role in lupus pathogenesis via osteopontin production.

Immune aging results in diminished adaptive immunity and increased risk for autoimmunity. We previously reported a unique PD-1(+) CD44(high)CD4(+) T cell population that increases with age in normal mice. In this study, we indicate that the age-dependent PD-1(+) CD44(high)CD4(+) T cells develop as unique T follicular (TF) cells in a B cell-dependent manner and consist of two subpopulations, as follows: CD153(+) cells preferentially secreting abundant osteopontin on TCR stimulation and CD153(-) cells that are apparently TCR anergic. These unique TF cells with essentially similar features increase much earlier and are accumulated in the spontaneous germinal centers (GCs) in lupus-prone female BWF1 (f-BWF1) mice. These TF cells showed characteristic cell-senescence features and developed in association with extensive CD4(+) T cell proliferation in vivo, suggesting replicative senescence. Although the CD153(+) TF cells were defective in proliferation capacity, they were quite stable and specifically responded to self GC-B cells to secret abundant osteopontin, which inhibited B cell receptor-induced GC-B cell apoptosis in f-BWF1 mice. Transfer of CD153(+) PD-1(+) CD4(+) T cells promoted the growth of spontaneous GCs, whereas administration of anti-osteopontin Ab suppressed GC enlargement and anti-nuclear Ab production and ameliorated clinical lupus nephritis of f-BWF1 mice. Current results suggest that senescent CD153(+) TF cells generated as a consequence of extensive endogenous CD4(+) T cell proliferation play an essential, if not sufficient, role in lupus pathogenesis in lupus-prone genetic background and may also contribute to an increased autoimmunity risk with age.

Calcium binding protein S100a9 protects male lupus-prone BWF1 mice from disease development (IRC4P.448)

Abstract Testosterone protects from SLE pathogenesis, yet the mechanism of protection remains unknown. Using the (NZB x NZW)F1 (BWF1) mouse model of SLE, we have previously shown that immunosuppressive Gr1+CD11b+ cells are elevated in protected male BWF1 mice compared with females. Female and male Gr1+ cells use different mechanisms of suppression: reactive oxygen and nitrogen species (ROS/NOS) (female), or a secreted component independent of ROS/NOS production (male). Furthermore, female Gr1+ cells lose their capacity to suppress as the mice age. In support hereof, in vivo depletion of Gr1+ cells accelerated disease development in male mice only. Calcium-binding protein S100a9 is induced by inflammation and has been shown to exert either proinflammatory or immunosuppressive functions. Finding that male-derived Gr1+ cells express 3-4 fold higher levels of S100a9 mRNA than females, we hypothesized that S100a9 is the immunosuppressive mechanism used by male Gr1+ cells. Using S100a9-/- BWF1 mice we show that male-derived Gr1+ cells indeed use S100a9 to suppress plasma cell differentiation in vitro. Moreover, BWF1.S100a9-/- males developed increased serum ANA levels and IgG-IC deposition in the kidney glomeruli, and accelerated renal failure, while only the kidney phenotype was somewhat affected in BWF1.S100a9-/- female mice. In conclusion, differential regulation of S100a9 by male and female Gr1+ cells may contribute to the differential development of lupus in male and female BWF1 mice.

Male microbiota protects female BWF1 mice from lupus and death by restoring CD103DC function possibly via metabolite(s) production (THER5P.914)

Abstract Alterations in microbiota composition are associated with autoimmune diseases. Little is known about the effect of microbial metabolite production on disease development. CD103 dendritic cells (CD103DC) induce regulatory T cells (iTregs) in the periphery. We have found that lupus-resistant male BWF1 mice exhibit increased disease incidence/mortality when treated with anti-CD103 antibody. Female CD103DC exhibit lower tolerogenic function than male cells in vitro and in vivo (i.e., oral tolerance) possibly due to a defect in the retinoic acid synthesis pathway. We also found that microbiota composition differed significantly between female and male BWF1 mice, and transfer of male microbiota decreased autoantibody production in female BWF1 mice. Here we report that transfer of male microbiota prevents kidney disease and dramatically increases survival of female BWF1 mice which correlates with enhanced female CD103DC function in both in vitro and in vivo assays, and increased iTregs. Furthermore, our preliminary study suggests that treatment with anti-CD103 antibody prevents male microbiota-mediated protection. Finally, metabolomic analysis of feces from female and male BWF1 mice revealed significant quantitative differences in 17 metabolites, including one that exhibits retinoid X receptor agonist activity. Taken together, these data suggest that male microbiota may protect against lupus through the production of metabolite(s) that enhance the retinoic acid synthesis pathway.

