Therapeutic Effect of Modulating TREM-1 via Anti-inflammation and Autophagy in Parkinson's Disease

Abstract

Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases, and neuroinflammation has been identified as one of its key pathological characteristics. Triggering receptors expressed on myeloid cells-1 (TREM-1) amplify the inflammatory response and play a role in sepsis and cancer. Recent studies have demonstrated that the attenuation of TREM-1 activity produces cytoprotective and anti-inflammatory effects in macrophages. However, no study has examined the role of TREM-1 in neurodegeneration. We showed that LP17, a synthetic peptide blocker of TREM-1, significantly inhibited the lipopolysaccharide (LPS)-induced upregulation of proinflammatory cascades of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, and nuclear factor-kappa B. Moreover, LP17 enhanced the LPS-induced upregulation of autophagy-related proteins such as light chain-3 and histone deacetylase-6. We also knocked down TREM-1 expression in a BV2 cell model to further confirm the role of TREM-1. LP17 inhibited 6-hydroxydopamine-induced locomotor deficit and iNOS messenger RNA expression in zebrafish. We also observed therapeutic effects of LP17 administration in 6-hydroxydopamine-induced PD syndrome using a rat model. These data suggest that the attenuation of TREM-1 could ameliorate neuroinflammatory responses in PD and that this neuroprotective effect might occur via the activation of autophagy and anti-inflammatory pathways.

Keywords: Parkinson’s disease; TREM-1; autophagy; neuroinflammation; rat; zebrafish.

FIGURE 1

The treatment of LP17 for 16 h attenuated LPS-induced upregulation of TREM-1 mRNA and protein expressions in BV2 murine microglial cell line (n = 4/group). As shown on (A) quantitative PCR, (B) at the protein level by Western blotting. Co-treatment with 10 μg/ml LP17 significantly attenuated the LPS-induced upregulation of TREM-1 in mRNA and protein level. The bars represent the mean ± SEM. The data were analyzed using two-way ANOVA. ^∗∗P < 0.05; ^∗∗∗P < 0.001 versus the control group; ^###P < 0.001 versus the LPS group.

FIGURE 2

The treatment of LP17 inhibited LPS-induced upregulation of expressions of proinflammatory cytokines iNOS, COX-2, IκB, and NF-κB protein in LPS-stimulated BV-2 murine microglial cell line (n = 4/group). (A) Western blotting for iNOS, COX-2 expression after treatment of BV-2 microglial cells with 0.01, 0.1, 1, and 10 μg/ml LP17 plus 100 ng/ml LPS for 16 h, showed that 10 μg/ml LP17 significantly inhibited LPS-induced increase in iNOS and COX-2 protein expressions. (B) Western blotting for IκB, and nuclear NF-κB after treatment of BV-2 microglial cells with 10 μg/ml LP17 plus 100 ng/ml LPS for 6 h, showed that LP17 significantly reversed the LPS-induced downregulation of IκB protein expression and significantly attenuated the LPS-induced upregulation of nuclear NF-κB protein expression. The bars represent the mean ± SEM. The data were analyzed using two-way ANOVA. ^∗∗P < 0.05; ^∗∗∗P < 0.001 versus the control group; ^##P < 0.05, ^###P < 0.001 versus the LPS group.

FIGURE 3

Treatment with LP17 enhanced LC3-II/LC3-I, HDAC6, and p-mTOR expression and inhibited p-ERK and p-Akt protein expression in an LPS-stimulated murine BV-2 microglial cell line (n = 4/group). (A) Quantitative PCR for LC3A and LC3B in the control, LPS, LPS plus LP17, and LP17-only groups after BV-2 microglial cells were treated with 10 μg/ml LP17 plus 100 ng/ml LPS for 6 h. Neither LP17 nor LPS affected LC3A and LC3B mRNA expression. (B) Western blot analysis for LC3, HDAC6, and p-mTOR expression in the control, LPS, LPS plus LP17, and LP17-only groups after the treatment of BV-2 microglial cells with 10 μg/ml LP17 plus 100 ng/ml LPS for 16, 16, and 2 h, respectively. LP17 significantly enhanced the LPS-induced increase in the LC-3-II/LC-3-I ratio, attenuated the LPS-induced down-regulation of p-mTOR, and enhanced the LPS-induced upregulation of HDAC6 protein expression. (C) Western blot analysis for LC3-II/LC3-I ratio in the control, LPS, LPS plus LP17, and LPS plus LP17 + pretreatment of bafilomycin groups after BV-2 microglial cells were treated with 10 μg/ml LP17, 100 ng/ml LPS plus 5 nM bafilomycin for 16 h. Pretreatment with bafilomycin for 1 h significantly inhibited the LP17-induced increase of the LC3-II/LC3-I ratio. (D) Western blot analysis for p-Akt and p-ERK after the treatment of BV-2 microglial cells with 10 μg/ml LP17 plus 100 ng/ml LPS for 16 h. The results showed that LP17 significantly inhibited the LPS-induced upregulation of the p-Akt and p-ERK proteins. The bars represent means ± SEM. The data were analyzed using two-way ANOVA. ^∗∗P < 0.05; ^∗∗∗P < 0.001 versus the control group; ^##P < 0.05 versus the LPS group; ⁺⁺P < 0.05 versus the LPS plus LP17 group.

