Nekeithia S. Wade

Mycophenolate mofetil but not atorvastatin attenuates atherosclerosis in lupus-prone LDLr−/− mice

Rationale Recent clinical and preclinical studies have demonstrated that systemic lupus erythematosus (SLE) is associated with an increased risk for cardiovascular disease (CVD). However, unlike in the general population, little is known regarding the efficacy of atheroprotective interventions in patients with SLE. The current study aims to determine the benefit of lymphocyte inhibition on reducing the atherosclerotic burden in SLE-susceptible LDLr-deficient mice. Methods Female LDLr−/− mice were lethally irradiated and reconstituted with bone marrow from C57Bl/6 mice (LDLr.B6) or the SLE-susceptible B6.Sle1.2.3 mice (LDLr.Sle). At 16 weeks post transplant, mice were treated with atorvastatin (10 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or both (MMF-A) for 8 weeks, after which the extent of atherosclerosis and the presence of SLE were assessed. Results Following 8 weeks of treatment, we observed that atorvastatin-mediated reduction in cholesterol levels attenuated atherogenesis in LDLr.B6 mice but failed to significantly reduce atherosclerotic lesion size in LDLr.Sle mice, in spite of a significant reduction in serum cholesterol levels. Treatment with MMF and MMF-A attenuated atherogenesis in LDLr.B6 and LDLr.Sle mice. In addition, MMF-containing regimens inhibited recruitment of CD4+ T cells to atherosclerotic lesions in LDLr.Sle mice. In these mice, MMF also reduced the proportion of activated splenic T cells, as well as interleukin 10 secretion by T cells. With regard to lupus activity, MMF had no overt effect on anti-double-stranded DNA (dsDNA) antibody titres or kidney function and pathology. Conclusions The current study demonstrates that reduction of cholesterol levels alone is not atheroprotective in lupus-mediated atherogenesis. This is the first study to demonstrate that MMF reduces the atherosclerotic burden in a model of lupus-accelerated atherosclerosis. Our results suggest that MMF treatment may prove beneficial in preventing CVD in patients with SLE.

The inhibitory FcγRIIb modulates the inflammatory response and influences atherosclerosis in male apoE−/− mice

BACKGROUND Atherosclerosis is widely accepted as an inflammatory disease involving both innate and adaptive immunity. B cells and/or antibodies have previously been shown to play a protective role against atherosclerosis. Aside from their ability to bind to antigens, antibodies can influence inflammatory responses by interacting with various Fcγ receptors on the surface of antigen presenting cells. Although studies in mice have determined that stimulatory Fcγ receptors contribute to atherosclerosis, the role of the inhibitory Fcγ receptor IIb (FcγRIIb) has only recently been investigated. METHODS AND RESULTS To determine the importance of FcγRIIb in modulating the adaptive immune response to hyperlipidemia, we generated FcγRIIb-deficient mice on the apoE-deficient background (apoE/FcγRIIb(-/-)). We report that male apoE/FcγRIIb(-/-) mice develop exacerbated atherosclerosis that is independent of lipid levels, and is characterized by increased antibody titers to modified LDL and pro-inflammatory cytokines in the aorta. CONCLUSIONS These findings suggest that antibodies against atherosclerosis-associated antigens partially protect against atherosclerosis in male apoE(-/-) mice by conveying inhibitory signals through the FcγRIIb that downregulate pro-inflammatory signaling via other immune receptors. These data are the first to describe a significant in vivo effect for FcγRIIb in modulating the cytokine response in the aorta in male apoE(-/-) mice.

Accelerated atherosclerosis is independent of feeding high fat diet in systemic lupus erythematosus-susceptible LDLr-/- mice