A role for hormones, gut microbiota and tolerogenic CD103DC in protection of male (NZBxNZW)F1 (BWF1) mice from lupus (MUC8P.805)

Abstract Mechanisms leading to gut homeostasis affect both mucosal and systemic immune function, and involve gut microbiota. Tolerogenic dendritic cells (CD103DC) play a role in gut homeostasis, including generation of induced regulatory T cells (iTregs). Previously, we found that treatment of male BWF1 mice with anti-CD103 antibody significantly increases disease incidence/mortality. Male CD103DC exhibit significantly greater tolerogenic function than either female or castrated male CD103DC in vitro. This study examines the mechanism of sex differences in CD103DC function, and the role of microbiota in lupus. Sex differences in CD103DC function correlated with increased expression of genes associated with tolerogenic/anti-inflammatory function in males, and pro-inflammatory function in females, and changes in iTreg frequency and function in vivo. Male BWF1 CD103DC expressed more Raldh2, an enzyme required for retinoic acid (RA) synthesis and CD103DC function, and supplementation with RA restored tolerogenic function in female CD103DC. Further, we found that male gut microbiota differed significantly from adult, but not immature, female microbiota, suggesting hormones influence the gut microbiota. Importantly, transfer of male gut microbiota to female BWF1 mice significantly reduced anti-dsDNA antibody production. These data suggest that an interplay between hormones, gut microbiota and tolerogenic CD103DC may play a critical role in providing protection to male BWF1 mice.

Gut CD103+ dendritic cells from female lupus-prone (NZBxNZW) F1 (BWF1) mice are much less effective than male BWF1 mice at inducing iTregs (P4087)

Abstract Regulatory CD4+Foxp3+ cells (Tregs) either develop in the thymus (nTregs) or are converted from non-Tregs in the periphery (iTregs). Lupus is much more prevalent in females than males, and although we have found that female BWF1 mice have lower Treg% than males and Treg% are increased in androgen-treated females and decreased in castrated males, it is unclear whether it is the nTregs or iTregs that are affected. In this study, we compared the ability of female and male BWF1 cells to mediate conversion. We found no differences in the ability of female and male BWF1 in vitro generated (bone marrow-derived) dendritic cells (DC) treated with TGFβ to induce Foxp3+ iTregs in vitro. Next, we analyzed CD103+DC from mesenteric lymph nodes (MLN) that can spontaneously induce iTregs. CD103+DC from female BWF1 mice exhibited a dramatically decreased ability to induce iTregs in vitro. CD103+DC from castrated male mice also had a similarly reduced ability to induce Tregs compared to sham-castrated males. Differences in CD103+DC function correlated with Treg% in MLN which were lower in females than males. There were no differences in CD103+DC% in MLN between female and male BWF1 mice. Interestingly, treatment in vivo with a blocking anti-CD103 antibody induced/accelerated disease and increased mortality in male BWF1 mice. Our data suggest 1) androgens may increase tolerogenic function of CD103+DC and 2) CD103+DC may induce a population of iTregs that are important for controlling lupus.