FIGURE 4

TREM-1 knockdown in BV2 cells decreased TREM-1 expression levels and the LPS-induced upregulation of iNOS, COX-2, and IκB expression and increased HDAC6 expression and the LC3-II/LC3-I ratio. (A) Western blot analysis of TREM-1 expression in the control siRNA and TREM-1 siRNA groups for 7 h (n = 4/group). The results demonstrate that the transfection of TREM-1 siRNA significantly attenuated the expression of TREM-1. (B) Western blot analysis for iNOS, COX-2, and IκB expression in TREM-1 knockdown BV2 cells in the control (control siRNA), LPS-treated (control siRNA), control (TREM-1 siRNA), and LPS-treated (TREM-1 siRNA) groups after treatment with 100 ng/ml LPS for 16 h (n = 4/group). The knockdown of TREM-1 in BV2 cells attenuated the LPS-induced upregulation of iNOS and COX-2 and reversed the LPS-induced downregulation of IκB expression. (C) Western blot analysis for HDAC-6 expression and LC3-II/LC3-I ratio in TREM-1 knockdown BV2 cells in the control (control siRNA), LPS-treated (control siRNA), control (TREM-1 siRNA), and LPS-treated (TREM-1 siRNA) groups after treatment with 100 ng/ml LPS for 16 h (n = 4/group). Knockdown of TREM-1 also enhanced the LPS-induced upregulation of HDAC6 and increased the LC3-II/LC3-I ratio. “–” represents control siRNA and “+” represents TREM-1 siRNA. The bars represent means ± SEM. ^∗∗∗P < 0.001 versus the control group (TREM-1 siRNA); ^##P < 0.05 versus the LPS group (control siRNA); ^###P < 0.001 versus the LPS group (control siRNA).

FIGURE 5

The treatment of TREM-1 agonist antibody reduced the anti-inflammatory effects of LP17 on iNOS and COX-2 expressions in LPS-stimulated murine BV-2 microglial cell line (n = 4/group). Western blotting for iNOS and COX-2 expression of control, LPS, LPS plus LP17, LPS plus LP17 + agonist antibody, and agonist antibody group after the BV-2 microglial cells were treated with 10 μg/ml LP17 plus 100 ng/ml LPS for 6 h and 30 min, followed by the addition of 5 μg/ml TREM-1 agonist antibody to the solution for 16 h. The agonist antibody significantly inhibited the anti-inflammatory effect of LP17 on LPS-induced upregulation of iNOS and COX-2 expressions. The bars represent the mean ± SEM. The data were analyzed using two-way ANOVA.^∗∗P < 0.05 versus the control group; ^##P < 0.05 versus the LPS group.

FIGURE 6

LP17 attenuated the 6-OHDA-induced upregulation of TREM-1 and iNOS expressions in a zebrafish PD model (n = 3/group). (A–C) The quantitative PCR of TREM-1, iNOS and calpain-1 expression of control, 6-OHDA, 6-OHDA plus LP17, and LP17 group after the zebrafish was treated with 0.1 μg/ml of LP17 (from 9 hpf to 4 dpf) and 250 μM 6-OHDA (from 2 to 4 dpf) at 4 dpf. LP17 attenuated the 6-OHDA-induced increase in TREM-1, iNOS, and calpain-1 mRNA expressions. Each sample contained 20 zebrafish heads. The bars represent the mean ± SEM. The data were analyzed using two-way ANOVA. ^∗∗P < 0.05 versus the control group; ^##P < 0.05 versus the 6-OHDA group.

