B cell and/or autoantibody deficiency do not prevent neuropsychiatric disease in murine systemic lupus erythematosus

Abstract

Background: Neuropsychiatric lupus (NPSLE) can be one of the earliest clinical manifestations in human lupus. However, its mechanisms are not fully understood. In lupus, a compromised blood-brain barrier may allow for the passage of circulating autoantibodies into the brain, where they can induce neuropsychiatric abnormalities including depression-like behavior and cognitive abnormalities. The purpose of this study was to determine the role of B cells and/or autoantibodies in the pathogenesis of murine NPSLE.

Methods: We evaluated neuropsychiatric manifestations, brain pathology, and cytokine expression in constitutively (JhD/MRL/lpr) and conditionally (hCD20-DTA/MRL/lpr, inducible by tamoxifen) B cell-depleted mice as compared to MRL/lpr lupus mice.

Results: We found that autoantibody levels were negligible (JhD/MRL/lpr) or significantly reduced (hCD20-DTA/MRL/lpr) in the serum and cerebrospinal fluid, respectively. Nevertheless, both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice showed profound depression-like behavior, which was no different from MRL/lpr mice. Cognitive deficits were also observed in both JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice, similar to those exhibited by MRL/lpr mice. Furthermore, although some differences were dependent on the timing of depletion, central features of NPSLE in the MRL/lpr strain including increased blood-brain barrier permeability, brain cell apoptosis, and upregulated cytokine expression persisted in B cell-deficient and B cell-depleted mice.

Conclusions: Our study surprisingly found that B cells and/or autoantibodies are not required for key features of neuropsychiatric disease in murine NPSLE.

Keywords: Autoantibodies; B cells; Lupus; Neuropsychiatric SLE; SLE.

Fig. 1

B cell counts and antibody titers are markedly reduced in the serum and CSF of JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice. FACS analysis of CD19 positive cells in the peripheral blood of JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice is shown in (a). The % CD19+ cells is provided in the red box in each panel. The MRL/lpr strain is used as a positive control. Total IgG levels (b) and anti-dsDNA IgG titers (c) in the serum and CSF of JhD/MRL/lpr and hCD20/MRL/lpr were measured by ELISA. Anti-NMDA receptor antibodies in the serum of JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice are shown in (d). (JhD and control mice at 12–15 weeks of age: LPR mice, n = 5–8; JhD mice, n = 5–8; MPJ mice, n = 5–8; hCD20-DTA and control mice at 15 weeks of age: LPR mice, n = 7; hCD20-DTA mice, n = 7; MPJ mice, n = 7; except for the CSF ELISA experiments, where the number of mice in the LPR, hCD20-DTA ,and MPJ group were 7, 4, and 4, respectively). The CSF ELISA was done once; all other experiments were repeated twice, with similar results

Fig. 2

JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice display depression-like behavior and cognitive dysfunction. The Porsolt swim test (a) was performed to evaluate behavioral despair in JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice. (Left panels: LPR mice, n = 10; JhD mice, n = 17; MPJ mice n = 4; Right panels: LPR mice, n = 7; hCD20-DTA mice, n = 7; MPJ mice, n = 12). Object placement (b) and object recognition (c) tests were employed to assess cognitive function in JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice. The dotted line indicates 53 % preference score. (Left panels: LPR mice, n = 5; JhD mice, n = 5; MPJ mice n = 5; Right panels: LPR mice, n = 7; hCD20-DTA mice, n = 7; MPJ mice, n = 10–11). Since testing affects subsequent performance in neurobehavioral analysis, all behavior tests in a–c were only conducted once. The JhD/MRL/lpr mice were tested at 7–12 weeks of age, and hCD20-DTA/MRL/lpr mice at 16–18 weeks of age. The control MRL/lpr and MRL/MPJ mice used for comparison were separately age matched to the JhD/MRL/lpr and hCD20-DTA/MRL/lpr strains, respectively

Fig. 3

JhD/MRL/lpr mice exhibit increased general locomotor activity. General locomotor activity was assessed by open field test in JhD/MRL/lpr at 7–12 weeks of age and hCD20-DTA/MRL/lpr mice at 16 weeks of age including measurement of a total track length, b rears, c center track length (in cm), d center time (in seconds (s)), and e relative center track length (center track length/total track length). (Left panels: LPR mice, n = 11; JhD mice, n = 11–12; MPJ mice, n = 5–8; Right panels: LPR mice, n = 7; hCD20-DTA mice, n = 7; MPJ mice, n = 7–17). Open field tests were conducted once. The control MRL/lpr and MRL/MPJ mice used for comparison were separately age matched to the JhD/MRL/lpr and hCD20-DTA/MRL/lpr strains, respectively

Fig. 4

Blood-brain barrier permeability and microglial activation in B cell-deficient and B cell-depleted lupus mice. a Albumin staining was performed in the brain sections from MRL/lpr (n = 6), JhD/MRL/lpr (n = 4), hCD20-DTA/MRL/lpr (n = 7), and MRL/MPJ (n = 7) mice, all at 19 weeks of age. Representative images of albumin leakage around the vessels (indicated by arrows) in the cortex are shown. Percentages (%) of mice in each group that are positive for albumin leakage were displayed. Albumin staining was repeated twice, with similar results. b Iba-1 staining was performed on the brain sections of MRL/lpr (n = 6), JhD/MRL/lpr (n = 4), hCD20-DTA/MRL/lpr (n = 7), and MRL/MPJ (n = 7) mice, all at 19 weeks of age. Representative images of Iba-1 staining in the cortex are shown. Fold changes of fluorescence intensity are shown in the bottom panel, with the mean of MRL/MPJ set at 1. Iba-1 staining was repeated twice, with similar results. The scale bar in each image represents 20 μm

Fig. 5

Brain cell apoptosis and hippocampal gliosis in JhD/MRL/lpr and hCD20-DTA/MRL/lpr mice. TUNEL and GFAP staining was performed in MRL/lpr (n = 6), JhD/MRL/lpr (n = 4), hCD20-DTA/MRL/lpr (n = 7), and MRL/MPJ (n = 7) mice, all at 19 weeks of age. Representative images of TUNEL staining in periventricular areas and choroid plexus are shown in (a, top panels). Quantitation of the average number of TUNEL positive cells in the two brain sections from each mouse is indicated in the bottom panel. b Representative images of GFAP staining in the hippocampus. Fold changes of fluorescence intensity are shown in the bottom panel, with the mean of MRL/MPJ set at 1. TUNEL and GFAP staining were repeated twice, with similar results. The scale bar in each image represents 40 μm

Fig. 6

Cytokine expression in the brain of B cell-deficient and B cell-depleted lupus mice. Real-time PCR was performed on the brain RNA samples, extracted from randomly selected MRL/lpr (n = 6), JhD/MRL/lpr (n = 4), hCD20-DTA/MRL/lpr (n = 7), and MRL/MPJ (n = 7) mice, all at 19 weeks of age. Fold changes of each gene were calculated and normalized to the mean of each gene in MRL/MPJ group, which was set at 1. Real-time PCR was repeated twice, with similar results