Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

Abstract

The choroid plexus (ChP) serves as the principal origin of cerebrospinal fluid (CSF). CSF hypersecretion due to ChP inflammation has emerged as an important pathogenesis of hydrocephalus recently. Nevertheless, the precise mechanisms of ChP inflammation and the ensuing CSF hypersecretion in hydrocephalus remain ill-defined. In the present study, we elucidate the critical role of macrophages in the pathogenesis of ChP inflammation. Specifically, we identify the chemokine CCL2, released by ChP epithelial cells, recruits CCR2⁺ monocytes to the ChP thereby inciting hydrocephalus pathogenesis. The accumulated ChP macrophages increase the inflammation in ChP epithelial cells through TNF-α/TNFR1/NF-κB signaling cascade, thereby leading to CSF hypersecretion. Strikingly, augmentation of ChP‒CCL2 using an adeno-associated viral approach (AAV) exacerbates macrophage recruitment, activation, and ventriculomegaly in rat PHH models. Systemic application of Bindarit, a specific CCL2 inhibitor, significantly inhibits ChP macrophage infiltration and activation and reduces CSF secretion rate. Furthermore, the administration of CCR2 antagonist (INCB 3284) reduces ChP macrophage accumulation and ventriculomegaly. This study not only unveils the ChP CCL2‒CCR2 signaling in the pathophysiology of hydrocephalus but also unveils Bindarit as a promising therapeutic choice for the management of posthemorrhagic hydrocephalus.

Keywords: CCL2; CCR2; Cerebrospinal fluid hypersecretion; Choroid plexus; Crosstalk; Epithelial cells; Hydrocephalus; Macrophages.

Graphical abstract

Figure 1

IVH triggers macrophage accumulation and migration in posthemorrhagic hydrocephalus. (A) Representative T2-weighted images depicting enlarged lateral ventricles in hydrocephalus rats. (B) Schematic of integrated transcriptome analysis. (C) Heat map visualizing the differential expressed genes in the ChP between normal and PHH rats. (D) Gene ontology (GO) analysis of biological processes associated with the differentially expressed genes. (E) Inferred composition of 25 immune cell subsets within the ChP of normal and PHH rats. (F) Cibersort algorithm analysis hinted the number of monocytes significantly increased in ChP after IVH. (G) The protein expression of IBA-1 and CD68 in ChP after IVH. (H) Western blot analysis of CD68 and IBA-1 (n = 3 animals per condition). (I) Representative fluorescence photos showing immunolabeling for IBA1 (green) positive cells in the ChP after IVH (n = 5 animals per condition). Scale bars = 250 μm. (J) Statistical analysis of IBA1+ macrophages. (K) Migration of ChP IBA1+ macrophages (green) observed within the subependymal zone of ctrl and PHH rats. Scale bars = 50 μm. All data were presented as mean ± SD,∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control/model.

Figure 2

ChP CCL2-CCR2 signaling plays a crucial role in regulating macrophage recruitment and activation. (A) The M1 macrophage markers in ChP of PHH. (B, C) Representative merged and magnified merged and single-channel immunofluorescence of Ki67 (yellow), IBA1 (blue), and DAPI in Ctrl and PHH (n = 5 animals per condition). Scale bars = 50 μm. (D) Immunofluorescence of CD68⁺ (red); IBA1⁺ (blue) macrophages (white arrow) in Ctrl and PHH (n = 5 animals per condition). Scale bars = 50 μm. (E) Quantitative analysis of immunofluorescence findings (CD68⁺; Iba1⁺; % of total Iba1⁺ and % Ki67⁺ cells). (F) ChP mRNA expression of TNF-α, IL-1α, and IL-1β mRNA in Ctrl or PHH individuals (n = 5 animals per condition). (G) KEGG pathway analysis of differentially expressed genes. (H) The chemokines and receptors sequencing related to monocyte-macrophages (∗P < 0.05). (I) Volcano plots illustrating differentially expressed genes. (J) QPCR analysis of ChP CCL2, CCL7, and CXCL14 expression in Ctrl or PHH rats (n = 5 animals per condition). (K) Western blot of ChP CCL2 in Ctrl and PHH rats (n = 3 animals per condition). (L) ELISA results of CCL2 expression in CSF (n = 3 animals per condition). (M) Correlation between ChP CCL2 expression and ventricular size severity (r² = 0.7266, P = 0.0311). (N) QPCR analysis of ChP CCR2 expression (n = 5 animals per condition). (O) Immunofluorescence staining revealing CCR2⁺ macrophages (white arrow) (n = 5 animals per condition), scale bar = 50 μm. (P) Quantitative evaluation of CCR2⁺ macrophages in all cells. All data were presented as mean ± SD,∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control/model.

Figure 3

ChP epithelial cells contribute to CSF CCL2 and macrophages enhance the expression of p-NKCC1 in ChP epithelial cells in vitro. (A) Quantification of CCL2 expression in ChP epithelial cells following LPS stimulation, via qPCR analysis. (B) Western blot analysis illustrating CCL2 expression levels. (C) In situ hybridization of CCL2 mRNA exclusively expressed in ChP epithelial cells in PHH. Scale bar = 50 μm. (D, E) Immunohistochemical staining of CCL2 distribution in ChP of ctrl and PHH rats. Scale bar = 50 μm. (F) Immunofluorescence analysis of pNKCC1 (red) in ChP. Scale bar = 50 μm. (G) Western blot results of CCL2 in Bindarit-treated ChP epithelial cells. (H) Western blot analysis comparing protein profiles among different groups. (I) Schematic diagram of 2D-transwell experiment. (J) Observation of NR8383 vertical migration in the co-culture system at 24 h using transwell images. (K) A 2D-transwell system to study the effect of macrophages on ChP epithelial cells. (L) Immunofluorescence staining of pNKCC1(green) in ChP epithelial cells in different groups. All data were presented as mean ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control/model.

