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. 2008 Jul 1;181(1):660-8.
doi: 10.4049/jimmunol.181.1.660.

Potent anti-inflammatory and neuroprotective effects of TGF-beta1 are mediated through the inhibition of ERK and p47phox-Ser345 phosphorylation and translocation in microglia

Li Qian  1 , Sung-Jen Wei, Dan Zhang, Xiaoming Hu, Zongli Xu, Belinda Wilson, Jamel El-Benna, Jau-Shyong Hong, Patrick M Flood
Affiliations

Potent anti-inflammatory and neuroprotective effects of TGF-beta1 are mediated through the inhibition of ERK and p47phox-Ser345 phosphorylation and translocation in microglia

Li Qian et al. J Immunol. .

Abstract

TGF-beta1 is one of the most potent endogenous immune modulators of inflammation. The molecular mechanism of its anti-inflammatory effect on the activation of the transcription factor NF-kappaB has been well-studied; however, the potential effects of TGF-beta1 on other proinflammatory signaling pathways is less clear. In this study, using the well-established LPS and the 1-methyl-4-phenylpyridinium-mediated models of Parkinson's disease, we demonstrate that TGF-beta1 exerts significant neuroprotection in both models via its anti-inflammatory properties. The neuroprotective effects of TGF-beta1 are mainly attributed to its ability to inhibit the production of reactive oxygen species from microglia during their activation or reactivation. Moreover, we demonstrate that TGF-beta1 inhibited LPS-induced NADPH oxidase (PHOX) subunit p47phox translocation from the cytosol to the membrane in microglia within 10 min. Mechanistic studies show that TGF-beta1 fails to protect dopaminergic neurons in cultures from PHOX knockout mice, and significantly reduced LPS-induced translocation of the PHOX cytosolic subunit p47phox to the cell membrane. In addition, LPS-induced ERK phosphorylation and subsequent Ser345 phosphorylation on p47phox were significantly inhibited by TGF-beta1 pretreatment. Taken together, our results show that TGF-beta1 exerted potent anti-inflammatory and neuroprotective properties, either through the prevention of the direct activation of microglia by LPS, or indirectly through the inhibition of reactive microgliosis elicited by 1-methyl-4-phenylpyridinium. The molecular mechanisms of TGF-beta1-mediated anti-inflammatory properties is through the inhibition of PHOX activity by preventing the ERK-dependent phosphorylation of Ser345 on p47phox in microglia to reduce oxidase activities induced by LPS.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
TGFβ1 protects DA neurons against LPS-induced toxicity. Rat primary mesencephalic neuron-glia cultures were seeded in a 24-well culture plate at 5×105 , then pretreated with various concentrations of TGFβ1 for 1 h before the addition of 5 ng/ml LPS. Seven days later, the LPS-induced DA neurotoxicity was quantified by the [3H]-DA uptake assay (A); the immunocytochemical analysis, including TH-IR neuron counts (B); and the representative pictures of immunostaining (C). Results were expressed as a percentage of the vehicle-treated control cultures and were the means ± SE. from three independent experiments in triplicate. *P<0.05, **P<0.01 compared with the LPS-treated cultures.
FIGURE 2
FIGURE 2
TGFβ1 protects DA neurons against MPP+-induced toxicity in neuron-glia cultures. Different doses of TGFβl were added to the neuron-glia cultures (A), or neuron-enriched cultures (B: open bars) and neuron-astrocyte cultures (B: closed bars) for 1 h prior to the addition of 0.2 µM MPP+ treatment. The [3H]-DA uptake measurements were performed 7 days following MPP+ treatment. Results were expressed as a percentage of the vehicle-treated control cultures and were the means ± SE from 3 independent experiments in triplicate. *P<0.05 compared with the MPP+ -treated cultures.
FIGURE 3
FIGURE 3
TGFβl inhibits ROS production and pro-inflammatory factors by activated microglia. Microglia-enriched cultures were seeded at a density of 1×105 /well. Cells were pretreated with different concentrations of TGFβl for 30 min followed by the addition of LPS (10 ng/ml). The production of ROS included extracellular superoxide (A) and iROS (B). Extracellular superoxide was measured as SOD-inhibitable reduction of WST-1, and iROS was determined by probe DCFH-DA. Primary rat mesencephalic neuron-glia cultures were pretreated for 1 h with vehicle or TGFβl before treatment with 0.2 µM of MPP+ .Two and 4 days after MPP+ treatment, TGFβl was added again to the TGFβ1-treated cultures. On day 4, the release of superoxide was determined as described above (C). TGFβ’s effect on LPS-induced production of TNFα and nitrite were shown in Fig.3D and Fig. 3E. Results were expressed as mean ± SE from three to seven independent experiments in triplicate. *P<0.05, **P<0.01 compared with the LPS-treated cultures.
