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Pon1 Deficiency Promotes Trem2 Pathway–Mediated Microglial Phagocytosis and Inhibits Pro-inflammatory Cytokines Release In Vitro and In Vivo

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Abstract

Paraoxonase 1 (PON1) plays an anti-inflammatory role in the cardiovascular system. Levels of serum PON1 and polymorphisms in this gene were linked to Alzheimer’s disease (AD) and Parkinson disease (PD), but its function in the neuroimmune system and AD is not clear. To address this issue, we used Pon1 knockout rats previously generated by our lab to investigate the role of Pon1 in microglia. Knockout of Pon1 in rat brain tissues protected against LPS-induced microglia activation. Pon1 deficiency in rat primary microglia increased Trem2 (triggering receptor expressed in myeloid cells 2) expression, phagocytosis, and IL-10 (M2-phenotype marker) release, but decreased production of pro-inflammatory cytokines such as IL-1β, IL-6, and IL-18 especially TNF-α (M1-phenotype markers) induced by LPS. Pon1 deficiency in rat primary microglia activated Trem2 pathway but decreased LPS-induced ERK activation. The phagocytosis-promoting effect of Pon1 knockout could be reversed by administration of recombinant PON1 protein. The interaction between PON1 and TREM2 was verified by co-immunoprecipitation (co-IP) using rat brain tissues or over-expressed BV2 cell lysates, which might be involved in lysosomal localization of TREM2. Furthermore, Pon1 knockout also enhanced microglial phagocytosis and clearance of exogenous Aβ by an intrahippocampal injection and decrease the transcription of cytokines such as IL-1β, IL-6, and TNF-α in vivo. These results suggest that Pon1 knockout facilitates microglial phagocytosis and inhibits the production of proinflammatory cytokines both in vivo and in vitro, in which the interaction between Pon1 and Trem2 may be involved. These findings provide novel insights into the role of PON1 in neuroinflammation and highlight TREM2 as a potential target for Alzheimer’s disease therapy.

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Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Aβ:

Amyloid β

PON1:

Paraoxonase 1

TREM2:

Triggering receptor expressed in myeloid cells 2

LPS:

Lipopolysaccharide

APOE:

Apolipoprotein E

TNF-α:

Tumor necrosis factor-α

IL-1β:

Interleukin-1 β

IL-6:

Interleukin-6

IL-12:

Interleukin-12

IL-18:

Interleukin-18

IL-10:

Interleukin-10

iNOS:

Inducible nitric oxide synthase

co-IP:

Co-immunoprecipitation

KO:

Knockout

SDS-PAGE:

Dodecyl sulfate, sodium salt -polyacrylamide gel electrophoresis

HRP:

Horseradish peroxidase

RT-PCR:

Reverse transcription-polymerase chain reaction

DSS:

Disuccinimidyl suberate

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Funding

The present work was supported by the National Science Foundation of China (31900380, 31970508), CAMS Innovation Fund for Medical Sciences (CIFMS, 2021-I2M-1–034), and Beijing Municipal Natural Science Foundation (7172135).

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Authors and Affiliations

  1. Beijing Engineering Research Center for Experimental Animal Models of Human, Diseases, Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, 100021, China

    Li Zhang, Yuanwu Ma, Lin Bai & Xu Zhang

  2. Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, 100021, China

    Wei Dong, Caixian Sun, Jingwen Li & Lianfeng Zhang

  3. Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, 100730, China

    Lianfeng Zhang

  4. Building 5, Panjiayuan Nanli, Chaoyang District, Beijing, 100021, China

    Lianfeng Zhang

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  1. Li Zhang
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Contributions

LFZ and LZ designed the study and wrote the paper, LZ, WD, YWM, LB, CXS, XZ, and JWL performed the experiments. All the authors have read and approved the final manuscript.

Corresponding author

Correspondence to Lianfeng Zhang.

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The experimental protocol was approved by the Animal Care and Use Committees of the Institute of Laboratory Animal Science of Peking Union Medical College.

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All authors read and approved the final manuscript.

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The authors declare no competing interests.