Figure 4

Macrophages enhance CSF hypersecretion in ChP epithelial cells via the TNF-α/TNFR1/NF-κB pathway. (A) Schematic diagram of mechanisms of crosstalk between ChP epithelial cells and macrophages. (B) Representative images showed that TNFR1 but not TNFR2 is expressed by ChP in PHH rats. Scale bar = 20 μm. (C) The expression of NF-κB and pNF-κB in ChP epithelial cells treated with NR8383 derived conditional medium (NCM), TNF-α, and R7050. (D) CCL2 and IL-1β mRNA expression in various groups. (E) The expression of NF-κB, pNF-κB, and CCL2 in epithelial cells treated with NCM or/and TNF-α neutralizing antibody. (F) The mRNA expression of proinflammatory cytokines such as CCL2, GM-CSF, IL-1β, IL-6, and IL-1α in ChP epithelial cells treated with TNF-α or/and Bay117082. (G) The expression of NF-κB, pNF-κB, and CCL2 in epithelial cells treated with TNF-α or/and Bay117082. (H) QPCR of CCL2 and NF-κB in epithelial cells treated with LPS or/and Bay117082. (I) The expression of NF-κB pathway, and CCL2 expression in ChP epithelial cells treated with LPS or/and TNF-α. (J) Western blot results of M1 marker (iNOS) and NF-κB signaling in rat macrophages NR8383 cells treated with LPS or/and Bindarit. (K) QPCR of M1 markers (iNOS and CD86) and inflammatory cytokines such as TNF-α, IL-1β, and GM-CSF in NR8383 cells treated with LPS or/and Bindarit. All data were presented as mean ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001 vs. control/model.

Figure 5

ChP CCL2 overexpression aggravates CSF secretion and exacerbates macrophage accumulation, activation, and CSF cytokines. (A) Schematic display of time nodes for AAV injection in vivo. (B) Representative image of AAV-mCherry (red) labeled brain tissue on Day 21 post-infection. (C) QPCR assessment of CCL2 expression in AAV-Cherry and AAV-CCL2 groups (n = 5 animals per condition). (D) Immunohistochemical staining of CCL2 expression (n = 5 animals per condition). (E) Representative T2-weighted MRI images of lateral ventricles in both groups (n = 5 animals per condition). (F) 3D reconstruction showcasing lateral ventricles. (G) Measurement of lateral ventricle volumes (n = 5 animals per condition) and quantification of CSF secretion rates (n = 3 animals per condition). (H) Increased expression of CD68⁺(red); and IBA1⁺ macrophages (green) in the ChP in the AAV-CCL2 group (n = 5 animals per condition). Scale bar = 100 μm. (I) Immunofluorescence of p-NKCC1 (red) in the ChP (n = 5 animals per condition). Scale bar = 50 μm. (J) The number of CCR2⁺ macrophages (green) in ChP of AAV-mCherry and AAV-CCL2 groups (n = 5 animals per condition). Scale bar = 100 μm. (K) Quantitative analysis of phagocytic ChP macrophages (CD68⁺; Iba1⁺; % of total Iba1⁺), p-NKCC1 expression, and % of CCR2⁺ macrophages (green) at the ChP. All data were presented as mean ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control/model.

Figure 6

Knockdown of ChP CCL2 using Bindarit relieves hydrocephalus and inhibits macrophage activation. (A) Representative T2-weighted images of lateral ventricles in the PHH group and Bindarit-treated group (n = 5 animals per condition). (B) 3D reconstruction showcasing the lateral ventricles. (C) Quantification volumes of the lateral ventricle based on the T2-weighted images (n = 5 animals per condition) and evaluation of CSF secretion rates (n = 3 animals per condition). (D) Bindarit significantly inhibited the CCL2 mRNA expression in ChP and CSF CCL2 after Bindarit treatment (n = 3 animals per condition). (E, F) The number of IBA1⁺ macrophages (green) in PHH and Bindarit-treated groups (n = 5 animals per condition). Scale bar = 20 μm. (G) Bindarit decreased the number of phagocytic ChP macrophages (CD68⁺; Iba1⁺; % of total Iba1⁺) (n = 5 animals per condition), Scale bar = 50 μm. (H) Quantitative analysis of the percent of macrophages in ChP cells, % of CD68⁺ macrophages in total macrophages, % of p65⁺ macrophages in ChP cells, and pNKCC1 expression in ChP, as well as the expression of TNF-α in CSF. (I) Bindarit reduced the expression of p-NKCC1 (red) in the ChP. Scale bar = 50 μm (n = 5 animals per condition). (J) NF-κB p65 signals (red) are seen in the cytoplasm of ChP epithelial cells (green arrow, b, and d) and cytoplasm-nuclear area in macrophages (white arrow, a and c) (n = 5 animals per condition). Scale bar = 20 μm. All data were presented as mean ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control/model.

Figure 7

INCB 3284, a CCR2 inhibitor, significantly relieves hydrocephalus and macrophage infiltration. (A) Representative T2-weighted images of lateral ventricles in the PHH group and INCB 3284 treated group (n = 3 animals per condition). (B) 3D reconstruction showcasing lateral ventricles. (C) Lateral ventricles volume analysis. (D, E) The number of IBA1⁺ macrophages (green) in PHH and INCB 3284 treated groups, Scale bar = 50 μm (n = 4–5 animals per condition). (F) Quantitative analysis of ChP macrophages. All data were presented as mean ± SD, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. control/model.