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PNAS Nexus
PNAS Nexus
pnasnexus
PNAS Nexus
2752-6542
Oxford University Press US

10.1093/pnasnexus/pgae334
pgae334
Biological, Health, and Medical Sciences
AcademicSubjects/MED00010
AcademicSubjects/SCI00010
AcademicSubjects/SOC00010
PNAS_Nexus/immun
CYP7B1 deficiency impairs myeloid cell activation in autoimmune disease of the central nervous system
https://orcid.org/0009-0003-4115-6546
Song Huanhuan Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Lv Aowei Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Zhu Zhibao Department of Neurology, Fujian Medical University Union Hospital, Fujian Key Laboratory of Molecular Neurology and Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Li Runyun Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Zhao Qiuping Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Yu Xintong Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Jiang Junyi Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China

Lin Xiang Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China
Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China

Zhang Cunjin Department of Neurology, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu 610072, China

Li Rui Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China
Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China
Institute of Immunotherapy, Fujian Medical University, Fuzhou 350122, China

https://orcid.org/0000-0002-1714-2318
Yan Yaping Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry (the Ministry of Education), National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an 710000, China

Chen Wanjin Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China
Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China

Wang Ning Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China
Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China

https://orcid.org/0000-0002-1590-3945
Fu Ying Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, Fujian Medical University, Fuzhou 350005, China
Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China

Bonini Nancy Editor
To whom correspondence should be addressed: Email: fuying@fjmu.edu.cn; ningwang@fjmu.edu.cn; wanjinchen75@fjmu.edu.cn
H.S., A.L., and Z.Z. contributed equally to this work.

Competing Interest: The authors declare no competing interests.

9 2024
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11 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.
2024
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Abstract

Dysregulation of cholesterol metabolism underlies neurodegenerative disease and is increasingly implicated in neuroinflammatory diseases, such as multiple sclerosis (MS). Cytochrome P450 family 7 subfamily B member 1 (CYP7B1) is a key enzyme in alternative cholesterol metabolism. A recessive mutation in the gene CYP7B1 is known to cause a neurodegenerative disease, hereditary spastic paraplegia type 5 and oxysterol accumulation. However, the role of CYP7B1 in neuroinflammation has been little revealed. In this study, we induced experimental autoimmune encephalomyelitis (EAE), as a murine model of MS, using CYP7B1 homozygous knockout (KO) mice. We found that CYP7B1 deficiency can significantly attenuate EAE severity. CYP7B1 deficiency is sufficient to reduce leukocyte infiltration into the central nervous system, suppress proliferation of pathogenic CD4+ T cells, and decrease myeloid cell activation during EAE. Additionally, live-animal imaging targeting translocator protein expression, an outer mitochondrial membrane protein biomarker of neuroinflammation, showed that CYP7B1 deficiency results in suppressed neuroinflammation. Using human monocyte-derived microglia-like cellular disease model and primary microglia of CYP7B1 KO mice, we also found that activation of microglia of CYP7B1 deficiency was impaired. These cumulative results suggest that CYP7B1 can regulate neuroinflammation, thus providing potential new targets for therapeutic intervention.

cytochrome P450 family 7 subfamily B member 1
cholesterol metabolism
neuroinflammation
experimental autoimmune encephalomyelitis
myeloid cell
National Natural Science Foundation of China 10.13039/501100001809 U2005201 U21A20360 82371349 Department of Science and Technology of Fujian Province 10.13039/501100005270 2020Y9129 Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China 2022ZQNZD005
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pmcSignificance Statement

Abnormal cholesterol metabolism drives the development of neurodegenerative disease and neuroinflammatory diseases, such as multiple sclerosis (MS). Cytochrome P450 family 7 subfamily B member 1 (CYP7B1) controls the first reaction in the cholesterol catabolic pathway of central nervous system, which mutation caused neurodegenerative disease hereditary spastic paraplegia type 5 and oxysterol accumulation. In this study, we report that CYP7B1 adjusts neuroinflammation by suppressing myeloid cell activation in experimental autoimmune encephalomyelitis, an animal model of MS. Additionally, we verified the above results by developing a live imaging strategy that targets translocator protein and using a human microglia-like cellular disease model. Our findings provide a new perspective to understand the relationship between neuroinflammation and cholesterol metabolism.

Introduction

Cholesterol homeostasis is vital for normal central nervous system (CNS) function and development, impacting processes such as neuronal dendrite and axon formation, astrocyte proliferation, microglia survival, nerve repair, and signaling transmission crucial for neurodevelopment (1). Dysregulated cholesterol metabolism is associated with neurological disorders such as multiple sclerosis (MS) and Alzheimer's disease (AD) (2). Oxidized cholesterol links cholesterol metabolism in peripheral circulation to the CNS and affects immune cell growth, proliferation, activation, and function via the nuclear receptors liver X receptors (LXRs)/sensor response element binding protein 2 axis (3) or Epstein–Barr virus-induced G-protein-coupled receptor 2 (EBI2) (4). However, how cholesterol metabolism influences CNS immunity is currently unclear.

Cytochrome P450 family 7 subfamily B member 1 (CYP7B1) encodes oxysterol 7-alpha-hydroxylase, a key enzyme in alternative cholesterol metabolism (5). CYP7B1 deficiency leads to congenital bile acid metabolism abnormalities in infancy and hereditary spastic paraplegia type 5 (SPG5) in later life (6). It plays essential roles in cellular development, predominantly expressed in the brain, and is up-regulated in response to pro-inflammatory stimuli, particularly in macrophages and innate immune cells (7). CYP7B1 is crucial for EBI2 ligand generation in lymphoid tissue, early B-cell formation, and plasma cell reactivity following immunization (8). Additionally, CYP7B1 is significantly expressed in macrophages during COVID-19 and mycobacterium tuberculosis (TB) infection. Furthermore, studies on SPG5 patients reveal widespread white matter injury (9–11), suggesting CYP7B1 involvement in neuroinflammation beyond lipid homeostasis maintenance.

MS is a common chronic neurological disease of the CNS that is driven by autoimmune, inflammatory, and neurodegenerative processes, often accompanied by systemic lipid metabolism dysfunction (12). An increasing body of evidence supports a relationship between cholesterol metabolism and clinical outcomes in MS (12, 13). In mice with experimental autoimmune encephalomyelitis (EAE), a model for MS (14), oxysterols regulate encephalitogenic CD4+ T cell trafficking into inflamed tissues (15) and CYP7B1 expression was significantly up-regulated in the CNS of EAE-induced mice (16). Here, in this study, we hypothesized that CYP7B1 could participate in regulating neuroinflammation associated with autoimmune disease.