FIGURE 4
FIGURE 4
Microglia NADPH oxidase is the target of TGFβ1 inhibition in LPS-induced neurotoxicity. PHOX+/+ and PHOX–/– mice neuron-glia cultures were pretreated with vehicle or TGFβ1 for 1 h followed by LPS treatment. Neurotoxicity was assessed by DA uptake. Results are expressed as % of the control culture, and are the means ± SE of 3 individual experiments in triplicate in each experiment. **P < 0.01 compared with LPS treated culture.
FIGURE 5
FIGURE 5
Effects of TGFβ1 on cytosolic p47phoxprotein translocation. (A): HAPI cells seeded in dish at 5 × 104 cells/well were treated with LPS for 10 min in the absence or presence of TGFβ1 pretreatment for 1 h. Cells were fixed with 3.7% paraformaldehyde in PBS for 10 min. After washing with PBS, cells were incubated with rabbit polyclonal antibody against p47phox . Cells were then washed and incubated with FITC-conjugated goat anti-rabbit antibodies. Focal planes spaced at 0.4-µm intervals were imaged with a Zeiss 510 laser scanning confocal microscope (63 × PlanApo 1.4 numerical aperture objective) equipped with LSM510 digital imaging software. Three adjacent focal planes were averaged using Metamorph software. The signal of p47phox (FITC-p47phox ; green) are shown. (B): HAPI cells were pretreated with vehicle or TGFβ1 (3 ng/ml) for 1 h, followed by LPS treatment for 10 min. Subcellular fractions were isolated to perform western blot analysis. c: cytosolic extract; m: membrane extract. GAPDH is as an internal cytosolic control, gp91phox as an internal membrane control. Each experiment has been performed three times.
FIGURE 6
FIGURE 6
Western blot analysis of p47phoxand ERK phosphorylation. Enriched microglial cells were treated with LPS (10 ng/ml) in the presence or absence of TGFβ1 (3 ng/ml) for 10 min, cells were then harvested, proteins were analyzed by SDS-PAGE and immunoblotting with anti-phospho-Ser345–p47phox Ab (p-p47-Ser345) or anti-p47phoxAb.The Western blots from different experiments were scanned, phosphorylated, total p47phoxwas quantified by densitometry, and the intensity of phosphorylated p47phoxwas corrected for the amount of p47phox(A); The levels of phosphorylated ERK relative to total ERK were determined by western blot using specific antibodies against phosphorylated or total ERK, respectively (B). Representative western blots for Ser345– p47phox, and ERK1/2 phosphorylation are shown from 3 independent experiments.
FIGURE 7
FIGURE 7
ERK1/2 activation is required for LPS-induced DA neurotoxicity, microglial superoxide production and p47phoxtranslocation. Rat primary mesencephalic neuron-glia cultures were seeded in a 24-well culture plate at 5×105, then pretreated with various concentrations of U0126 for 30 min before the addition of 5 ng/ml LPS. Seven days later, LPS-induced DA neurotoxicity was quantified by the [3H]-DA uptake assay (A); Microglia-enriched cultures were seeded at a density of 1×105/well. Cells were pretreated with different concentrations of U0126 for 30 min followed by the addition of LPS (10 ng/ml). The production of extracellular superoxide was measured as SOD-inhibitable reduction of WST-1 (B). HAPI cells were pretreated with U0126 (5 µM) or vehicle for 30 min, followed by LPS (10 ng/ml) treatment for 10 min. Cellular membrane fractions were isolated for western blot analysis using antibodies against either p47phoxor gp91phoxas described in material and methods (C). ImageJ software was used to quantitate the intensity of the p47phoxand gp91phoxbands in western blot, and the results given in figure D represents the percentage difference of the ratio of p47phoxcompared with gp91phoxnormalized to the vehicle-treated control (D). Each experiment has been performed three times. Results in A, B, and D were expressed as mean ± SE from three independent experiments in triplicate. *P<0.05, compared with the LPS-treated cultures, while the results in C show the results of 1 representative western blots.
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