Laquinimod suppression of lupus nephritis in BWF1 mice associates in renal tissue with increase of myeloid suppressor cells and switch of macrophages to anti-inflammatory type II (P5193)

Abstract Background: Laquinimod (LAQ) is a novel drug that has been in clinical trial in systemic lupus erythematosus (SLE), an autoimmune disease characterized by nephritis mediated in part by autoantibodies. Aim: We investigated the immunomodulatory properties of LAQ on lupus nephritis in NZB x NZW F1 (BWF1) mice, a murine model for SLE. Methods: Six-month old BWF1 female mice were treated orally 3 times a week with LAQ 25 mg/kg or water. Results: All LAQ-treated animals survived up to 10 weeks after treatment vs 0% survival in controls. No LAQ-treated mouse had proteinuria by week 10 while it was significantly high (3 g/L) in 100% of controls by week 7 after treatment. Examination of kidney specimens showed significantly decreased histologic inflammation, damage, immunoglobulin and C3 deposition in LAQ-treated vs controls. LAQ significantly reduced macrophages (CD11b+) and their expression of MHC, and these cells produced higher levels of IL-10 and lower TNF-alfa vs controls, a phenotype characteristic of anti-inflammatory type II macrophages. In addition, increased frequency of CD11b+Ly6C+Ly6G+ and CD11b+Ly6C+Ly6G- cells was found in LAQ-treated vs controls and these cells suppressed CD4+ T cell proliferation, what characterizes them as myeloid suppressor cells. Conclusion: LAQ suppressed lupus nephritis, improved animals’ survival and increased both anti-inflammatory macrophages and myeloid suppressor cells. Thus, LAQ is a promising therapeutic for human lupus nephritis.

Germline deletion of β2 microglobulin or CD1d reduces anti-phospholipid antibody, but increases autoantibodies against non-phospholipid antigens in the NZB/W F1 model of lupus

Introductionβ2-microglobulin (β2m) is required for the surface expression of MHC class I and class I-like proteins such as CD1d, Qa1 and neonatal Fc receptor (FcRn), all of which may impact the development of autoimmunity. Since CD1d is known to bind and present phospholipid antigens to T cells, we asked if the deficiency of β2m or CD1d will impact the development of anti-phospholipid antibodies as compared to other aspects of lupus autoimmunity.MethodsWe introgressed the β2m-null genotype onto the NZB and NZW backgrounds for 12 to 14 generations to generate genetically lupus-susceptible (NZB/NZW)F1 (BWF1) mice that are β2m-deficient (β2m°). Circulating immunoglobulins (Ig), rheumatoid factor (RF), anti-DNA and anti-cardiolipin (anti-CL) antibodies, and renal disease were analyzed in these and CD1d-deficient (CD1d°) BWF1 mice that we had previously generated.ResultsWhereas β2m° BWF1 mice had reduced serum IgG, they had increased mortality, nephritis, serum IgG anti-DNA antibody and RF as compared to heterozygous and wild-type littermates. These effects were recapitulated in CD1d° BWF1 mice, except that they also had increased serum IgG as compared to control littermates. Intriguingly, both β2m° and CD1d° mice had lower serum anti-CL antibody levels than in control littermates. Such CD1d dependence of anti-CL antibody production is not mediated by CD1d/glycolipid-reactive iNKT cells, as these cells reduced the production of RF and anti-DNA antibodies but had no effect on anti-CL antibodies.ConclusionsWe report a novel dichotomous role of β2m and CD1d, whereby these molecules differently regulate autoimmunity against phospholipid versus non-phospholipid autoantigens.

Metabolic Alterations and Increased Liver mTOR Expression Precede the Development of Autoimmune Disease in a Murine Model of Lupus Erythematosus

Although metabolic syndrome (MS) and systemic lupus erythematosus (SLE) are often associated, a common link has not been identified. Using the BWF1 mouse, which develops MS and SLE, we sought a molecular connection to explain the prevalence of these two diseases in the same individuals. We determined SLE- markers (plasma anti-ds-DNA antibodies, splenic regulatory T cells (Tregs) and cytokines, proteinuria and renal histology) and MS-markers (plasma glucose, non-esterified fatty acids, triglycerides, insulin and leptin, liver triglycerides, visceral adipose tissue, liver and adipose tissue expression of 86 insulin signaling-related genes) in 8-, 16-, 24-, and 36-week old BWF1 and control New-Zealand-White female mice. Up to week 16, BWF1 mice showed MS-markers (hyperleptinemia, hyperinsulinemia, fatty liver and visceral adipose tissue) that disappeared at week 36, when plasma anti-dsDNA antibodies, lupus nephritis and a pro-autoimmune cytokine profile were detected. BWF1 mice had hyperleptinemia and high splenic Tregs till week 16, thereby pointing to leptin resistance, as confirmed by the lack of increased liver P-Tyr-STAT-3. Hyperinsulinemia was associated with a down-regulation of insulin related-genes only in adipose tissue, whereas expression of liver mammalian target of rapamicyn (mTOR) was increased. Although leptin resistance presented early in BWF1 mice can slow-down the progression of autoimmunity, our results suggest that sustained insulin stimulation of organs, such as liver and probably kidneys, facilitates the over-expression and activity of mTOR and the development of SLE.