In this study, we investigated whether CYP7B1 regulates neuroinflammation in acute EAE pathogenesis. CYP7B1 knockout (KO) mice exhibited significantly reduced EAE severity, CNS inflammation, and demyelination compared with wild-type (WT) mice. Using a novel fluorescent probe targeting translocator protein (TSPO), we observed suppressed neuroinflammation in CYP7B1 KO mice, accompanied by decreased myeloid cell numbers and activation. Analysis of CYP7B1-deficient myeloid cells in vivo further supported these findings. Our study suggests that targeting CYP7B1 could offer a promising approach for regulating neuroinflammation therapeutically.

Results

EAE disease progression is attenuated under CYP7B1 deficiency

Since CYP7B1 deficiency results in dysfunction of cholesterol metabolism and accumulation of 27-hydroxycholesterol (27-OHC) and 25-hydroxycholesterol (25-OHC) in the peripheral and the CNS (6), we investigated its possible role in neuroinflammation and EAE pathogenesis. CYP7B1 KO mice were made via CRISPR/Cas-mediated editing of exon 2 (Figure S1A). Pups were genotyped by PCR and Sanger sequencing (Figure S1B and C). Plasma samples were analyzed using ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) showed that total oxidative cholesterols showed an increasing trend compared with that in WT mice (Figure S1D). And both 27-OHC and 25-OHC levels were remarkably higher in KO mice (Figure 1A). Brain MRI showed no apparent abnormalities in KO mice compared to controls (Figure 1B; Figure S1E).

Fig. 1. EAE disease progression is attenuated under CYP7B1 deficiency. A) The plasma levels of 27-OHC and 25-OHC in WT and KO mice were analyzed using UPLC-MS/MS and quantitated with a stable isotope dilution method as in our previous study (17). The number of each group is as follows: WT (n = 4), KO (n = 3). B) MRI brain images of WT and KO mice at 6 months in vivo. n = 4 per group. C) WT and KO mice, aged 6 to 8 weeks, were immunized with MOG35–55 combined with CFA and PT. Behavioral evaluation for WT and KO mice was scored daily on a 0 to 5 scale. The number of each group is as follows: WT (n = 7), KO (n = 6). Data are representative of four independent experiments. D) Days of disease onset post-transfer (p.t.), E) cumulative clinical score, and F) peak score for WT and KO mice after EAE immunization. The number of each group is as follows: WT (n = 7) and KO (n = 6). Data are representative of four independent experiments. G–I) The slices of lumbar spinal cords were stained with H&E (top) and LFB (middle) to assay for inflammation and demyelination. Representative immunohistochemistry images of MBP (bottom) on lumbar spinal cord sections of EAE mice. The number of each group is as follows: WT (n = 5) and KO (n = 8). Data are representative of four independent experiments. The error bars indicate means ± SEM. Statistical analysis performed by Student's t test (A, D, E, F, H, and I). Two-way ANOVA followed by Bonferroni’s post hoc test (C). ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm in G top and middle and 40 μm in G bottom.

To gain a deeper understanding of the role of the CYP7B1 gene in neuroinflammation, we examined the immunological function of CYP7B1 KO mice. By tracking their weights and evaluating their behavior, we discovered that CYP7B1 KO mice's motor function was unaffected (Figure S2A–D). The detection of specific subsets of immune cells was performed on spleen samples from KO and WT mice. No abnormalities were detected (Figure S2E and F). In summary, under physiological conditions, the immunological state is essentially unaffected by CYP7B1 deficiency.

We then investigated the potential role of CYP7B1 in EAE, an animal model of MS (14). Both KO and WT mice were immunized with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55)/complete Freund's adjuvant (CFA)/pertussis toxin (PT) and observed the clinical behavior for 60 days. KO mice exhibited a delayed onset and initially lower clinical severity scores compared to WT mice, although disease progression ultimately became comparable (Figure 1C). Analysis of disease evolution revealed a significant decrease in overall disease severity in KO mice (Figure 1D–F).

Hematoxylin and eosin (H&E) staining in thin sections of the spinal cord showed that lymphocyte infiltration was significantly lower in KO mice than that in WT mice (Figure 1G top and H), while Luxol fast blue (LFB) staining also indicated that demyelination was significantly less severe in the spinal cord samples of KO mice than in WT mice (Figure 1G middle and H). Further staining for the myelin-specific marker, myelin basic protein (MBP) in lumbar slices verified the significantly lower severity of demyelination in KO mice compared to WT mice (Figure 1G bottom and I). These results indicated that KO mice generally developed milder EAE with delayed onset, less inflammatory cell infiltration, and fewer demyelinated foci.

CYP7B1 deficiency lowers inflammatory cells infiltration of CNS

Leukocyte infiltration into the CNS is a key driver of inflammation and tissue degradation in EAE (18). Then, we tested whether immune cell activation could explain the above findings. To this end, we conducted cell counts and flow cytometry analysis of CNS-infiltrating mononuclear cells to characterize mononuclear cells populations in the CNS. The data show that CD45+ immune cell counts were lower in the lumbar spinal cord of KO mice compared to the WT mice (Figure 2A–D). T cells play pivotal roles in the pathogenesis of MS and EAE; we found a robust decrease in CD3+ T cells and CD3 + CD4+ T cells in both the CNS and the periphery in KO mice (Figure S3A–C). In contrast, no significant differences were observed between KO mice and WT mice in CD3 + CD8+ T-cell, CD3−CD19+ B-cell, or CD3−NK1.1+ NK cell counts in the CNS (Figure S3A and B). We also noted that samples of the spleen showed extensive mobilization. In addition to general CD3+ T cell and CD4+ T subset, the CD8+ T subset and NK cell populations were both significantly higher in KO mice (Figure S3A and C). Immunostaining of sections of lumbar spinal cord showed that significantly less area contained CD4+ cells in KO mice than in WT mice, further suggesting that CYP7B1 deficiency could inhibit inflammatory cells infiltration into CNS lesions (Figure S3D and E).