Individuals suffering from systemic lupus erythematosus (SLE) are predisposed to accelerate cardiovascular disease. Our laboratory has recently developed an animal model of SLE-accelerated atherosclerosis. We have shown that, following 8 weeks feeding high fat Western diet, radiation chimeras consisting of SLE-derived haematopoietic cells transferred to low-density lipoprotein (LDL)r−/− mice (LDLr.Sle) have increased atherosclerosis compared with C57Bl/6 bone marrow recipients (LDLr.B6). However, this feeding regimen resulted in significant mortality in SLE-susceptible mice compared with controls with surviving animals having extremely elevated serum cholesterol (>500 mg/dL) and increased serum markers of kidney pathology. To test the hypothesis that SLE-associated autoimmune dysregulation can exacerbate atherosclerosis under more mild serum cholesterol conditions (approximately 200 mg/dL), we examined SLE and lesion development in radiation chimeras fed either a normal chow or high fat Western diet for 8 weeks. High fat fed LDLr.Sle mice exhibited increased mortality and were significantly more hypertensive. LDLr.Sle mice had greater titres of antibodies against dsDNA, oxLDL and phospholipid compared with controls. Lupus-susceptibility increased the atherosclerotic lesions and the percentage of CD4+ T cells in the lesions of proximal aortas, independent of diet. These data show that increased dyslipidemia resulting from high-fat feeding can exacerbate autoimmunity and associated vascular complications. Conversely, they also show that autoimmune dysregulation can accelerate atherosclerosis in LDLr-deficient animals independent of feeding high fat diet. Collectively this study provides additional evidence that the accelerated atherosclerosis observed in SLE is autoimmune associated.

Neutrophils Contribute to Excess Serum BAFF Levels and Promote CD4 + T Cell and B Cell Responses in Lupus-Prone Mice

Despite increased frequencies of neutrophils found in autoimmune diseases such as systemic lupus erythematosus (SLE), how they contribute to disease pathogenesis and the mechanisms that affect the accumulation of neutrophils are poorly understood. The aim of this study was to identify factors in autoantibody-mediated autoimmunity that controls the accumulation of spleen resident neutrophils and to determine whether neutrophils contribute to abnormal B cell responses. Increased levels of the cytokine BAFF have been linked to loss of B cell tolerance in autoimmunity, but the cellular source responsible for excess BAFF is unknown. B cell maturation antigen (BCMA) is a receptor for BAFF and is critical for the survival of bone marrow plasma cells. Paradoxically, BCMA deficiency exacerbates the formation of autoantibody-secreting plasma cells in spleens of lupus-prone mice and the reasons for this effect are not understood. Here we analyzed the phenotype, localization and function of neutrophils in spleens of healthy mice and congenic lupus-prone mice, and compared mice sufficient or deficient in BCMA expression. Neutrophils were found to be significantly increased in frequency and activation status in spleens of lupus-prone mice when BCMA was absent. Furthermore, neutrophils localized within T cell zones and enhanced CD4+ T cell proliferation and IFNγ production through the production of BAFF. Reduced BAFF and IFNγ serum levels, decreased frequencies of IFNγ-producing T cells, germinal center B cells, and autoantibody production after neutrophil depletion indicated the involvement of neutrophils in these autoimmune traits. Thus, we have identified a novel role for BCMA to control excess BAFF production in murine lupus through restraining the accumulation of BAFF-producing neutrophils. Our data suggests that devising therapeutic strategies to reduce neutrophils in autoimmunity may decrease BAFF levels and ameliorate disease.

BAFF regulates follicular helper t cells and affects their accumulation and interferon-γ production in autoimmunity.

Follicular helper T (Tfh) cells are critical for the development of protective antibodies via germinal center (GC) B cell responses; however, uncontrolled Tfh cell expansion activates autoreactive B cells to produce antibodies that cause autoimmunity. The mechanisms that control Tfh cell homeostasis remain largely unknown. The aim of this study was to determine the contribution of BAFF to Tfh cell responses in autoimmunity.

Dysregulated CD4+ T cells from SLE-susceptible mice are sufficient to accelerate atherosclerosis in LDLr−/− mice

