Microglia activation in the presence of intact blood-brain barrier and disruption of hippocampal neurogenesis via IL-6 and IL-18 mediate early diffuse neuropsychiatric lupus

Abstract

Introduction: Inflammatory mediators are detected in the cerebrospinal fluid of systemic lupus erythematosus patients with central nervous system involvement (NPSLE), yet the underlying cellular and molecular mechanisms leading to neuropsychiatric disease remain elusive.

Methods: We performed a comprehensive phenotyping of NZB/W-F1 lupus-prone mice including tests for depression, anxiety and cognition. Immunofluorescence, flow cytometry, RNA-sequencing, qPCR, cytokine quantification and blood-brain barrier (BBB) permeability assays were applied in hippocampal tissue obtained in both prenephritic (3-month-old) and nephritic (6-month-old) lupus mice and matched control strains. Healthy adult hippocampal neural stem cells (hiNSCs) were exposed ex vivo to exogenous inflammatory cytokines to assess their effects on proliferation and apoptosis.

Results: At the prenephritic stage, BBB is intact yet mice exhibit hippocampus-related behavioural deficits recapitulating the human diffuse neuropsychiatric disease. This phenotype is accounted by disrupted hippocampal neurogenesis with hiNSCs exhibiting increased proliferation combined with decreased differentiation and increased apoptosis in combination with microglia activation and increased secretion of proinflammatory cytokines and chemokines. Among these cytokines, IL-6 and IL-18 directly induce apoptosis of adult hiNSCs ex vivo. During the nephritic stage, BBB becomes disrupted which facilitates immune components of peripheral blood, particularly B-cells, to penetrate into the hippocampus further augmenting inflammation with locally increased levels of IL-6, IL-12, IL-18 and IL-23. Of note, an interferon gene signature was observed only at nephritic-stage.

Conclusion: An intact BBB with microglial activation disrupting the formation of new neurons within the hippocampus represent early events in NPSLE. Disturbances of the BBB and interferon signature are evident later in the course of the disease.

Keywords: Autoimmunity; Cytokines; Lupus Erythematosus, Systemic.

Figure 1

Hippocampus-linked behavioural alterations including impaired cognition, depressive-like behaviour and increased rates of anxiety in female NZB/W-F1 lupus mice at the prenephritic and nephritic stages of the disease. (A) Schematic representation of experimental design to assess behavioural phenotype; the same female Lupus (n=13) and WT (n=14) underwent a comprehensive behavioural test battery at 3 and 6 months of age. (B) Novel object recognition evaluates visual recognition memory; expressed as discrimination index (time spent sniffing novel object-time spent sniffing familiar object/total time spent sniffing) and (C) novel object location evaluates spatial recognition memory; expressed as discrimination index (time spent sniffing object in novel location-time spent sniffing object in familiar location/total time spent sniffing). (D) Elevated plus maze evaluates anxiety-like phenotype; expressed as time spent (%) in the open arms. (E) Tail suspension test and (F) sucrose preference test evaluate depressive-like behaviour. (G) Rotarod assesses motor performance/coordination. (H) Prepulse inhibition evaluates sensorimotor gating (n=7–8/group). (I) Social novelty and (H) Social preference evaluated with the sociability test. Lupus, NZB/W-F1 stain; WT, C57BL/6; S, seconds; Bars, mean±SD data were analysed: (B–H) Student’s t-test, (I, H) two-way ANOVA, Bonferroni post-hoc test; *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; WT, wild-type.

Figure 2

Hippocampal neurogenesis in NZB/W-F1 lupus mice. (A) Schematic illustration of adult neurogenesis in mice. Radial glia-like (RGL) cells express GFAP and not Sox2 under quiescent state. On activation, GFAP+RGL cells express Sox2. Fast proliferating neural progenitors at early stages of neurogenesis express Sox2. Neuronal progenitors express doublecortin (DCX) and are divided into early and late progenitors based on their morphology. (B) Representative images of immunohistochemical detection of DCX+neuronal progenitors in the DG of 3-month-old mice; Scale bar: 100 mm. (C) Quantification of DCX+cells in the DG of WT and Lupus mice at 3 and 6 months of age (n=5/group). (D) Representative image of DCX+early and late neuronal progenitors and the index used to indicate differentiating rate of neuronal progenitors. Scale bar: 10 mm. (E) Quantification of differentiating rate of DCX+neuronal progenitors in the DG of WT and Lupus mice at 3 and 6 months of age (n=5/group). (F) Representative images of immunohistochemical detection of GFAP+RGL and Sox2+cells in the DG of 3-month-old mice; Scale bar: 10 mm. (G–I) Quantification of (G) GFAP+RGL neuronal precursors, (H) Sox2+fast proliferating progenitors and (I) the proliferation rate of RGL neuronal precursors; expressed as the percentage of Sox2+/GFAP+activated RGL of total RGL neuronal precursors in the DG of WT and Lupus mice at 3 and 6 months of age (n=5/group); DAPI stains nuclei. Lupus, NZB/W-F1 stain; WT, C57BL/6; Bars, mean±SD data were analysed: C, G-I: Student’s t-test, E: two-way ANOVA, Bonferroni post-hoc test; *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; DG, dentate gyrus; WT, wild-type.