Fig. 2. CYP7B1 deficiency lowers inflammatory cells infiltration of CNS. WT and KO mice were sacrificed between days 20 and 30 after EAE induction. Mononuclear cells were isolated from the brain and spinal cord or spleen. Lumbar spinal cord sections of EAE mice were harvested for immunofluorescent staining. A) Representative immunohistochemistry images of CD45 on spinal cord sections of EAE mice. B) Quantitative analysis of CD45 expression using Image-Pro. We conducted measurements on 8–10 areas in each mouse, which encompassed nearly the whole white matter region. Subsequently, calculate the mean value and generate statistical analysis. The number of each group is as follows: WT (n = 7) and KO (n = 6). Data are representative of three independent experiments. C), D) Representative flow cytometry plots and the quantification of leukocytes gated on all living mononuclear cells are shown. The number of each group is as follows: WT (n = 5) and KO (n = 6). Data are representative of three independent experiments. E–G) Subpopulations of CD4+ T cells in the CNS and spleen were analyzed by flow cytometry. Mononuclear cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin in the presence of 5 μg/mL BFA for 5 h at a density of 1 × 106 cells/mL in RPMI 1640 complete medium. The subsets of Th1 and Th17 cells in the CD4+ T-cell gate were analyzed by combining surface staining for CD4 with intracellular staining for IFN-γ and IL-17A. The number of each group is as follows: WT (n = 5) and KO (n = 5) in CNS or WT (n = 12) and KO (n = 10) in spleen. Data are representative of three independent experiments. The error bars indicate means ± SEM. Two-tailed unpaired Student's t test. ns, no significant difference. *P < 0.05, **P < 0.01. Scale bars, 40 μm in A and 4 μm in the magnification.

We then assessed the number of Th1 (CD4 + IFN-γ+) and Th17 (CD4 + IL-17A+) cells in the CNS and peripheral organs. We found that the number of Th1 cells was significantly lower in CNS of KO mice than in WT mice, consistent with the results of Th17 cell counts (Figure 2E and F). However, CYP7B1 deficiency did not impact Th1 and Th17 cell counts in the spleen (Figure 2E and G). These results suggested that CYP7B1 contributes to shaping the immune microenvironment in the CNS of EAE mice.

CYP7B1 deficiency does not impact the antigen-specific sensitization of T cells

To initiate inflammation in the CNS, myelin-specific T cells must be activated in the periphery, gain access to the CNS, and then be reactivated by local antigen-presenting cells (APCs) that have the required self-antigen (19). Therefore, we compared the antigen-specific responses of T cells in WT and KO mice to MOG35–55 peptide. Detection of the proliferation marker, Ki67, showed that KO mice had significantly fewer CD3 + Ki67+ cells compared to WT mice (Figure 3A and B). We then isolated mononuclear cells from brain and spinal cord of KO mice and WT mice between days 20 and 30 after immunization. MOG35–55 restimulated the isolated mononuclear cells for 72 h in vitro. Flow cytometry analysis with carboxyfluorescein diacetate succinimidyl ester (CFSE) chemical marker of cell proliferation indicated that T-cell populations responsive to MOG35–55 in KO mice were significantly suppressed in both the brain (Figure 3C and D), compared to WT mice. Flow cytometry quantified the Th1 and Th 17 cells from the CNS expressing pro-inflammatory IL-17A and IFN-γ cytokines after restimulation with MOG35–55 peptide and phorbol 12-myristate 13-acetate (PMA)–ionomycin. Results showed that KO mice had significantly fewer MOG-reactive Th1 cells and Th17 cells in the CNS (Figure 3E and F).

Fig. 3. CYP7B1 deficiency does not impact the antigen-specific sensitization of T cells. WT and KO mice were immunized with MOG35–55/CFA/PT and sacrificed between days 20 and 30 after immunization. Spleen, brain, and spinal cord were harvested, and mononuclear cells were isolated. Lumbar spinal cord sections of EAE mice were analyzed by immunohistochemistry. A) Double immunostaining of CD3 and Ki67 (for proliferation) showing the number of growing CD3+ T cells in the slices of lumbar spinal cords. B) Quantitative analysis of CD3 + Ki67+ cell expression using Image-Pro. The number of each group is as follows: WT (n = 7) and KO (n = 6). Data are representative of three independent experiments. C), D) Mononuclear cells in the CNS were restimulated with MOG35–55 for 72 h. Expansion of CD4+ T cells was assessed by CFSE dilution. The number of each group is as follows: WT (n = 6) and KO (n = 6). Data are representative of three independent experiments. E), F) Mononuclear cells were restimulated with MOG35–55 for 72 h and cultured with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of 5 μg/ml BFA for the last 5 h. IFN-γ- and IL-17A-expressing CD4+ T cells were measured by intracellular staining. The number of each group is as follows: WT (n = 7) and KO (n = 9). Data are representative of three independent experiments. G) Schematics of adoptive transfer EAE design. We extracted spleen and lymph node from both KO mice and WT mice after immunized EAE for 11 – 12 days. Following a 72-h cell culture period induced by MOG35–55, CD4+ T cells were administered via intravenous injection into WT or KO mice that had received sublethal doses of radiation. PT was administered intraperitoneally on days 0 and 2 after infusion to induce EAE. H) Clinical scores of adoptive transfer EAE mice. The number of each group is as follows: KO → WT (n = 7), WT → KO (n = 8), and KO → KO (n = 8). Data are representative of two independent experiments. The error bars represent the means ± SEM. Two-tailed unpaired Student's t test (B, D, and F). Two-way ANOVA followed by Bonferroni’s post hoc test (H). ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 40 μm in A and 1 μm in the magnification.

Subsequently, we examined the impact of CYP7B1 deficiency on the pathogenicity of MOG-specific T cells. We took spleen from KO and WT mice in EAE, and they had just started exhibiting behavioral alterations. After 72 h of cell culture stimulation by MOG35–55, CD4+ T cells were sorted and intraperitoneally administered into WT or KO mice, which were irradiated with sublethal doses. PT was injected intraperitoneally on days 0 and 2 following infusion to cause EAE. Daily behavioral assessments were conducted and recorded (Figure 3G). The transfer of CD4+ T cells from KO mice immediately started the immunological response in WT mice, causing behavioral changes on the 10th day and worsening the disease. However, infusion of WT mouse-derived CD4+ T cells into KO mice attenuated the disease progression. The results showed that MOG-specific T cells of KO mice remained pathogenic (Figure 3H).