Lupus-prone (NZBxNZW)F1 mice exhibit decreased ability to induce iTregs due to defects in both antigen presenting cells and T cells (123.1)

Abstract Quantitative and qualitative defects in regulatory T cells (CD4+CD25+Foxp3+; Tregs) are associated with many autoimmune diseases, including systemic lupus erythematosus (SLE). SLE-prone (NZBxNZW)F1 (BWF1) mice have very low frequencies of CD4+Foxp3+ in lymphoid organs compared to non-autoimmune strains, and adoptive transfer of Tregs prevents SLE presumably by restoring the “proper” Treg:effector T cell ratios. In this study, we investigated the mechanisms that could affect Treg frequencies in BWF1 mice. There were no differences in either thymic production or peripheral proliferation of Tregs between BWF1 and control mice. However, in vitro induction of Treg conversion (iTregs) was defective using either BWF1 antigen presenting cells, i.e., TGFβ-treated bone marrow-derived dendritic cells (DC) or untreated CD103+ DC (DC able to induce conversion constitutively), or BWF1 T cells. Furthermore, BWF1 mesenteric lymph nodes contained decreased CD103+ DC concomitant with decreased Helios-CD4+Foxp3+ cells (a potential marker of iTregs) frequencies compared to controls. Taken together, these data suggest that defects in both DC and T cells in BWF1 mice lead to defective conversion of iTregs and may contribute to the decreased Treg frequencies, and consequently, to the development of disease. Identifying the mechanisms that cause these defects could lead to the development of therapeutic strategies that restore an optimal Treg compartment, and thereby treat lupus.

Higher numbers of Gr1hiCD11b+ cells suppress B cell differentiation and autoantibody production in male BWF1 mice (72.7)

Abstract (NZBxNZW) F1 (BWF1) mice spontaneously develop a lupus-like disease characterized by elevated levels of anti-nuclear autoantibodies (ANA) and systemic inflammation and with a similar female bias as observed in humans. Bone marrow (BM) chimera studies have shown intrinsic disease-driving properties of female BM cells and subsequent flow-based analyses identified significantly less Gr1hiCd11b+ myeloid cells in BWF1 female mice as compared to males. As Gr1hiCD11b+ cells have been suggested to be immunosuppressive, we hypothesized that these cells suppress B cell activation and differentiation and protect against lupus-like disease in male BWF1 mice. We analyzed the effect of male and female Gr1hiCD11b + cells on 1) cytokine-driven B cell differentiation in vitro, 2) antibody responses to immunization, and 3) spontaneous autoantibody production. We found that Gr1hiCD11b+ cells from both male and female 4 wk old BWF1 mice significantly suppressed cytokine-driven plasma cell differentiation in vitro in a dose dependent manner. Likewise, treatment of male BWF1 mice with depleting anti-Gr1 antibody resulted in elevated antibody response to Np-CGG immunization. And finally, spontaneous production of ANA were significantly increased in male BWF1 mice during and after anti-Gr1 antibody treatment, with ANA levels comparable to those of unmanipulated BWF1 females. These data suggest a new and important role for immunosuppressive Gr1hiCD11b+ cells during antibody-mediated autoimmunity.

