Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway - PubMed (original) (raw)

Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway

Juan Ji et al. Aging Cell. 2018 Aug.

Abstract

Microglia-mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)-induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS-stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL-4, IGF-1, TGF-β1, TGF-β2, TGF-β3, G-CSF, and GM-CSF, and reduced the expression of M1 markers, such as CD86, Cox-2, iNOS, IL-1β, IL-6, TNF-α, IFN-γ, and CCL2, thereby inhibiting NFκB-IKKβ activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3-II/LC3-I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1-STRAD-MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1-to-M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1-AMPK signaling and inhibited NFκB-IKKβ activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1-to-M2 phenotypic shift in LPS-induced microglia, which might be due to improved autophagy via the activation of the LKB1-AMPK signaling pathway.

Keywords: autophagy; liver kinase B1; microglial polarization; peroxisome proliferator-activated receptor γ.

© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1

Antagonizing

PPAR

γ prevents

LPS

‐induced M1 microglial activation and facilitates microglial polarization to the M2 phenotype. Microglia were pretreated 15 min with

LPS

(0.01 μg/

ml

) and then incubated with

PPAR

γ antagonist T0070907 (0.1 μ

m

) for 24 hr. (a) The

PPAR

γ antagonist T0070907 suppressed the amoeboid “activated” morphology of microglia induced by

LPS

. The

PPAR

γ antagonist T0070907 reversed

LPS

‐induced

mRNA

expressions of M1 markers (

CD

86, Cox‐2,

iNOS

,

IFN

‐γ,

IL

‐1β,

TNF

‐α, and

CCL

  1. (b–i) and

mRNA

expressions of M2 markers (

CD

206,

IL

‐4, G‐

CSF

,

GM

‐

CSF

,

IGF

‐1,

TGF

‐β1,

TGF

‐β2, and

TGF

‐β3) in microglial cells (j–q). The ratios of p‐

NF

κB to

NF

κB (r, s) and p‐

IKK

β to

IKK

β (t, u) in microglial cells were determined by Western blotting. Data are presented as mean ±

SEM

, n ≥ 4, *p < .05, **p < .01, ***p < .001, compared to the Con group; # p < .05, ## p < .01, ### p < .001, compared to the LPS group

Figure 2

Figure 2

The

PPAR

γ antagonist T0070907 improves autophagy in microglia. (a–d) The

PPAR

γ antagonist T0070907 prevented

LPS

‐induced decreases in

LC

3‐

II

/

LC

3‐I, Beclin1, and Atg5 and

LPS

‐induced increases in P62. Data are presented as mean ±

SEM

, n ≥ 4, *p < .05, **p < .01, compared to the Con group; # p < .05, ## p < .01, compared to the

LPS

group. (e) Representative confocal images of

LC

3 puncta formation;

LC

3 (green) was colocalized with Iba1 (red) in cells treated with the

PPAR

γ antagonist T0070907 and

LPS

. Nuclei counterstained with Hoechst 33342. Scale bar = 20 μm. (f) Representative

TEM

images. Scale bar = 2 μm. High magnification of the boxed areas is shown below. Scale bar = 500 nm. Autophagosomes (blue arrows), autolysosomes (red arrows), and multilamellar body (yellow arrow). (g) Confocal microscopy analysis was used to measure autophagosomes and autolysosomes by monitoring the distribution and alteration of

mC

herry and

EGFP

fluorescent signals from

mC

herry‐

EGFP

‐

LC

3B. Scale bar = 20 μm. Microglia were pretreated with 3‐

MA

(500 μ

m

) or vehicle for 15 min, followed by treatment with 0.01 μg/

ml LPS

and 0.1 μ

m PPAR

γ antagonist T0070907 for 24 hr. (h) Immunofluorescence staining of

LC

3 (green),

CD

63 (red), and nuclei (blue) in

LPS

,

LPS

+T0070907, and

LPS

+T0070907 + 3‐

MA

groups. Scale bar = 20 μm. The protein expressions of

LC

3 (i), Beclin1 (j), p62 (k), and Atg (l) were determined by Western blotting. Data are presented as mean ±

