Cerebral nitric oxide represses choroid plexus NFκB-dependent gateway activity for leukocyte trafficking

Abstract

Chronic neuroinflammation is evident in brain aging and neurodegenerative disorders and is often associated with excessive nitric oxide (NO) production within the central nervous system (CNS). Under such conditions, increased NO levels are observed at the choroid plexus (CP), an epithelial layer that forms the blood-cerebrospinal fluid barrier (BCSFB) and serves as a selective gateway for leukocyte entry to the CNS in homeostasis and following injury. Here, we hypothesized that elevated cerebral NO levels interfere with CP gateway activity. We found that induction of leukocyte trafficking determinants by the CP and sequential leukocyte entry to the CSF are dependent on the CP epithelial NFκB/p65 signaling pathway, which was inhibited upon exposure to NO. Examining the CP in 5XFAD transgenic mouse model of Alzheimer's disease (AD-Tg) revealed impaired ability to mount an NFκB/p65-dependent response. Systemic administration of an NO scavenger in AD-Tg mice alleviated NFκB/p65 suppression at the CP and augmented its gateway activity. Together, our findings identify cerebral NO as a negative regulator of CP gateway activity for immune cell trafficking to the CNS.

Keywords: BCSFB; NFκB; choroid plexus; nitric oxide.

Figure 1

Intracerebroventricular administration of TNFα induces synchronized CP-mediated leukocytes trafficking to the CSF

1. A Gating strategy and representative flow cytometry plots of CD4⁺ and CD11b⁺ leukocytes (CD45⁺) in CSF aspirated from mice, 24 h following ICV injection of TNFα (100 ng/mouse), or from naïve mice (n = 4 per group; SSC, side scatter; FSC, forward scatter).

2. B–D Quantitative analysis of leukocytes (total CD45⁺ leukocytes (B); CD4⁺ and CD11b⁺ (C, D) cells out of total CD45⁺ cells) in the CSF, 24 h following ICV injection of 50, 100, or 150 ng of TNFα (n = 4 per group; cells per μl; one-way ANOVA followed by Newman–Keuls post hoc test).

3. E Representative flow cytometry plots and fraction analysis of the kinetics of CSF cellular composition during the first 24 h following ICV administration of TNFα (100 ng/mouse; n = 4 per group).

4. F, G Representative microscopic images of the brain's third ventricle (3v), immunostained for the myeloid markers, Mac-2 or IBA-1, together with cytokeratin or E-cadherin, 24 h following ICV injection of TNFα (100 ng/mouse; n = 5 per group; scale bar, 50 μm; inserts show PBS-injected controls and higher magnification of the boxed area; arrows indicate Mac-2-positive myeloid cells).

Data information: In all panels, bars represent mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Nitric oxide represses NFκB/p65 nuclear translocation in choroid plexus epithelial cells

1. A Schematic representation of the experimental groups. CP epithelial cells were cultured for 4 days (reaching confluence). On day 4, groups were either treated with the NO donor, DETA/NONOate (150 μM) or left untreated. On day 6, the groups were either stimulated with TNFα (20 ng/ml) or left untreated. Next, cell cultures were dissociated into a single-cell suspension and fixed for intracellular staining (M/M, medium only; D/D, DETA/NONOate only; M/T, medium followed by TNFα; D/T, DETA/NONOate followed by TNFα).

2. B Similarity index between NFκB/p65 and nuclear (Hoechst) localization, which is a measure of NFκB/p65 nuclear translocation, examined by ImageStream (see Materials and Methods) (n > 2,000 cells per group; one-way ANOVA followed by Newman–Keuls post hoc test).

3. C Representative images of NFκB/p65 cellular localization in the different experimental groups (cytokeratin in green; p65 in red; Hoechst nuclear staining in blue; scale bar, 10 μm).

4. D, E Representative visualization (D) and quantitative analysis (E) of S-nitrosylated proteins in CP epithelial cells cultures, which were either exposed to DETA/NONOate or left untreated (S-nitrosylation in green, Hoechst nuclear staining in blue) (scale bar, 25 μm; n = 10 per group; Student's t-test).

5. F Quantitative analysis of intracellular NO, measured by flow cytometry of DAF-2 DA florescence intensity, in DETA/NONOate-treated and untreated cells (n > 2,000 cells per group; Student's t-test).

6. G Negative correlation between NFκB/p65 nuclear translocation and intracellular NO, as assessed by DAF-2 DA florescence intensity, on a single-cell level (Pearson's r = −0.2968, P < 0.0001). Arrows indicate representative images of the cellular spatial localization of NFκB/p65 (in orange), DAF-2 DA (in green), cytokeratin (in red), and Hoechst nuclear staining (in blue), as well as a brightfield (BF) overview of the cell.

Data information: In all panels, bars represent mean ± s.e.m.; ***P < 0.001.