Disparate effects of depletion of CD1d-reactive T cells during early versus late stages of disease in a genetically susceptible model of lupus

Some T cells react with lipid antigens bound to antigen-presenting molecule CD1d. Numbers and functions of a subset of such lipid-reactive T cells are reduced in patients with systemic lupus erythematosus (SLE) and their relatives, as well as in genetically susceptible and chemically induced animal models of lupus-like disease. We have reported that the germline deletion of CD1d exacerbates lupus, suggesting a protective role of these cells in the development of lupus. The use of a knockout mouse model in this study, however, did not allow examination of the role of these cells at different stages of disease. Here, we describe an approach to deplete CD1d-dependent T cells, which allowed us to investigate the role of these cells at different stages of disease in genetically lupus-prone NZB/NZW F1 (BWF1) mice. Repeated intravenous injections of large numbers of CD1d-transfected cells resulted in ∼50-75% reduction in these cells, as defined by the expression of CD4, NK1.1 and CD122, and lack of expression of CD62 ligand. TCR γδ (+)NK1.1(+) cells were also reduced in the recipients of CD1d-transfected cells as compared with control recipients. Such depletion of CD1d-reactive T cells in preclinical BWF1 mice resulted in disease acceleration with a significant increase in proteinuria and mortality. In older BWF1 mice having advanced nephritis, however, such depletion of CD1d-reactive T cells resulted in some disease improvement. Taken together, these data as well as our published studies suggest that CD1d-reactive T cells protect against the development of lupus in animal models. However, these cells appear to be unable to suppress established lupus nephritis in these animals, and might even play a disease aggravating role in late stages of disease.

Brief Treatment with iNKT Cell Ligand α-Galactosylceramide Confers a Long-term Protection Against Lupus

CD1d presents glycolipid antigens such as α-galactosylceramide (αGalCer) to invariant natural killer T cells (iNKT). We have reported that activated iNKTs inhibit IL-10-producing autoreactive B cells, while promoting or leaving intact the normal B cell responses, making iNKT modulation an attractive therapeutic modality. Here, we report that a brief treatment of young lupus-prone (NZB/NZW)F1 (BWF1) mice with two injections of αGalCer conferred a long-term protection against lupus. Long-term repeated administrations of αGalCer, however, afforded no clinical benefit. These disparate clinical effects correlated with iNKT responsiveness. While a brief treatment with αGalCer enhanced iNKT responses upon in vitro recall, the long-term αGalCer treatment resulted in reduced iNKT responses in BWF1 mice. The improvement in disease with αGalCer treatment was associated with the reduced IL-10 production. Furthermore, iNKTs directly inhibited IL-10-secreting cells in vivo in reconstituted SCID mice and inhibited IL-10-secreting B cells in vitro in co-cultures. Thus, a brief treatment with a CD1d-binding glycolipid enhances iNKT responses, reduces IL-10 production, and delays the onset of lupus, whereas long-term repeated treatments induce marked iNKT hyporesponsiveness and do not affect disease outcome in BWF1 mice. Identifying glycolipid regimens that can modulate iNKT responsiveness will have important implications for developing iNKT-based therapies for autoimmune diseases.

Depletion of Autoreactive Plasma Cells and Treatment of Lupus Nephritis in Mice Using CEP-33779, a Novel, Orally Active, Selective Inhibitor of JAK2

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC(50) = 1.3 nM; JAK3 enzyme IC(50)/JAK2 enzyme IC(50) = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1β, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138(+) plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.

Decreased frequencies of CD4+CD25+Foxp3+ cells and the potent CD103+ subset in peripheral lymph nodes correlate with autoimmune disease predisposition in some strains of mice