SEM

, n ≥ 4, *p < .05, **p < .01, ***p < .001, compared to the Con group; # p < .05, ## p < .01, ### p < .001, compared to the

LPS

group; † p < .05, †† p < .01, ††† p < .001, compared to the

LPS

+T0070907 group

Figure 3

Figure 3

Antagonizing

PPAR

γ reverses

LPS

‐mediated inhibition of autophagy in microglial cells by activating

AMPK

. Microglial cells were pretreated with 0.01 μg/

ml LPS

, followed by treatment with 0.1 μ

m PPAR

γ antagonist T0070907 for 24 hr. The upstream regulatory protein levels of autophagy,

AMPK

(a, b),

ULK

1 (a, c), and

mTOR

(a, d) were analyzed by Western blotting. Data are presented as mean ±

SEM

, n ≥ 4, *p < .05, **p < .01, ***p < .001, compared to the Con group; # p < .05, ## p < .01, ### p < .001, compared to the

LPS

group; † p < .05, †† p < .01, ††† p < .001, compared to the

LPS

+T0070907 group. (e–h) The

PPAR

γ antagonist T0070907 increased the phosphorylation of

LKB

1, but it did not change the phosphorylation of Ca

MKK

β and

TAK

1 in the microglial cells treated with

LPS

. (i) An anti‐

LKB

1 antibody was used for Dynabeads Protein G immunoprecipitation, and it detected the immunoprecipitates of

MO

25 and

STRAD

by Western blotting. (j) An anti‐

MO

25 antibody was used for Dynabeads Protein G immunoprecipitation, and it detected the immunoprecipitates of

LKB

1 and

STRAD

by Western blotting

Figure 4

Figure 4

Phosphorylation of

LKB

1–

AMPK

is necessary for T0070907‐mediated upregulation of autophagy. (a, b) Below the concentration of 0.05 μ

m

, radicicol did not influence the viability of the microglia**.** After

LPS

stimulation for 15 min, the

LKB

1 inhibitor radicicol (0.025 and 0.05 μ

m

) was applied for 15 min before treatment with

PPAR

γ antagonist T0070907 for 24 hr. (c–g) Radicicol turned over the protein expressions of

LKB

1,

AMPK

,

LC

3, Beclin1, and p62 in the microglia treated with

LPS

and T0070907. Data are presented as mean ±

SEM

, n ≥ 4, *p < .05, **p < .01, ***p < .001, compared to the Con group; # p < .05, ## p < .01, ### p < .001, compared to the

LPS

group; † p < .05, †† p < .01, compared to the

LPS

+T0070907 group. (h) Immunofluorescence staining with of

LC

3 (green),

CD

63 (red), and nuclei (blue) in the

LPS

,

LPS

+T0070907, and

LPS

+T0070907 + radicicol groups. Scale bar = 20 μm. (i) Quantitation of Western blotting data showing declines in

LKB

1, which was used to observe the efficiency of transfection. Microglia were pretreated with si‐

LKB

1 (500 n

m

) or vehicle for 24 hr and then stimulated with

LPS

for 15 min and treated with

PPAR

γ antagonist T0070907 for 24 hr.

LKB

1 si

RNA

prevented T0070907‐induced increases in

LC

3‐

II

/

LC

3‐I (j), Beclin1 (k), and Atg5 (m) and T0070907‐induced decreases in P62 (l). Data are presented as mean ±

SEM

, n ≥ 4, **p < .01, ***p < .001 compared to the

LPS

group in the Ctrl; ## p < .01, ### p < .001, compared to the

LPS

+T0070907 group in the Ctrl

Figure 5

Figure 5

Blocking

LKB

1 reverses the T0070907‐mediated transition between M1 and M2 phenotypes by suppressing

NF

κB. After

LPS

stimulation for 15 min, the

LKB

1 inhibitor radicicol (0.025 and 0.05 μ

m

) was applied 15 min before treatment with

PPAR

γ antagonist T0070907 for 24 hr. (a) Fluorescence‐activated cell sorting analysis of the microglia in the