Figure 3

Nitric oxide inhibits induction of leukocyte trafficking determinants by the CP

1. A, B mRNA levels of the genes ifngr2, ccl2, cxcl10, and icam1, measured by qPCR, in CP epithelial cultures, treated as described in Fig2A. Expression levels are relative to untreated (M/M) cultures (n = 6 per group; one-way ANOVA followed by Newman–Keuls post hoc test; data are representative of at least three independently performed experiments in each case).

2. C, D Representative microscopic images (C) and quantitative analysis (D) of CP cell cultures treated as described in Fig2A, immunostained for ICAM-1 (in red), ZO-1 (in green) and Hoechst nuclear staining (in blue) (n = 6 per group; one-way ANOVA followed by Newman–Keuls post hoc test).

3. E Representative microscopic images of ZO-1 epithelial tight junction's disturbance in CP cell cultures, treated as described in Fig2A.

Data information: In all panels, bars represent mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; M/M, medium only; D/D, DETA/NONOate only; M/T, medium followed by TNFα; D/T, DETA/NONOate followed by TNFα.

Figure 4

Elevated NO levels in the CP of AD transgenic mice are involved in repression of NFκB signaling pathway

1. A Flow cytometry gating strategy to identify cytokeratin-positive CP epithelial cells, and measurement of the similarity index between NFκB/p65 and Hoechst nuclear staining, which is a measure for NFκB/p65 nuclear translocation, as assessed by ImageStream analysis, in AD-Tg and WT mice at various age groups following ex vivo stimulation with 20 ng/ml TNFα (n > 5,000 cells per group; one-way ANOVA followed by Newman–Keuls post hoc test).

2. B Representative images (ImageStream) of NFκB/p65 cellular localization in CP epithelial cells from 9-month-old AD-Tg or WT mice, following ex vivo stimulation with 20 ng/ml TNFα (cytokeratin in green; p65 in red; Hoechst nuclear staining in blue; scale bar, 10 μm).

3. C–E Representative microscopic images (C) of the brain of 10-month-old AD-Tg mice treated with either rutin or vehicle. Brain slices (6 μm) were immunostained for amyloid beta (Aβ) plaques (in red), GFAP (in green), and Hoechst nuclear staining (in blue). Mean Aβ plaque area (D) in the hippocampal dentate gyrus (HC) and the cortex (5^th layer) were quantified. Astrogliosis was assessed in the cortex (5^th layer) by GFAP immunoreactivity (E) (n = 8 per group; Student's t-test; scale bar, 250 μm).

4. F Quantitative analysis of intracellular NO, measured by flow cytometry of DAF-2 DA mean florescence intensity (MFI), in CP of 4-month-old AD-Tg mice and age-matched WT controls, which were treated with either rutin or vehicle (drinking water) (n = 3–6 per group; one-way ANOVA followed by Newman–Keuls post hoc test).

5. G Similarity index analysis, examined by ImageStream, of NFκB/p65 nuclear translocation, in CP of 4-month-old AD-Tg mice, which were treated with either rutin or vehicle (drinking water), and untreated WT controls (n = 3–4 per group; one-way ANOVA followed by Newman–Keuls post hoc test).

6. H mRNA expression levels of the genes icam1, ccl2, and cxcl10, measured by qPCR, in CP of 4-month-old AD-Tg mice, which were treated with either rutin or vehicle (drinking water) (n = 5–6 per group; Student's t-test).

7. I Representative flow cytometry plots and quantitative analysis of cells isolated from the brains of 10-month-old AD-Tg mice, treated with either rutin or vehicle (drinking water). CD11b^high/CD45^high mo-MΦ were gated and quantified (n = 5–7 per group; Student's t-test).

Data information: In all panels, bars represent mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Proposed model for CP gateway dysfunction for leukocyte trafficking following cerebroventricular nitric oxide elevation Schematic illustration of the intracellular cascade of events at the CP compartment, following stimulation with TNFα, in physiology, and under pathological conditions of chronic exposure to nitric oxide. (1) In the steady state, the CP senses CSF-borne danger/pro-inflammatory signals, derived from the brain parenchyma. TNFα, as a particular example of those signals, is sensed by the CP via the TNFα receptor (TNF-R). (2) Upon TNFα stimulation, TNF-R signaling cascade is funneled, through the NFκB pathway, into translocation of the p65 subunit to the nucleus, which initiates a cellular response involving the upregulation of IFNγ-R on the CP epithelium, leukocyte trafficking determinant expression, and disruption of the epithelial tight junctions. (3) IFNγ, secreted by CP stromal Th1 cells, acts in synergy with the NFκB signaling pathway, in inducing the expression of specific leukocyte trafficking molecules such as ICAM-1, CCL2, and CXCL10. Together, these events support CP-mediated leukocyte entry to the CNS. (4) Under neuroinflammatory conditions, which involve cerebroventricular elevation of nitric oxide levels and its accumulation at the CP, nitrosative modifications of the NFκB complex prevent its nuclear translocation following TNFα stimulation.