Taken together, this suggests that the alterations in disease status observed in CYP7B1 KO mice subsequent to the development of the EAE model are attributable to anomalies within the CNS.

CYP7B1 deficiency attenuates the activation of myeloid cells

Neuroinflammation is a hallmark of EAE that involves myeloid cells activation. We designed and synthesized a fluorescent probe targeting TSPO, a representative neuroinflammation marker (20), to define the neuroimmune cells landscape by in vivo chemiluminescence imaging. At the peak of EAE disease, when EAE disease scores remained stable across time points, TSPO signal intensity increased more slowly in KO mice than in WT mice at 5 min after intravenous tail injection (Figure 4A and B). Moreover, KO mice had lower signal intensity than WT mice at several detection time points (Figure 4A and B).

Fig. 4. CYP7B1 deficiency attenuates the activation of myeloid cells. A), B) Visualization and quantification of neuroinflammation by in vivo chemiluminescence imaging at days 20–30 after immunization using IVIS Lumina X5 Spectrum System. Results are expressed as the ROI measurements of brain by Loess regression statistical analyses. n = 6 per group. Data are representative of three independent experiments. C), D) Mononuclear cells were isolated from the brain and spinal cord of WT and KO mice. The percentages of microglia (CD11b + CD45int) and macrophage (CD11b + CD45hi) were record and analyzed by flow cytometry. The number of each group is as follows: WT (n = 5) and KO (n = 8). Data are representative of three independent experiments. E)–G) The lumbar spinal cords were obtained from EAE mice between days 20 and 30 after immunization and immunofluorescent stained with monoclonal antibodies against IBA-1 and CD68 to assess the activation of microglia. The number of each group is as follows: WT (n = 7) and KO (n = 6). Data are representative of three independent experiments. H), I) Double immunostaining of Lamp 2 (green) and IBA-1 (red) showing microglia phagocytosis in the slices of lumbar spinal cords. The number of each group is as follows: WT (n = 7) and KO (n = 6). Data are representative of three independent experiments. The error bars represent the means ± SEM. Two-way ANOVA followed by Bonferroni’s post hoc test (B). Two-tailed unpaired Student's t test (D, F, G, and I). ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 40 μm in E) and H) and 4 μm in the magnification.

TSPO expression is known to occur early in myeloid cells activation and persists throughout the different stages of EAE (21). CNS-infiltrating macrophages also overexpress TSPO during neuroinflammation (20). We first investigated the number of microglia and macrophage at the peak of EAE disease. Flow cytometry indicated that the number of microglia was significantly reduced in KO mice, as well as the number of macrophages (Figure 4C and D). Furthermore, the activation of microglia and macrophage in KO mice was weakened (Figure 4E top and F). To confirm those results, we examined changes in major histocompatibility complex class II (MHC-II) and galectin-3 (Gal-3) proteins to investigate inflammatory myeloid cells (Figure S4A–D). In concordance with the above results, the expression of MHC-II and Gal-3 was lower in the lumbar spinal cord of KO mice than in WT mice (Figure S4A–D). At the same time, the expression of CD68 and lysosomal-associated membrane protein 2 (Lamp2), two specific markers of activated phagocytic microglia and macrophage, was down-regulated in KO mice (Figure 4E bottom, G–I). In our model, we observed that EAE was delayed onset in KO mice, further indicating that suppression of myeloid cells activation was correlated with neuroinflammation.

CYP7B1 role in EAE severity is dependent on myeloid cells

As CYP7B1 deficiency affects the activation of myeloid cells, we next investigated whether myeloid cells depletion could impact the development of EAE in KO mice. To this end, we first used flow cytometry to examine whether treatment with the colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397, resulted in depleting myeloid cells and attenuating neuroinflammation (22). For this experiment, WT mice and CYP7B1 KO mice were treated for 30 days with 40 mg/kg/day i.g. PLX3397 or phosphate buffered saline (PBS) vehicle control, with EAE induced at 14 days after the start of PLX3397 treatment. Following 21 days from the date of the PLX3397 intervention, brain and spinal cord tissue were obtained from the WT mice via post-anesthesia perfusion. After the extraction of individual cells, microglia (CD11b + CD45int) and macrophage (CD11b + CD45hi) in CNS were examined using flow cytometry (Figure 5A). The results showed that about 75% of microglia were depleted during a 21-day intervention with PLX3397 in comparison with normal mice, and macrophages exhibited minimal alteration (Figure 5B; Figure S5A).

Fig. 5. CYP7B1 role in EAE severity is dependent on myeloid cells. A) Schematics of microglia depletion and experimental design. Mice were treated with daily 40 mg/kg body weight CSF1R inhibitor, PLX3397, or PBS vehicle control for 14 days before EAE induction and continued until the end of experiments. Following 21 days from the date of the PLX3397 intervention, brain and spinal cord tissue were obtained from the WT mice via post-anesthesia perfusion. The proportion of microglia (CD11b + CD45int) and macrophage (CD11b + CD45hi) in CNS was measured by flow cytometry to detect the efficiency of PLX3397 in deleting myeloid cells. Mice were immunized with MOG/CFA/PT at day 14 and sacrificed between days 25 and 30 after immunization. Single cells were isolated from spleen, brain, and spinal cord. B) After PLX3397 treatment for 21 days, EAE mice were sacrificed and the percentage of microglia and macrophage in CNS was analyzed by flow cytometry. n = 4 per group. C) Effect of myeloid cell depletion on the development and progression of EAE. EAE clinical scores were recorded daily on a 0 to 5 scale. The number of each group is as follows: WT mice with PLX3397 treatment (n = 13), WT mice (n = 14), KO mice with PLX3397 treatment (n = 8), and KO mice (n = 14). Data are representative of three independent experiments. D), E) Single-cell suspensions were prepared from brain and spinal cord tissues of WT and KO mice at days 25 – 30 after immunization. Flow cytometry plots show gating of CD11b + CD45int, CD11b + CD45hi, and CD11b−CD45hi cell subsets which gated in living mononuclear cells. The bar graph shows the expression of indicated cell subsets in two groups. The number of each group is as follows: WT mice with PLX3397 treatment (n = 7) and KO mice with PLX3397 treatment (n = 6). Data are representative of three independent experiments. F), G) Mononuclear cells in the periphery and in the CNS were restimulated with MOG35–55 for 72 h. Expansion of CD4+ T cells was assessed by CFSE dilution. The number of each group is as follows: WT (n = 6) and KO (n = 6) in CNS or WT (n = 15) and KO (n = 15) in spleen. Data are representative of three independent experiments. H), I) Mononuclear cells were restimulated with MOG35–55 for 72 h and cultured with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of 5 μg/ml BFA for the last 5 h. Th1(CD4 + IFN-γ+) and Th17(CD4 + IL-17A+) cells were measured by intracellular staining. The number of each group is as follows: WT (n = 7) and KO (n = 7) in CNS or WT (n = 12) and KO (n = 12) in spleen. Data are representative of three independent experiments. Data are presented as means ± SEM. Two-tailed unpaired Student's t test (B, E–I). Two-way ANOVA followed by Bonferroni’s post hoc test (C). ns, no significant difference. *P < 0.05, **P < 0.01.