The CD4+CD25+Foxp3+ cells are essential for regulation of the immune response, and the integrin, CD103 (αEβ7), identifies a potent subset of these cells. Defects in CD4+CD25+Foxp3+ cells are thought to contribute to susceptibility to autoimmune disease in predisposed individuals. Studies evaluating the quality and quantity of CD4+CD25+Foxp3+ regulatory cell populations in the context of autoimmune disease susceptibility have been inconclusive, and few if any, have analyzed the CD103 subset. In this study, we analyzed regulatory T cells (Tregs) from different strains of mice with varying degrees of susceptibility to autoimmune disease. We found no differences in the ability of CD4+CD25+ or the CD103+ subset of Tregs from young female (NZB × NZW)F1 (BWF1), SJL, C57BL/6, or BALB/c mice to suppress CD4+CD25− responders in vitro. Analysis of CD4+Foxp3+ and CD4+CD25+CD103+ cell frequencies in lymphoid organs revealed that BWF1 mice had dramatically lower percentages of both populations in the lymph node (LN) than the other strains, and lower percentages in the spleen in all but the C57BL/6 strain. We next determined whether these findings extended to another autoimmune-prone strain. Similar to BWF1 mice, percentages of CD4+Foxp3+ and CD4+CD25+CD103+ cells were significantly lower in predisease NOD mice. The low frequencies of CD4+Foxp3+ and CD4+CD25+CD103+ cells in BWF1 and NOD mice were not due to deficiencies in either thymic production or homeostatic proliferation. These data indicate that decreased percentages of CD4+Foxp3+ cells and particularly, CD4+CD25+CD103+ cells in LN correlate with the predisposition to spontaneous development of autoimmune disease.

Blockade of Programmed death-1 induces functional CD4+ regulatory T cells to resist apoptosis in (New Zealand Black x New Zealand White)F1 lupus-prone mice (115.25)

Abstract PD-1 is a negative regulatory signal in T cells. We hypothesize that regulation of PD-1 signaling is required for maintenance of functional regulatory T cells (Treg) and subsequent control of autoimmunity in BWF1 mice, and that regulatory capacity of the cells is sustained in part by resistance to apoptosis, resulting in Treg survival. By toleration with an artificial peptide pCons, we have shown induction of CD4+ and CD8+ Foxp3+Treg with reduced of PD-1 expression decrease anti-dsDNA and nephritis and prolong survival. The same outcomes are observed when PD-1 expression is blocked by a neutralizing antibody. Here, anti-PD1 or pCons treatments maintained total numbers of functional suppressive Foxp3+CD4+CD25+ T cells between 10 and 40 weeks of age. The decline in IL-2 was significantly delayed by anti-PD1, and TGF-β production was increased. These CD4+Treg were resistant to spontaneous apoptosis, suppressed CD4+CD25- T cell proliferation, and induced apoptosis in B cells. In particular, Fas and FasL are two candidate molecules in the apoptotic pathways that were upregulated in splenic CD4+Treg upon pCons-tolerization or PD-1 blockade. To conclude, PD-1 is central in control of some regulatory T cells that suppress autoimmunity; their PD-1 expression may need fine tuning to permit the cells to survive and retain suppressive capacities. A mechanism by which PD-1 sustains these Treg is by reducing their susceptibility to apoptosis, which may relate to altering Fas/FasL pathway.

Administration of adenovirus encoding anti-CD20 antibody gene induces B-cell deletion and alleviates lupus in the BWF1 mouse model

Increasing evidence demonstrates that pathological B cells play an essential role in the triggering and development of human systemic lupus erythematosus (SLE). A rational strategy for treating SLE might be to delete B cells thereby suppressing autoimmunity. Commercial monoclonal anti-CD20 antibody is widely used for treatment of B cell-related autoimmune disorders. However its long term use is limited by several factors including short half-life, high cost, and possible side effects of antibody protein therapy. Therefore, we constructed a recombinant adenovirus encoding the murine anti-CD20 antibody gene, and used it to immunize lupus-prone (BWF1) mice. Our data demonstrated that administration of adenovirus encoding the murine anti-CD20 antibody gene generated murine anti-CD20 antibody, which resulted in elimination of B cells in BWF1 mice. In addition, the anti-CD20 reduced serum anti-dsDNA antibody levels, impeded the development of proteinuria and improved the survival of BWF1 mice. These findings suggested that the adenovirus encoding murine anti-CD20 antibody gene might provide an alternative strategy for B cell-mediated diseases.

