PON1 is expressed in both human and rat brain tissues and rat primary microglia.
Given the important function of PON1 in macrophages (31), we proposed that PON1 might play a role in brain macrophages, microglia. PON1 mRNA was detected in both human and rat brain tissues and the level in rat liver was approximately 3.8 times higher than in rat brain (Fig. 1A, B). The absence of PON1 protein in PON1 KO rats was confirmed by western blot of total proteins from brain tissues and primary microglia (Fig. 1C, D). PON1 was also detected in HM1900 and BV2 microglia cell lines (Fig. S1). To identify the location of PON1 protein in microglia, we stained brain tissue sections from WT rats at two months of age. Immunofluorescence analysis indicated that PON1 protein was primarily localized in neurons, but also expressed in a small number of Iba1-positive microglia (Fig. 1E). Unlike the results from brain tissue sections, immunofluorescence staining of primary microglia from neonatal rats showed PON1 co-localized with Iba1 in almost all microglia. PON1 immunostaining was not observed in microglia from PON1-/- rat (Fig. 1F). PON1 also co-localized with MAP2 in cultured neurons and with GFAP in cultured astrocytes (Fig. 1G).
PON1 deficiency reduces LPS lethality
Exposure to bacterial lipopolysaccharide (LPS) induces tumor necrosis factor (TNF-α) production and causes death in rodents (32-34). Intravenous injection of 20 mg/kg LPS into WT rats resulted in death of 100% (5/5, female) of the rats within 48 h after injection. In contrast, only 40% of the PON1-/- (2/5, female) rats were dead within 48 h at the same dose. Intravenous injection of 5 mg/kg LPS into WT rats resulted in death of 26.7% (4/15) of the rats within 48 h. In contrast, just 6.7% (1/15) of PON1-/- rats was dead within 48 h after injection of 5 mg/kg LPS. This significant (p<0.05) difference in LPS mortality demonstrates that PON1 deficiency protects against LPS-induced death (Fig. 2A). The appearance of WT and PON1-/- rats at 1 h and 6 h after injection of 5 mg/kg LPS is shown in Fig. 2B.
TNF-α is considered to be the cytokine responsible for LPS lethality. Inhibition of the LPS-induced serum levels of TNF-α by the absence of PON1 is expected to make rats less susceptible to LPS. The level of TNF-α in the peripheral blood of PON1-/- rats (n = 4) and WT rats (n = 4) was determined up to 3 h after injection of a lethal dose of LPS (20 mg/kg) and the results indicated that both WT and KO rats showed an increase in TNF-α starting at 30 min after injection, peaking at 90 min and declining between 120 and 180 min (Fig. 2C). TNF-α levels in PON1-/- rats declined significantly faster than in WT rats (p <0.01, two way ANOVA), indicating that the absence of PON1 protects against the toxic effects of LPS in rats. An ELISA was also performed and confirmed that the absence of PON1 decreased TNF-α (Fig. 2D, p <0.05，n = 4/group) and increased the release of IL-10 (Fig. 2E, p <0.05, n = 4/group) in serum at 6 h after injection of 5 mg/kg LPS.
PON1 deficiency inhibits LPS-induced microglial activation in vivo
PON1 inhibits monocyte-to-macrophage differentiation and PON1 KO caused obvious morphological changes of macrophages in mice (35). The results of Iba1 immunohistochemistry showed that the microglia in brain sections from PON1-/- rats were larger (Fig. 3A-C) and had fewer and shorter branches (Fig. 3D, E) than those from WT rats without LPS treatment. The microglia from WT rats with LPS treatment showed M1-like activation and polarization with amoeboid shape with no long branches (Fig. 3A, B). In contrast, the microglia from PON1-/- rats still had large cell bodies with several longer branches than those from WT rats after LPS stimulation.