We next examined the impact of CYP7B1 on neuroinflammation in mice with myeloid cell deletion. We found that EAE onset was significantly delayed and disease severity was significantly alleviated in WT mice lacking myeloid cell (Figure 5C), which was in agreement with previous reports (23). However, CYP7B1 deficiency reversed this trend and only insignificant changes were observed in the clinical behavior of KO mice with or without myeloid cell deletion (Figure 5C). Furthermore, microglial and macrophage cell counts, as well as myeloid cell proliferation, were all significantly decreased in WT mice and KO mice treated with PLX3397 compared with them in EAE. We also found that the number of microglia and macrophages did not differ between WT and KO mice after PLX3397 intervention (Figure 5D and E), while the abundance of CNS-infiltrating lymphocytes was lower in KO mice after myeloid cell depletion than that in WT mice (Figure 5D and E). These data showed the efficacy of myeloid cell depletion using PLX3397 and that depleting myeloid cell can inhibit the development of EAE in WT mice, not in KO mice.

To further dissect CYP7B1 function in myeloid cells during inflammatory responses, we examined the effects of reactivating pathogenic CD4+ T cells in EAE mice treated with or without PLX3397. We found that the disparity in pathogenic CD4+ T-cell proliferation between WT mice and KO mice was abrogated by PLX3397 treatment, both in the CNS and in peripheral organs (Figure 5F and G; Figure S5B). Consistent with these results, MOG-reactive Th1 and Th17 cell levels were similar between WT and KO mice (Figure 5H and I; Figure S5C). Taken together, these data demonstrated that CYP7B1 function in suppressing neuroinflammation at least partially depends on myeloid cells.

Myeloid cell dysfunction induced by CYP7B1 deficiency can be treated with bone marrow transplantation

Bone marrow transplantation (BMT) is an excellent model for researching the function of myeloid cells because myeloid cells produced from bone marrow hematopoietic stem cells (HSC) are mostly dispersed in the bone marrow and CNS (24). To further demonstrate the effect of CYP7B1 deletion on myeloid cell activity, we acquired bone marrow cells from healthy adult KO and WT mice and separated and purified Lin−CD4−CD8− cells. We injected them into the adult KO and WT mice that were exposed to lethal dose irradiation through the tail vein. Standard eating for 7–8 weeks enabled the restoration of the immune system. The EAE model was built using MOG35–55 in conjunction with CFA and PT. Daily behavioral alterations were tracked and recorded, and peripheral immune organs were kept for the eventual detection of Th1 and Th17 cell subsets (Figure 6A). Compared to WT mice transfused with Lin−CD4−CD8− cells derived from WT, behavioral scores indicated that WT mice transfused with KO murine-derived cells were noticeably less sick (Figure 6B). The incidence and progression of EAE in KO mice were facilitated by the administration of WT murine-derived cells (Figure 6B). The results suggest that myeloid cells in KO mice have a diminished capacity.

Fig. 6. Myeloid cell dysfunction induced by CYP7B1 deficiency can be treated with BMT. Lin−CD4−CD8− cells were isolated from femur and tibia bone marrow of 6 – 8W healthy WT and KO mice. 2 × 106 Lin−CD4−CD8− cells were administered via intravenous injection into WT or KO mice that had undergone sublethal irradiation. EAE was induced in bone marrow chimeric mice by administering MOG35–55 in combination with CFA and PT. Flow cytometry analysis was performed on day 24 after transfer. A) The schematic diagram illustrates the experimental design. B) Clinical scores of bone marrow chimeric mice after EAE induced. The number of each group is as follows: WT → WT (n = 9), KO → WT (n = 8), WT → KO (n = 7), KO → KO (n = 8). Data are representative of two independent experiments. C), D) Bone marrow chimeric mice were anesthetized and euthanized at day 24 after induced EAE. After gathering the brain and spinal cord, a single-cell solution was created. The cells were stimulated with MOG35–55 for 72 h and cultured with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of 5 μg/ml BFA for the last 5 h. Th1(CD4 + IFN-γ+) and Th17(CD4 + IL-17A+) cells were quantified via intracellular staining. n = 6 per group. Data are representative of three independent experiments. Data are presented as means ± SEM. Two-way ANOVA followed by Bonferroni’s post hoc test (B). Two-tailed unpaired Student's t test (D). ns, no significant difference. *P < 0.05, **P < 0.01.

The brain and spinal cord tissues of mice were taken on day 24 after induction, and single-cell suspensions were prepared in order to quantify the quantity of Th 1 and Th17 cells in the CNS using flow cytometry. The findings demonstrated that, in comparison with the transfusion of WT murine-derived HSC, the number of Th1 cells and Th17 cells of WT mice dramatically decreased following the transfusion of KO murine-derived HSC (Figure 6C and D). When WT murine-derived HSC was infused into KO mice, the quantity of Th1 and Th17 cells rose noticeably in comparison with KO mice that received HSC from KO mice (Figure 6C and D). These findings imply that the CYP7B1 gene deletion may affect myeloid cell function, which may be partially restored by infusing healthy HSC.