Figure 3

Increased immune cell trafficking and inflammatory response in hippocampus of NZB/W-F1 lupus mice. (A, B) Heatmaps of the expression of inflammatory associated genes in hippocampal tissue of WT and Lupus mice at (A) three and (B) 6 months of age (n=4–5/group). (C) Evans blue dye was intravenously injected followed by quantification of Evans blue in hippocampus. (D) Flow cytometry analysis; representative FACS plots and frequency of CD11b+CD45+ cells in myeloid (CD11b+) cells. (E) Flow cytometry analysis; representative FACS plots and frequency of CD11b+CD45+Ly6G-Ly6C+infiltrating monocytes in myeloid (CD11b+) cells. (F) Flow cytometry analysis; representative FACS plots and frequency of CD45+CD11b- lymphocytes in hippocampal cells. (G) Flow cytometry analysis; representative FACS plots and frequency of CD8+T cells and B220+B cells in lymphocytes (CD45+CD11b-). (H) Quantification of IL-6, IL-18, IL-12p40 and IL-23 in hippocampal tissue. All experiments (C–H) were performed in Lupus and WT mice at 3 and 6 months of age (n=4–6/group) and obtained from two independent experiments. Lupus, NZB/W-F1 stain; WT, C57BL/6; Bars, mean±SD data were analysed with Student’s t-test, *p<0.05, **p<0.01, ***p<0.001. WT, wild-type.

Figure 4

Activation of hippocampal microglia towards a proinflammatory state in NZB/W-F1 lupus mice. (A) Flow cytometry analysis; representative histogram and mean fluorescence intensity (MFI) of IBA1 in microglia cells (CD11b+CD45 low). (B) Representative images of immunohistochemical detection of IBA1+microglia cells in the DG of 3-month-old mice; Scale bar: 50 mm. (C) Quantification of IBA1+microglia cells in the subgranular zone (SGZ)/granule cell layer (GCL) and hilus of WT and Lupus mice at 3 and 6 months of age (n=5/group). (D) Flow cytometry analysis; representative FACS plots and frequency of iNOS+proinflammatory microglia cells (CD11b+CD45 low). (E) Flow cytometry analysis; representative FACS plots and frequency of Arginase1+anti-inflammatory microglia cells (CD11b+CD45 low). (F) Flow cytometry analysis; representative histogram and frequency of CD80+cells in microglia cells (CD11b+CD45 low). (G) Flow cytometry analysis; representative histogram and frequency of CD86+cells in microglia cells (CD11b+CD45 low). (H) Flow cytometry analysis; representative histogram and MFI of MHC-II+cells in microglia cells (CD11b+CD45 low). (I) Quantification of CCL17, CCL22 and CXCL1 mRNA levels of sorted microglia with real time RT-qPCR in 3 month-old mice. All experiments were performed in Lupus and WT mice at 3 and 6 months of age (n=4–6/group) and obtained from two independent experiments. Lupus, NZB/W-F1 stain; WT, C57BL/6; Bars, mean±SD data were analysed with Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Interleukin-6 and Interleukin-18 directly promote proliferation and increase apoptosis of adult hiNSCs in vitro. (A) Experimental design for the assessment of direct effects of Interleukin-6 and Interleukin-18 on proliferation and survival of adult hiNSCs. (B, D, F) Representative images of adult hiNSCs in culture stained against (B) BrdU and (D) PH3 or (F) subject to apoptosis with TUNEL assay after exposure to IL-6 or IL-18. (C, E, F) Quantification of the proliferative (C, E) and apoptotic (F) effects of IL-6 and IL-18 on adult hiNSCs. Data are representative of three independent experiments. DAPI stains nuclei; Scale bar: 10 mm; bars, mean±SEM. Data were analysed with Mann-Whitney U test, *p<0.05, **p<0.01. hiNSCs, hippocampal neural stem cells.

Figure 6

Proposed mechanism of early diffuse neuropsychiatric lupus. In early neuropsychiatric lupus, activation of microglia leads to the secretion of interleukin-6 and interleukin-18 which induce apoptosis to hippocampal neural stem cells (hiNSCs) leading to decreased formation of new neurons and neuropsychiatric changes. Of note, during the early stages of disease the blood–brain barrier (BBB) remains intact but activated microglia and the inflammatory cytokines may gradually impair its integrity. as the disease progresses, BBB is disrupted facilitating the entry of immune components of peripheral blood, particularly B-cells, to penetrate into the hippocampus exaggerating inflammation with locally increased levels of cytokines and an interferon signature. peripheral inflammatory mediators including interferon-α further compromise the integrity of the BBB and an interferon signature within the hippocampus becomes apparent. other pathogenetic aspects of NPSLE include the disturbance of choroid plexus and infiltration of antibodies.