Objective Accelerated atherosclerosis is a major source of morbidity in systemic lupus erythematosus (SLE). However, the cause of SLE-accelerated atherosclerosis remains unclear. Methods CD4+ T cells from C57/Bl/6 (B6) or SLE-susceptible B6.Sle1.2.3 (B6.SLE) mice were transferred into LDLr−/−, Rag−/− mice. T cells were examined for cytokine production and expression of interleukin-10 receptor (IL-10R) and functional markers. T cells were isolated based on FoxP3GFP expression and transferred to LDLr−/−, Rag−/− mice to establish a role for B6.SLE effector T cells (Teff) in atherosclerosis. Results Mice receiving whole B6.SLE CD4+ T cells displayed no other SLE phenotype; however, atherosclerosis was increased nearly 40%. We noted dysregulated IL-17 production and reduced frequency of IL-10R expression by B6.SLE regulatory T cells (Treg). Functional assays indicated resistance of B6.SLE Teff to suppression by both B6.SLE and B6 Treg. Transfer experiments with CD4+FoxP3− Teff and CD4+FoxP3+ Treg from B6.SLE and B6 mice, respectively, resulted in increased atherosclerosis compared with B6 Teff and Treg recipients. Treg isolated from mice receiving B6.SLE Teff with B6 Treg had increased production of IL-17 and fewer expressed IL-10R compared with B6 Teff and Treg transfer. Conclusions Transfer of B6.SLE Teff to LDLr−/−, Rag−/− mice results in accelerated atherosclerosis independent of the source of Treg. In addition, the presence of B6.SLE Teff resulted in more IL-17-producing Treg and fewer expressing IL-10R, suggesting that B6.SLE Teff may mediate phenotypic changes in Treg. To our knowledge, this is the first study to provide direct evidence of the role of B6.SLE Teff in accelerating atherosclerosis through resistance to Treg suppression.

Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection

BACKGROUND The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota. METHODS Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin. RESULTS Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment. CONCLUSIONS Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

The lupus susceptibility locus Sle3 is not sufficient to accelerate atherosclerosis in lupus-susceptible low density lipoprotein receptor-deficient mice

Cardiovascular disease risk is increased in individuals suffering from systemic lupus erythematosus. Understanding the mechanism(s) of systemic lupus erythematosus-accelerated atherosclerosis is critical for the development of effective therapies. Our laboratory previously demonstrated that radiation chimeras of systemic lupus erythematosus-susceptible B6.Sle1.2.3 and low density lipoprotein receptor (LDLr)—/— mice have augmented atherosclerosis, which is associated with increased T-cell burden and activation in the lesion. The goals of this study were to further define specific immune mechanisms that mediate accelerated atherosclerosis and to determine whether the gene interval Sle3, which is linked to lupus-associated T-cell dysregulation, was sufficient to modulate atherogenesis. We transferred B6.Sle3 or C57Bl/6-derived bone marrow cells into lethally irradiated LDLr —/— mice (hereafter referred to as LDLr.Sle3 and LDLr.B6, respectively). Sixteen weeks after transplantation, the mice were placed on a western-type diet for 8 weeks. Our analyses revealed that LDLr.Sle3 mice had increased auto-antibody production against double-stranded DNA and cardiolipin compared with LDLr.B6 controls. We also found an increase in atherosclerosis-associated oxLDL antibodies. Antibody isotypes and serum cytokine analysis suggested that the humoral immune response in LDLr.Sle3 mice was skewed toward a Th2 phenotype. This finding is consistent with lupus-associated immune dysregulation. Additionally, LDLr.Sle3 mice had decreased serum cholesterol and triglyceride levels. However, there was no difference in lesion area or cellular composition of lesions between the two groups. These data demonstrate that, despite no change in lesion area, transfer of Sle3-associated T-cell dysregulation alone to LDLr-deficient mice is sufficient to decrease serum cholesterol and to exacerbate humoral immune responses that are frequently associated with atherosclerosis. Lupus (2010) 19, 34—42.

BAFF regulates T follicular helper cells and affects accumulation and IFNγ production in autoimmunity

Follicular helper T (Tfh) cells are critical for the development of protective antibodies via germinal center (GC) B cell responses; however, uncontrolled Tfh cell expansion activates autoreactive B cells to produce antibodies that cause autoimmunity. The mechanisms that control Tfh cell homeostasis remain largely unknown. The aim of this study was to determine the contribution of BAFF to Tfh cell responses in autoimmunity.

BAFF Regulates Follicular Helper T Cells and Affects Their Accumulation and Interferon-γ Production in Autoimmunity: BAFF Regulates Follicular Helper T Cells

Follicular helper T (Tfh) cells are critical for the development of protective antibodies via germinal center (GC) B cell responses; however, uncontrolled Tfh cell expansion activates autoreactive B cells to produce antibodies that cause autoimmunity. The mechanisms that control Tfh cell homeostasis remain largely unknown. The aim of this study was to determine the contribution of BAFF to Tfh cell responses in autoimmunity.