The activation and phagocytosis of myeloid cells under CYP7B1 deficiency was impaired

We further investigated the action of CYP7B1 on myeloid cells. Microglia-like (iMG) cells, which can be induced from human peripheral blood cells, are useful for studying myeloid cells (25). Using fresh peripheral blood mononuclear cell (PBMC) of CYP7B1 mutation patients or healthy controls, we generated iMG cells through treatment with a cocktail of IL-34 (100 ng/ml) and GM-CSF (10 ng/ml), as previously described (25, 26).

Upon induction, iMG cells started to grow branches and ultimately displayed a typical morphology of widely nonactivated ramified microglia with a small cell soma and multiple branching processes, compared to the typical round morphology of untreated monocytes at day 0 (Figure 7A). Immunofluorescent staining of the transmembrane protein 119 (TMEM119), P2RY12, IBA-1, and CD163 was used to verify the specificity of iMG cells (Figure 7B). The results indicated that iMG cells mostly expressed microglia-specific markers, TMEM119 and P2RY12, as well as IBA-1 (Figure 7B). Innate immune cell activation can be measured by phagocytosis (27). Phagocytosis activity in iMG cells derived from CYP7B1 mutation patients was compared to iMG cells derived from healthy controls utilizing pHrodo-labeled E. coli particles. The quantity and phagocytic uptake of tagged E. coli particles per cell reduced in iMG cells derived from CYP7B1 mutation patients (Figure 7C and D; Figure S6A). Taken together, our findings indicate that a subpopulation of iMG cells derived from CYP7B1 mutation patients have defects in activation.

Fig. 7. The activation and phagocytosis of myeloid cells under CYP7B1 deficiency was impaired. PBMCs were isolated from fresh peripheral blood of CYP7B1 mutation patients or healthy controls within 2 h. PBMCs were cultured with RPMI-1640 Glutamax supplemented with 10 ng/ml recombinant human GM-CSF and 100 ng/ml recombinanthumanIL-34 for up to 14 days. A) Representative phase contrast images of iMG cells differentiated for 0, 7, and 14 days in culture. B) Immunofluorescence images of microglia-specific marker TMEM119, P2RY12, myeloid cells marker IBA1, and other cell types markers CD163 with counter-stain DAPI in iMG cultured on day 7 and day 14. C) At day 14, pHrodo-labelled E. coli particles were added in the culture conditions and incubated for 60 min. Representative images were showing the phagocytosis of iMG. D) The pHrodo-labelled E. coli particles uptake was quantified by flow cytometry. The number of each group is as follows: CYP7B1 mutation patients (n = 3) and healthy controls (n = 4). E) Cortices from P0-2 WT or KO mouse pups were dissected. A single-cell suspension was created following the removal of the meninges. Using two rounds of magnetic bead sorting, CD11b+ microglia were isolated from single-cell suspension and grown in Dulbecco's modified eagle medium (DMEM) complete culture media. Primary microglia were tested for morphology, activation, phagocytosis, and secretion following 10 ng/ml LPS stimulated in standard culture conditions for 24 h. Quantitative analysis of homeostatic microglia-specific marker TMEM119, activated microglia marker IBA1, CD68, Gal-3, Lamp2, MHC-II with counter-stain DAPI. Data are representative of three independent experiments. F) Cytokine secretion of primary microglia in response to LPS or not. n = 5 per group. Data are representative of three independent experiments. G) Primary microglia were incubated with sonicated fluorescent pHrodo-labelled E. coli particles for 2 h, and phagocytic activity was observed by fluorescent microscopy. H), I) The pHrodo-labelled E. coli particles uptake was quantified by flow cytometry. The number of each primary microglia group is as follows: WT (n = 5), WT after LPS stimulated (n = 8), KO (n = 7), and KO after LPS stimulated (n = 7). Data are representative of three independent experiments. Data are presented as means ± SEM. Two-tailed unpaired Student's t test (D and I). One-way ANOVA with multiple Tukey’s comparison tests (E and F). ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 40 μm in A–C and G and 1 μm in the magnification.

In the brain of EAE, antigen-specific T cells are restimulated by APCs, leading to disease induction and progression. While monocyte are professional APCs, microglia in the CNS are also important APCs. We extracted and cultured primary microglia from both WT and KO mice. Our studies found that lipopolysaccharide (LPS) altered the shape of microglia, transitioning them from a polarized baseline condition to an ameboid-like shape. Several cells exhibited bushy morphology and possess a greater number of branches (Figure S6B). However, in the cells derived from KO mice, less of the microglia were ameboid-like shape and bushy morphology, and most of them maintained polarized morphology (Figure S6B).

Apart from the evaluation of morphology, we also contrasted the reaction of primary microglia derived from WT and KO mice to stimulation by LPS. Unexpectedly, our findings revealed that primary microglia derived from KO mice displayed a less pro-inflammatory reaction to LPS stimulation compared to those from WT mice (Figure 7E; Figure S6B). We also analyzed the release of cytokines of microglia in response to LPS using a tailored multiple luminex assay. Primary microglia generated from WT mice displayed notably elevated secretion levels of pro-inflammatory cytokines, such as IL-1β and IL-6, when stimulated with 10 ng/ml LPS for 24 h, in comparison with the unstimulated control group (Figure 7F). However, primary microglia obtained from KO mice, when stimulated under the same condition, exhibited significantly reduced secretion of most pro-inflammatory cytokines compared to treated primary microglia derived from WT mice, although their secretion levels were marginally greater than those in untreated KO control group (Figure 7F). Subsequently, we investigated the phagocytosis activity in primary microglia obtained from KO mice and WT mice using E. coli particles tagged with pHrodo. In KO mice-derived primary microglia, phagocytic uptake of labeled E. coli particles per cell and the number of labeled E. coli particles in microglia both decreased, in comparison with the WT mice-derived primary microglia (Figure 7G–I). This agrees with the findings from the iMG experiment.

To summarize, our findings indicate that the deletion of the CYP7B1 gene impacts reactions and activation of myeloid cells in response to pathological environmental alterations.

Discussion

Cholesterol metabolism plays a crucial role in immune regulation, but the specific contribution of CYP7B1, a key enzyme in this pathway, to microglia-mediated neuroinflammation remains unclear. Our work provides initial evidence that CYP7B1 has an effect in the pathogenesis of neuroinflammation. We discovered that the homozygous CYP7B1 deficiency in EAE model mice delays disease onset and reduces severity. Interestingly, disease progression in CYP7B1 KO mice is not primarily due to impaired myeloid cells function but rather pathogenic alterations in MOG-specific T cells. Furthermore, two in vitro cell models, iMG cells derived from PBMC of CYP7B1 mutation patients and primary microglia derived from CYP7B1 KO mice, support the notion that CYP7B1 deficiency partly reduces neuroinflammation during EAE by affecting myeloid cells activation.