FIGURE 7

Treatment of LP17 reversed 6–OHDA–induced downregulation of TH expression, upregulation of TNF-α expression, and locomotor deficit. (A) Western blot analyses for TH and TNF-α expression at 5 dpf of control, 6-OHDA, 6-OHDA plus LP17, and LP17 group after the zebrafish were treated with 0.001, 0.01, and 0.1 μg/ml LP17 (from 9 hpf to 4 dpf) and with 250 μM 6–OHDA (from 2 to 4 dpf) (n = 3/group). It showed that LP17 significantly reversed the 6-OHDA-induced downregulation of TH and attenuated the 6–OHDA–induced upregulation of TNF-α expression. Each sample contained 20 zebrafish heads. (B) Determination of the typical swimming pattern and total swimming distance of zebrafish larvae at 5 dpf in the control, 6–OHDA, 6–OHDA plus LP17, and LP17 groups (n = 16/group) shows that 0.1 μg/ml of LP17 significantly reversed the 6–OHDA–induced deficiency of locomotor activity in 5 dpf. The bars represent the mean ± SEM. The data were analyzed using two-way ANOVA. ^∗∗P < 0.05, ^∗∗∗P < 0.001 versus the control group; ^##P < 0.05; ^###P < 0.001 versus the 6-OHDA group.

FIGURE 8

Treatment of LP17 rescued 6-OHDA-induced upregulation of the number of amphetamine-induced rotation behavior, downregulation of TH expression, and upregulation of plasma sTREM-1 in rat model (n = 5/group). (A) Analyses of the number of rotations at 14 days after development of lesions in the 6-OHDA group and the 6-OHDA plus LP17 group after the rats were treated with either the control peptide or 40 μg/kg of LP17 (1 μg/μl for a 10-μl solution) plus 5 μg 6-OHDA. It showed that LP17 significantly reversed the 6-OHDA-induced increase in the number of rotation behaviors. (B,C) Whole-mount immunohistochemistry and quantitative result of TH-positive cells for TH expression at 14 days after development of lesions in the control, 6-OHDA, 6-OHDA plus LP17, and LP17 alone groups. LP17 significantly reversed the 6-OHDA-induced downregulation of TH (scale bar = 100 μm). (D) Based on the plasma level of sTREM-1 at 14 days after development of lesions in the control, 6-OHDA, 6-OHDA plus LP17, and LP17 alone groups, LP17 significantly attenuated the 6-OHDA-induced upregulation of sTREM-1 in plasma. The bars represent the mean ± SEM. The data were analyzed using two-way ANOVA. ^∗∗P < 0.05; ^∗∗∗P < 0.001 versus the control group; ^##P < 0.05; ^###P < 0.001 versus the 6-OHDA group.

FIGURE 9

Treatment with LP17 attenuated the 6-OHDA-induced upregulation of OX-42 and GFAP expression in a rat model of PD (n = 5/group). (A) Analyses of GFAP and OX-42 immunoreactivity at 14 days after the development of lesions in the 6-OHDA group and the 6-OHDA plus LP17 group after treatment with either the control peptide or 40 μg/kg LP17 (1 μg/μl for a 10-μl solution) plus 5 μg 6-OHDA. The results showed that LP17 significantly attenuated 6-OHDA-induced increases in GFAP and OX-42 expression. (B) Quantitative results of immunoreactivity analyses for GFAP and OX-42 expression at 14 days after the development of lesions in the control, 6-OHDA, 6-OHDA plus LP17, and LP17-only groups. LP17 significantly decreased 6-OHDA-induced GFAP and OX-42 upregulation (scale bar = 25 μm). The bars represent means ± SEM. The data were analyzed using two-way ANOVA. ^∗∗∗P < 0.001 versus the control group; ^##P < 0.05 versus the 6-OHDA group.

FIGURE 10

Putative mechanism of action of TREM-1 in LPS-induced neuroinflammation and 6-OHDA-induced neuron cell death. LPS increases the upregulation of TREM-1 protein expression. Downstream MAPK proteins further activate microglial inflammatory processes such as the translocation of NF-κB and upregulation of iNOS and COX-2. Then, the overactivated microglia release various neurotoxic factors such as TNF-α that damage dopamine neurons. These damaged neurons release extracellular calpain-1, further stimulating the microglia and continuing the cycle of neurotoxic microglial activation in response to neuron injury. In our study, the LP17 peptide not only significantly attenuated LPS-induced inflammation process but also enhanced autophagy as indicated by an increase in the LC3-II/LC3-I ratio, upregulation of HDAC6 expression, and downregulation of mTOR. The diminishment of microglial activation may lead to the downregulation of neurotoxic factors and may protect cells against damage. Moreover, LP17 attenuated extracellular calpain-1 release to alleviate the further activation of microglia.