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Supplementary file1 (PDF 2970 KB) Figure S1. Expression of PON1 protein in microglial cell lines. The expression of PON1 protein in human microglia HM1900, mouse microglia BV2 and rat primary microglia was determined by western blot. GAPDH was used as loading control. MG, microglia. Figure S2. The identification of rat primary microglia cells. Staining of Iba1 (red) and Cd11b (green) was performed and immunofluorescence was observed by confocal microscopy (magnification ×630, scale bar = 50 μm). Figure S3. Cytokines regulated by Pon1 knockout in primary rat microglia. For IL-7 (A), IL-12 (B), MCP-1 (C), VEGF (D) and G-CSF (E), there was no significant difference between WT and Pon1-/- or between WT LPS and Pon1-/- LPS groups, (n = 3 per group), although all the five cytokines were significantly increased with LPS treatment. * p<0.05, ** p <0.01, *** p<0.001 indicate significance; NS, no significance. Figure S4. Effects of Pon1 KO on p38 and JNK signaling pathways. (A) Total protein lysates of WT, Pon1-/-, WT LPS and Pon1-/- LPS microglia were prepared and the levels of p-p38, p-JNK, t-p38 and t-JNK were determined by western blot (n = 3 per group). The relative protein expression of p-p38 (B) and p-JNK(C) were normalized to t-p38 and t-JNK. Values are expressed as mean ± S.D. NS indicate no significance. Figure S5. RNA sequencing and nervous system development enriched differentially expressed genes in rat primary microglia. Nervous system development (GO:0007399) enriched differentially expressed genes (fold change >2, p < 0.05) between WT and Pon1-/- microglia (A) or between WT LPS and Pon1-/-LPS microglia (B) were highlighted. * p<0.05 and ** p<0.01 indicate significance. GO enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package and GO terms with corrected P value less than 0.05 were considered significantly. Figure S6. RNA sequencing and GO enrichment analysis in rat primary microglia. Differentially expressed genes were classified into four categories using the SOTA function in the clValid package. Among them, two categories of genes were significantly up-regulated (A and B) and two categories were significantly down-regulated (C and D) in LPS-treated WT and Pon1-/- microglia compared to controls. The top Gene Ontology (GO) terms, corresponding to enrichment p values and gene numbers are shown on the right side. Figure S7. Effect of PON1 KO on LPS/TLR4/NFκB signaling pathway. (A) Total protein lysates of WT, Pon1-/-, WT LPS and Pon1-/- LPS microglia were prepared and the level of TLR4 was determined by western blot. The nuclear and cytoplasmic proteins of WT, Pon1-/-, WT LPS and Pon1-/- LPS microglia were extracted and the level of P65 was detected by western blot (n = 3 per group). Nucleolin and GAPDH were used as the markers of nucleus and cytosol, respectively. The relative protein expression of TLR4 (B), nucleus (C) and cytosol P65 (D) were normalized GAPDH, nucleolin and GAPDH, respectively. Values are expressed as mean ± S.D. NS indicate no significance. *** p<0.001 indicate significance. Figure S8. The microglia cells and phagocytosis 24h post-injection. Representative images (A) of Aβ oligomers (red) and quantitation analysis of microglia cells (B) at the injection sites of WT and Pon1-/- rat brains (n= 5 WT, n= 4 KO) 24h post-injection. DAPI (blue) stained the nucleus. Scale bar, 750 or 250 or 50 μm; A yellow circles indicate microglia hyperplastic focus. Representative confocal images of microglia cells labelled with Iba1 (green) merged with Aβ (red) at the injection sites of WT and Pon1-/- rats 24h post-injection (C). (WT, ①-⑧; KO, ①’-⑧’ ). Scale bar=25μm. * p<0.05 indicate significance. ① indicates resting microglia cells with long branches, ②’ indicates phagocytizing microglia cells with open mouth, others were microglia cells with uptake of Aβ. Figure S9. Aβ injection and Trem2 expression. The transcription of TREM2 mRNA in the hippocampus from WT and Pon1-/- rats 0-, 1- and 3- day post-injection was detected using real time PCR (A). n= 5 for WT and Pon1-/- rats 0-day post-injection; n= 4 for WT and Pon1-/- rats 1-day and 3-day post-injection. * p<0.05 indicate significance, WT versus Pon1-/-rats 3-day post-injection. The expression of TREM2 proteins in the hippocampus from WT and Pon1-/- rats 14- day post-injection was detected using western blot (B). n=3- 4 for WT and Pon1-/- rats 14-day post-injection. Figure S10. A preliminary model of the proposed mechanism of PON1 action in microglia.

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Zhang, L., Dong, W., Ma, Y. et al. Pon1 Deficiency Promotes Trem2 Pathway–Mediated Microglial Phagocytosis and Inhibits Pro-inflammatory Cytokines Release In Vitro and In Vivo. Mol Neurobiol 59, 4612–4629 (2022). https://doi.org/10.1007/s12035-022-02827-1

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