Cholesterol-metabolizing enzymes play a key role in regulating myeloid cell function within the CNS. Notably, the Ch25h gene, an upstream regulator of CYP7B1, is a characteristic gene of “disease-associated microglia (DAM)” (28, 29). Microglia, as key immune cells in the brain, actively clear cholesterol through phagocytosis. However, lipid accumulation within microglia can impair phagocytic function, leading to chronic activation. In animal models of AD, promoting lipid efflux in microglia via LXR agonists reduces reactive microglia activation, suppresses neuroinflammation, and mitigates Tau pathology (29, 30). These observations highlight the profound influence of cholesterol metabolism on modulating microglial function and emphasize its therapeutic potential in AD and other neurodegenerative diseases.

Previous research has shown that CYP7B1 insufficiency leads to the accumulation of oxysterols, particularly 25-OHC and 27-OHC. CYP7B1 deficiency causes congenital bile acid synthesis disorders in infants and hereditary SPG5 in adults, primarily due to impaired physiological processes. However, in pathological conditions, CYP7B1 deficiency may attenuate the development of inflammatory diseases. The pathophysiology of osteoarthritis was abolished when CYP7B1 knocked down (31). The CYP7B1 inhibitor clotrimazole demonstrates therapeutic efficacy in rheumatoid arthritis (29, 30). Our findings align with these studies, demonstrating that CYP7B1 deficiency significantly alleviates the severity of EAE. This indicates that further investigation is needed to explore the role of CYP7B1 in neuroinflammation and its potential therapeutic application in neuroinflammatory diseases.

Noninvasive imaging techniques are vital for assessing neuroimmune responses in neuroinflammatory diseases. Studies have explored various methods, such as reactive oxygen species (ROS), dihydroethidium, diffusion-weighted magnetic resonance imaging, positron emission tomography, and near-infrared (NIR) imaging techniques to identify neuroinflammation (32). NIR imaging offers distinct advantages including noninvasiveness, real-time feedback, high sensitivity, and spatial resolution (33). Several neuroinflammatory biomarkers such as sphingosine-1-phosphate receptor subtype 1 (Cx3Cr1), monoamine oxidase B, and soluble epoxide hydrolase are utilized to monitor microglial and astrocytic activity. The widely accepted myeloid marker TSPO serves as a standard for neuroinflammation assessment (34). Thus, we designed a TSPO-targeting fluorescent probe for monitoring neuroinflammation in EAE. Remarkably, CYP7B1 deficiency in EAE mice exhibited consistently lower fluorescence intensity compared to WT mice, suggesting a potential alleviation of neuroinflammation.

EAE/MS, primarily triggered by sensitized CD4+ T cells, is characterized by inflammation, myelin loss, and gliosis. IFN-γ produced by Th1 and Th17 cells in EAE stimulates Toll-like receptor-activated microglia, leading to neurotoxic phenotypes and neuronal dysfunction primarily mediated by ROS (35). The up-regulation of MHC-II is a crucial characteristic of microglial response to IFN-γ (36). Our research shows that CYP7B1 deficiency in EAE mice significantly reduces microglial inflammatory activation and diminishes MHC-II abundance and distribution in myeloid cells. This impediment hinders microglia's ability to efficiently present myelin autoantigens to effector T cells, inhibiting their activation, proliferation, and infiltration. Consequently, EAE symptoms are alleviated.

PLX3397, a CSF1R inhibitor, efficiently depletes microglia in the CNS when administered through diet (37, 38). Peripheral immune cells from lymphoid and nonlymphoid organs show minimal effects, except for the heart, and PLX3397 does not alter cytokine profiles in the brain or serum of naïve mice (39, 40). Furthermore, the other studies demonstrated that PLX3397-induced microglia reduction is a useful technique for understanding microglial functions without altering cognition or behavior (41, 42). Our research aligns with these observations, as we did not observe notable detrimental effects of PLX3397 in mice.

The BMT model emerges as an ideal platform for probing myeloid cell functionality (43). In this system, peripheral progenitors replace myeloid cells, potentially infiltrating the vulnerable CNS due to irradiation-induced effects. This phenomenon may stem from irradiation's direct impact on CNS-resident myeloid cells’ proliferation capacity and function (44). Traditional BMT using whole bone marrow cells is associated with high mortality due to low progenitor cell numbers. To overcome this, we developed a Lin−CD4−CD8− bone marrow cell-based BMT model, increasing progenitor cell proportions and reducing post-transplant immune responses, thereby improving survival rates (24). Notably, transplanting WT bone marrow-derived HSC into CYP7B1 KO mice exacerbated EAE rapidly, indicating that CYP7B1 deficiency impairs myeloid cell function.

Our work highlights the crucial involvement of the CYP7B1 gene in microglial phagocytic activity (43). CYP7B1 deficiency impairs this function regardless of inflammatory context. Microglia, as CNS phagocytes, maintain homeostasis by clearing cellular debris, dendritic spines, extracellular matrix, and pathogens. However, in pathology, they assume a dual role: protective through clearance of pathological protein aggregates, yet detrimental when excessive uptake compromises phagocytosis, leading to neuroinflammation and neurodegeneration. Evaluating the impact of CYP7B1 deficiency requires a nuanced perspective. Moreover, due to the design of the study, we were unable to distinguish the possible contribution of infiltrated monocytes and microglia. Future investigations utilizing conditional CYP7B1 KO in myeloid cells with the Cre-loxP system are necessary to validate these findings.

Taken together, our studies demonstrate the role of the CYP7B1 gene in the pathogenesis of neuroinflammation. However, additional research is required to analyze the subcellular mechanisms that elucidate the role of CYP7B1 in pathogenic cells, specifically in the activation of DAM, which is a significant outcome of lipid metabolism (45). If this is the case, our study suggests that identifying CYP7B1 action along the course of the disease (e.g. AD, amyotrophic lateral sclerosis, MS) could shed light on the molecular mechanisms of DAM regulation and potentially suggest new therapeutic targets.