LPS

,

LPS

+T0070907, and

LPS

+T0070907 + radicicol groups. Surface expression of

CD

86 and

CD

206 was detected in microglia by flow cytometry. The percentage of

CD

86 (b) and

CD

206 (c) cells in the microglia was determined.

pH

rodo™ Green Zymosan BioParticles were added to the cells and imaged after 30, 60, 90, 120, and 150 min. The green staining in the microglial cells was due to Cell Tracker™ Green. (d) The microglial cells showed the time course of red fluorescence increased, documenting the accumulation of

pH

rodo‐conjugated zymosan bioparticles (1 μm in diameter) in the intracellular acidic environment corresponding to phagosomes. (e) The proportion between the red‐stained cells and the total cells was calculated. Data are presented as mean ±

SEM

, n = 3, ### p < .001 compared to the

LPS

group in each time point; † p < .05, ††† p < .001, compared to the

LPS

+T0070907 group in each time point. Nonfluorescence appeared at a neutral

pH

outside of the cell. Scale bar = 20 μm. Radicicol (f) and knocking down

LKB

1 (i) reversed the

PPAR

γ antagonist T0070907‐induced changes in the protein expression of the M1 markers (

iNOS

, green) and the M2 markers (

CD

206, red) by immunofluorescence staining. Scale bar = 20 μm. (g, h) Western blotting showed that radicicol prevented the decreases in

iNOS

and the increases in

CD

206 induced by T0070907. Data are presented as mean ±

SEM

, n = 4, ***p < .001, compared to the Con group; ## p < .01, ### p < .001, compared to the

LPS

group; † p < .05, ††† p < .001, compared to the

LPS

+T0070907 group. (j and k) Knocking down

LKB

reversed the

PPAR

γ antagonist T0070907‐induced changes in the protein expressions of

iNOS

and

CD

206 by Western blotting. Data are presented as mean ±

SEM

, n = 4, ***p < .001, compared to Con group, respectively in Ctrl or in si‐

LKB

1; ### p < .001, compared to

LPS

group, respectively in Ctrl or in si‐LKB1

Figure 6

Figure 6

Knocking down

PPAR

γ reduces the inflammatory response by promoting

LKB

–

AMPK

activation. (a) Labeled (FM) with green were used to observe the efficiency of transfection reagent in BV2 cells. Scale bar = 1,000 μm. (b) Images of dissociated microglial cells exposed to scrambled (Con, top panels) or

PPAR

γ si

RNA

, and labeled with antibodies to Iba1 (red) and

PPAR

γ (green), and colabeled with a nuclear stain (blue). Scale bar = 100 μm. (c) Quantitation of Western blotting data showing declines in

PPAR

γ. Statistical analysis was performed using one‐way

ANOVA

followed by Bonferroni's post hoc test. Data are presented as mean ±

SEM

, n = 5, p < .01 compared to the

NC

group. Microglia were pretreated with si‐

PPAR

γ (500 n

m

) or vehicle for 24 hr and then stimulated with

LPS

for 15 min and treated with

PPAR

γ antagonist T0070907 for 24 hr. (d)

PPAR

γ antagonist T0070907 did not influence

PPAR

γ si

RNA

‐mediated protein expressions of the M1 marker (

iNOS

, green) and the M2 marker (

CD

206, red). Scale bar = 20 μm. The experiment was repeated three times. (e, f) Western blotting was used to quantify the expressions of

iNOS

and

CD

206 in each group. Data are presented as mean ±

SEM

, n = 4, *p < .05, ***p < .001, compared to the Con group, respectively in Ctrl or in si‐

PPAR

γ; ### p < .001, compared to the

LPS

group, respectively in Ctrl in Ctrl or in si‐

PPAR

γ.

PPAR

γ si

RNA

inhibited the activation of

NF

κB and

IKK

β (e, f, and g).

PPAR

γ si

RNA

was also associated with a rise in p‐

LKB

1 (e, h) and p‐

AMPK

(e, i). The

PPAR

γ antagonist T0070907 did not affect the efficacy of

PPAR

γ si

RNA

. Data are presented as mean ±

SEM

, n ≥ 5, **p < .01, ***p < .001, compared to the

LPS

group in the control

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