Blockade of Programmed Death-1 in Young (New Zealand Black × New Zealand White)F1 Mice Promotes the Activity of Suppressive CD8+ T Cells That Protect from Lupus-Like Disease

The programmed death-1 (PD-1)/programmed death-1 ligand 1 (PD-L1) pathway regulates both stimulatory and inhibitory signals. In some conditions, PD-1/PD-L1 inhibits T and B cell activation, induces anergy, and reduces cytotoxicity in CD8(+) T cells. In other conditions, PD-l/PD-L1 has costimulatory effects on T cells. We recently showed that induction of suppressive CD8(+)Foxp3(+) T cells by immune tolerance of lupus-prone (New Zealand black × New Zealand white)F(1) (BWF(1)) mice with the anti-DNA Ig-based peptide pConsensus (pCons) is associated with significantly reduced PD-1 expression on those cells. In this study, we tested directly the role of PD-1 by administering in vivo neutralizing Ab to PD-1 to premorbid BWF(1) and healthy control mice. Anti-PD-1-treated mice were protected from the onset of lupus nephritis for 10 wk, with significantly improved survival. Although the numbers of T cells declined in aging control mice, they were maintained in anti-PD-1-treated mice, including CD8(+)Foxp3(+) T cells that suppressed syngeneic CD4(+)CD25(-) T cell proliferation and IFN-γ production, reduced production of IgG and anti-dsDNA IgG, induced apoptosis in syngeneic B cells, and increased IL-2 and TGF-β production. The administration of anti-PD-1 Ab to BWF(1) mice after induction of tolerance with pCons abrogated tolerance; mice developed autoantibodies and nephritis at the same time as control mice, being unable to induce CD8(+)Foxp3(+) T suppressor cells. These data suggest that tightly regulated PD-1 expression is essential for the maintenance of immune tolerance mediated by those CD8(+)Foxp3(+) T cells that suppress both T(h) cells and pathogenic B cells. PD-1 regulation could represent a target to preserve tolerance and prevent autoimmunity.

Pro-inflammatory high-density lipoproteins and atherosclerosis are induced in lupus-prone mice by a high-fat diet and leptin

Atherosclerosis is accelerated in people with systemic lupus erythematosus, and the presence of dysfunctional, pro-inflammatory high-density lipoproteins is a marker of increased risk. We developed a mouse model of multigenic lupus exposed to environmental factors known to accelerate atherosclerosis in humans - high-fat diet with or without injections of the adipokine leptin. BWF1 mice were the lupus-prone model; BALB/c were non-autoimmune controls. High-fat diet increased total serum cholesterol in both strains. In BALB/c mice, non-high-density lipoprotein cholesterol levels increased; they did not develop atherosclerosis. In contrast, BWF1 mice on high-fat diets developed increased quantities of high-density lipoproteins as well as elevated high-density lipoprotein scores, indicating pro-inflammatory high-density lipoproteins; they also developed atherosclerosis. In the lupus-prone strain, addition of leptin increased pro-inflammatory high-density lipoprotein scores and atherosclerosis, and accelerated proteinuria. These data suggest that environmental factors associated with obesity and metabolic syndrome can accelerate atherosclerosis and disease in a lupus-prone background.

RETRACTED: A novel pseudovirus encoding Diphtheriatoxin A fragment eliminates autoantibody-producing B cells and inhibits lupus in the BWF1 mouse model

Lupus-associated anti-dsDNA Abs plays an important role in the triggering and development of human systemic lupus erythematosus (SLE). The method of targeting B cells that produce anti-dsDNA Abs has become a promising way to treat autoantibody-mediated SLE in the immunotherapy field. In this study, we reported a novel pseudovirus, which consisted of the peptide DWEYSVWLSN and a plasmid encoding the Diphtheriatoxin A fragment (DTA). Our data demonstrated that vaccination of lupus-prone (BWF1) mice with the pseudovirus could eliminate anti-dsDNA antibody-producing B cells, reduce serum anti-dsDNA antibody levels, impede the development of proteinuria and improve survival. These findings suggested that this novel pseudovirus ablated autoreactive B cells and might represent a promising therapeutic strategy for autoantibody-mediated diseases.