Material and methods

Detailed descriptions for all procedures are available in SI Appendix.

Animals

CYP7B1−/− KO mice were purchased from Cyagen Biosciences Inc. (Guangzhou, China). Mice were kept under pathogen-free conditions, with limited access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Fujian Medical University (IACUC FJMU 2022-0833) and conducted in accordance with the US Public Health Service's policy on the Humane Care and Use of Laboratory Animals. Investigators involved in pathological staining and analysis were blinded to the experimental groups during the experiments. Additional details are provided in Supplementary material.

Generation of iMG cells from PBMCs

We performed the process of distinguishing and cultivating iMG cells as previously described (46, 47). PBMCs were resuspended with RPMI-1640 medium (Gibco Laboratories, Grand Island, NY, USA) contained 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA). PBMCs were plated into 24-well plates at a density of 5 × 105 cells/mL. After 24-h incubation, culture supernatant and nonadherent cells were removed. For differentiation of PBMCs into iMG, IL-34 and GM-CSF were added to each well, and medium changes were performed every 3 days. iMG were harvested on day 14 for downstream experiments. Additional details are provided in Supplementary material.

Active EAE induction

Female mice (aged 8 weeks) were subcutaneously immunized with an emulsion containing 200 μg myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) (Genescript, Piscataway, NJ, USA) and 5 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI, USA). Pertussis toxin (List Biologic, Campbell, CA, USA) was injected intraperitoneally on day 0 and 2 post-immunization. Clinical EAE manifestations were evaluated daily from day 0 to 5. Additional details are provided in Supplementary material.

Bone marrow transplantation

Lin−CD4−CD8− bone marrow cells were isolated from healthy donor CYP7B1 KO and WT mice using a mouse hematopoietic progenitor cell isolation kit. The cells were collected, and recipient mice were irradiated before transplantation. After 4 h, the mice were given 2 × 106 Lin−CD4−CD8− cells via tail vein injection. After injection, the mice were fed antibiotics-containing water for 8 weeks. BMT mice were used to induce active EAE at 8 weeks after transplantation. Further experimental and instrument details are provided in Supplementary material.

Flow cytometric analysis

Human PBMCs were isolated and stained with fluorochrome-conjugated monoclonal antibodies to evaluate CD3, CD4, CD8, and CD19 counts and phenotype. Immune cells from EAE mice were incubated with antibodies to CD3, CD4, CD8, CD19, and NK1.1. Intracellular cytokine staining was performed on cells plated in 24-well plates and stimulated with PMA, ionomycin, and Brefeldin A (BFA). MOG-specific Th cells were also analyzed. Flow cytometry analysis was performed on CytoFLEX S. Further experimental and instrument details are provided in Supplementary material.

In vivo imaging systems optical imaging

In order to realize in vivo monitoring of brain tissue inflammation, we designed and synthesized a fluorescent small molecule probe target to TSPO. In brief, 0.4 mg/kg PEG3-CY5-DPA714 was administered to anesthetized mice through lateral tail vein injection. Bioluminescence images in live mice were collected by using an in vivo imaging systems (IVIS) Lumina X5 Spectrum System (PerkinElmer, Waltham, MA, USA) at several time points. A region of interest (ROI) tool was used to measure the fluorescent intensity. Data were collected as photons/s/cm2 using the Living Image software (Caliper Life Sciences, Hopkinton, MA, USA). Additional details are provided in Supplementary material.

Immunofluorescence

For immunofluorescent staining, lumbar spinal cords were dehydrated and embedded in optimal cutting temperature compound (Tissue-Tek, Sakura Finetek, Japan). Transverse sections of spinal cord were cut into 7 μm sections, and immunohistochemistry was performed using various antibodies. Sections were fixed, washed, and blocked with 5% BSA. Primary antibodies were used against various proteins, followed by species-specific secondary antibodies. Images were obtained using confocal microscopy and quantified by two independent observers in a blinded manner. Additional details are provided in Supplementary material.

Phagocytosis

Phagocytosis was examined by fluorescent microscopy according to the manufacturer's protocol. Briefly, 10 ng/ml LPS was incubated with primary microglia for 24 h, followed by adding sonicated pHrodo-labelled E. coli particles (Thermo Fisher, USA). The cells were then incubated for 60 min, washed three times, and stained with DAPI. Each well was analyzed by using a fluorescence microscope (Invitrogen). The uptake of pHrodo--labelled E. coli particles was quantified by flow cytometry. All parameters were kept constant throughout all experiments. Further experimental details are provided in Supplementary material.

Administration of PLX3397

PLX3397 (Abmole, CA, USA) was dissolved in 5% dimethyl sulfoxide, 25% polyethylene glycol 300 (PEG300) followed by dilution with PBS. As previously described (48), mice were treated daily with vehicle PBS or PLX3397 (40 mg/kg) by oral gavage for 14 days before EAE induction. The treatment was continued until the end of experiments.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 software (La Jolla, CA, USA). Investigators involved in pathological staining and analysis were blinded to the experimental groups during the experiments. The values are presented as the mean ± standard error of mean (SEM). For the EAE clinical score, two-way ANOVA followed by Bonferroni’s post hoc test were used. When two groups were compared, two-tailed unpaired Student's t test or Mann–Whitney test was used. One-way ANOVA with multiple Tukey’s comparison tests was performed to compare multiple groups. A significance criterion of P < 0.05 was used for all statistical analysis.

Supplementary Material

pgae334_Supplementary_Data

Supplementary Material

Supplementary material is available at PNAS Nexus online.

Funding

This study was supported by grants (U2005201, U21A20360, and 82371349) from the National Natural Science Foundation of China, Joint Funds for the Innovation of Science and Technology of Fujian Province (no. 2020Y9129), and Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (no. 2022ZQNZD005).

Author Contributions

Y.F., N.W., and H.S. conceived the project. Y.F. and N.W. secured funding. Y.F. and H.S. designed the experiments. A.L., R.L., Z.Z., J.J., Y.Y., and C.Z. collected the data. H.S., A.L., Q.Z., X.Y., and X.L. carried out the experiments. H.S. and A.L. analyzed the data. Y.F., H.S., and A.L. wrote the manuscript. Y.F., N.W., R.L., and W.C. edited the manuscript.

Data Availability

All data supporting the findings of this study are included in the main text.
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