PON1 deficiency in rat microglia decreased LPS-induced cytokine levels
The phenotypes of primary microglia were further analyzed by immunofluorescence staining and cytokine measurement. The microglial cells from PON1-/- rats were larger than those from WT rats and LPS treatment decreased the cell size in WT rats but not those in PON1-/- rats (Fig. 4A). To determine whether PON1 KO altered cytokine release from microglia, we quantified the levels of IL-1β, IL-6, IL-12, IL-18, TNF-α (M1-phenotype markers) and IL-10 (M2-phenotype marker) in cell supernatants following LPS administration (Fig. 4B). IL-1β, IL-6, and TNF-α were all significantly decreased (48.4%, 17.1% and 26.6% respectively) in PON1-/- microglia compared to WT. A significant 4.5-fold increase of IL-10 was observed in PON1-/- microglia compared to WT. The increase in LPS-induced M1-phenotype markers, IL-12, IL-18 and TNF-α was smaller in PON1-/- microglia than in WT; although IL-1β, IL-6, IL-12, IL-18, TNF-α, and IL-10 were all increased in WT and PON1-/- microglia after LPS. The increase in LPS-induced IL-10 (M2-phenotype marker) in PON1-/- microglia was larger than in WT. Additional seven cytokines of IL-4, IL-5, IL-7, IL-12, MCP-1, VEGF and G-CSF showed no significant difference between the two groups (Fig.S3). IL-4VInducible nitric oxide synthase (iNOS), a key inflammatory mediator, was also significantly increased in WT and PON1-/- microglia after LPS stimulation; but the increase of iNOS in PON1-/- microglia was smaller (Fig.4C, D). In accord with the cytokine results, the increase in P-ERK/ERK in PON1-/- microglia was less than in WT (Fig.4E), which might explain the differences in cytokine release. These data also suggest that PON1 KO promotes M2-Like polarization in rat microglia.
PON1 deficiency enhances phagocytosis in rat primary microglia
To determine whether PON1 deficiency affected phagocytosis, we measured the internalization of fluorescently labeled Aβ peptide, previously reported to be phagocytosed by microglia (36). The results demonstrated that PON1 depletion significantly enhanced intracellular levels of fluorescent Aβ (Fig. 5A, B). Consistent with the enhanced uptake, we found a similar effect using fluorescently labeled dextran (Fig. 5C, D), which targets phagocytosed compounds. These results show that PON1 depletion in microglia increased the overall phagocytic activity. A 24 h treatment with 200 ng/ml LPS inhibited the phagocytic ability of WT and PON1-depleted microglia (Fig. 5A-D). Addition of human recombinant PON1 protein to WT and PON1-/- microglia inhibited the phagocytic ability of both (Fig. 5E, F).
PON1 deficiency and LPS treatment changed the transcriptomic profiles in rat primary microglia
Twelve microglia samples treated with or without LPS (200 ng/ml) were subjected to RNA-seq. Differential expression analysis demonstrated that PON1 knockout down-regulated a number of genes associated with nervous system development, cell-cell signaling, glial cell differentiation and gliogenesis, while only two genes associated with nervous system development were up-regulated (Fig. 6A, B, Table 1). However, after LPS treatment, PON1 knockout microglia showed an increase in down-regulated and up-regulated genes associated with nervous system development and regulation of ion transport (Fig. 6 A, C, Table 1). LPS treatment resulted in similar changes in gene profile in PON1 KO and WT rats (Fig. 6D-G, fold change >2, p <0.01). The genes with the most significant variation were classified in four categories by GO analysis. The two categories of up-regulated genes induced by LPS treatment were associated with the defense response, the response to lipopolysaccharide, the response to molecule of bacterial origin, immune system processes and multi-organism processes (Fig. 6D, E, p <0.01). Additional categories of down-regulated genes induced by LPS treatment were associated with immune system processes, the dynein complex, leukocyte differentiation, extracellular matrix, chromosome segregation and the proteinaceous extracellular matrix (Fig. 6F, G, p <0.01). These results demonstrate that PON1 KO changed the transcriptome profiles of microglia, which might be partly responsible for the phenotypic changes of microglia from PON1 KO rats.
PON1 deficiency up-regulated TREM2 signal in rat primary microglia
To investigate the mechanism underlying PON1-mediated phagocytosis and cytokine release, we analyzed the protein expression of genes that are known to be involved in LPS response and phagocytosis (Fig.7, Fig.S4, Fig.S5). Triggering receptor expressed on myeloid cells 2 (TREM2) is a receptor expressed in microglia. The TREM2/DAP12 signaling pathway reduces inflammatory responses and promotes phagocytosis. As expected, TREM2 (Fig. 7A, B) and the phosphorylated tyrosine kinase, P-Syk, (Fig. 7A, C) were significantly increased in PON1-/- microglia compared to WT, but decreased with LPS treatment of both WT and PON1-/- microglia, similar to the results of the phagocytosis assay. Expression of the guanine nucleotide exchange factors, Vav2 (Fig.7A, D) and Vav3 (Fig.7A, E), and actin-related protein, Arp2 (Fig.7A, F), were increased in PON1-/- microglia compared to WT, but were not affected by LPS. The data suggested that PON1 deficiency enhanced phagocytosis by activation of the TREM2 signaling pathway and up-regulation of the actin cytoskeleton. As shown above, TREM2 protein levels were significantly increased in PON1-/- microglia compared to WT (Fig. 7A, B) even though TREM2 mRNA was not altered by PON1 deficiency (Fig. 7G). However, when human PON1 recombinant protein was added to PON1-/- microglia, the protein level of TREM2 and P-DAP12 was decreased dependent on the PON1 protein concentration (Fig. 7H-J).
PON1 protein interacts with TREM2 and promotes its lysosome localization in microglial cells
Co-immunoprecipitation (co-IP) was employed to identify the mechanism of TREM2 regulation by PON1. The results confirmed that there was a direct interaction between endogenous PON1 and TREM2 proteins in rat brain tissues (Fig. 8A, B) and between over-expressed human PON1 and TREM2 proteins in BV2 cells (Fig. 8C, D). Immunofluorescence assays verified the co-localization of TREM2 and the lysosomal marker LAMP1 in primary microglia (Fig. 8E). PON1-/- microglia showed a highly increased, clustered distribution of TREM2 on the cell surface compared to WT. After administration of recombinant human PON1 protein, the distribution of TREM2 decreased but there was increased co-localization of TREM2 with LAMP1 in WT and PON1-/- microglia. APOE is a novel ligand for TREM2 (37), and we used DSS crosslinking to test whether the binding of APOE and TREM2 was disrupted by PON1. The crosslinking between APOE and TREM2 was increased in PON1-/- rat brain tissues relative to WT (Fig. 8F-H), suggesting that PON1 might compete with APOE for binding to the TREM2 receptor on microglia.
Taken together, the results suggested that PON1 interacts with TREM2 and promotes its internalization and degradation in lysosomes. Conversely, PON1 deficiency increases TREM2 and causes clustering on the surface of the microglial cells (Fig. 8I), which might promote TREM2 binding to APOE and phagocytosis.
PON1 deficiency facilitated microglial Aβ42 clearance in vivo
We investigated the effects of PON1 knockout or not on microglial phagocytosis or Aβ clearance in a rat model of AD produced by an intrahippocampal injection of amyloid-β1-42 (Aβ1-42) (Fig. 9A). The results indicated that PON1 knockout enhanced the clearance of Aβ1 –42 (Fig. 9B, C). Aβ1 –42 injected into the hippocampus of WT and PON1-/- rats induced microglial activation, proliferation, accumulation and phagocytosis, and the amount of Aβ1 –42 then gradually decreased along with the time increase (Fig. s6 and Fig. 9D, E). PON1 knockout significantly increased the number of microglia cells around the injection sites and Aβ1 –42 uptake of single microglia cell, finally reduced the deposit of Aβ42 inside hippocampus neurons (Fig. s6 and Fig. 9F, G). On another hand, PON1 knockout decreased Aβ-induced increase of cytokines including IL-1β, TNF-α, iNOS, IL-10 and IL-6，and increased the expression of Mrc-1, one receptor mediated endocytosis (Fig. 9H). In contrast with LPS, Aβ1 –42 injection increased the transcription of TREM2 in both WT and PON1-/- rats along with the time increase, and the protein level of TREM2 in PON1-/- rats was still higher than WT rats (14 days post-injection) (Fig. s7). These results suggest that PON1 knockout and increased TREM2 protein level may facilitate microglial clearance of exogenic Aβ in rat brains.