Roflupram, a Phosphodiesterase 4 Inhibitior, Suppresses

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Roflupram, a phosphodiesterase 4 inhibitior, suppresses inflammasome activation through autophagy in microglial cells Tingting You, Yu-Fang Cheng, Jiahong Zhong, Bingtian Bi, Bingqing Zeng, Wenhua Zheng, Hai-Tao Wang, and Jiang-Ping Xu ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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ACS Chemical Neuroscience

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Roflupram, a phosphodiesterase 4 inhibitior, suppresses inflammasome

2

activation through autophagy in microglial cells

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Tingting Youa,b, Yufang Chenga, Jiahong Zhonga, Bingtian Bia,c, Bingqing Zenga,

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Wenhua Zhengd, Haitao Wanga,*, Jiangping Xua, *

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a

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Sciences, Southern Medical University, Guangzhou 510515, China

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b

Department of Neuropharmacology and Drug Discovery, School of Pharmaceutical

Department of Pharmacology, Guangdong Key Laboratory for R&D of Natural Drug,

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Guangdong Medical University, Zhanjiang 524023, China

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c

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Guangzhou 510060, China

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d

Department of Clinical Trial Center, Cancer Center, Sun Yat-sen University,

Faculty of Health Sciences, University of Macau , Taipa, Macau, China

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*Correspondence:

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Prof. Jiangping Xu, M.D., Ph.D.

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Department of Neuropharmacology and Drug Discovery,

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School of Pharmaceutical Sciences,

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Southern Medical University, Guangzhou 510515, China.

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Tel.: +86-20-61648236, Fax: +86-20-61648236

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E-mail: [email protected]

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Dr. Haitao Wang, Ph.D.

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Department of Neuropharmacology and Drug Discovery,

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School of Pharmaceutical Sciences,

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Southern Medical University, Guangzhou 510515, China.

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Tel.: +86-20-61648594, Fax: +86-20-61648236

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Inhibition of phosphodiesterase 4 (PDE4) suppressed the inflammatory responses in

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the brain. However, the underlying mechanisms are poorly understood. Roflupram

4

(ROF) is a novel PDE4 inhibitor. In the present study, we found that ROF enhanced

5

the level of microtubule-associated protein 1 light chain 3 II (LC3-II) and decreased

6

p62 in microglial BV-2 cells. Enhanced fluorescent signals were observed in BV-2

7

cells treated with ROF by Lysotracker red and acridine orange staining. In addition,

8

immunofluorescence indicated a significant increase in punctate LC3. Moreover, β

9

amyloid25-35 (Aβ25-35) or lipopolysaccharide (LPS) with ATP were used to activate

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inflammasome. We found that both LPS plus ATP and Aβ25-35 enhanced the

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conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of

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mature IL-1β in BV-2 cells. Interestingly, these effects were blocked by the treatment

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of ROF. Consistently, Knocking down the expression of PDE4B in primary microglial

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cells led to enhanced level of LC-3Ⅱ and decreased activation of inflammasome.

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What's more, Hoechst staining showed that ROF decreased the apoptosis of neuronal

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N2a cells in conditioned media from microglia. Our data also showed that ROF

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dose-dependently enhanced autophagy, reduced the activation of inflammasome and

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suppressed the production of IL-1β in mice injected with LPS. These effects were

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reversed by inhibition of microglial autophagy. These results put together demonstrate

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that ROF inhibits inflammasome activities and reduces the release of IL-1β by

21

inducing autophagy. Therefore, ROF could be used as a potential therapeutic

22

compound for the intervention of inflammation-associated diseases in the brain.

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Keywords: Phosphodiesterase 4; Roflupram; Autophagy; Inflammasome; Microglia;

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Neuroinflammation.

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Abbreviations

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3-MA, 3-methyladenine; Aβ, β amyloid protein; AD, Alzheimer’s disease; AO,

3

acridine orange; ATG, autophagy-related gene; ATG16L1, autophagy-related gene 16

4

like 1; ATG7, autophagy related protein 7; ATP, adenosine triphosphate; cAMP,

5

3',5'-cyclic monophosphate; CASP1, caspase-1; CASP3, caspase-3; cGMP, cyclic

6

guanosine monophosphate; CM, conditioned media; CNS, central nervous system;

7

CQ, lysosome inhibitor chloroquine; ELISA, enzyme linked immunosorbent assay;

8

fAβ, amyloid β-protein fibrils; FORS, forskolin; IL-1β, interleukin beta; LC3,

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microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LYT,

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lysotraker red; NLR, NOD-like receptor; NLRP3, pyrin domain containing 3;

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SQSTM1,

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phosphodiesterase-4; PD, Parkinson’s disease; pro-IL-18, precursor interleukin-18;

13

pro-IL-1β, precursors interleukin-1β; PSD95, postsynaptic density-95; RAPA,

14

rapamycin; ROF, Roflupram; ROLI, Rolipram; TNF-α, tumor necrosis factor alpha.

sequestosome

1

protein;

PDEs,

phosphodiesterases;

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3


p62/ PDE4,


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Introduction

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Sustained neuroinflammation contributes to the pathogenesis of various neurological

3

and psychiatric disorders, including Alzheimer’s disease (AD), Parkinson’s disease

4

(PD), and depression1. A variety of factors, such as toxic metabolites, infection, and

5

traumatic brain injury, potentially activate the glial cells that secrete proinflammatory

6

cytokines and eventually contribute to the initiation and progression of

7

neuroinflammation 2. However, activated glia can initiate both protective and toxic

8

inflammatory responses according to the severity and duration of the activation 3.

9

When inflammation is rampant, persistent activation of glial cells leads to neuronal

10

injury 4. Hence, limiting the inflammatory responses is useful in ameliorating chronic

11

inflammation-related diseases.

12

Autophagy is a critical quality control system for cellular homeostasis regulation 5.

13

Recent studies have established that autophagy plays a pivotal role in regulating

14

immune responses and controlling excessive inflammation

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effects of autophagy in the pathology of inflammation is the modulation of the

16

inflammasome activation and the production of interleukin-1β (IL-1β)

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inflammasome is a multimeric protein complex composed of a sensor protein (such as

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an NOD-like receptor, NLR), an adaptor protein ASC, and the inflammatory protease

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caspase-1 that enzymatically processes pro-IL-1β and pro-IL-18 into their mature

20

forms9. Autophagy deficiency might contribute to the exaggeration of inflammation 10.

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For example, fetal macrophages obtained from autophagocytosis-deficient ATG16L1-/-

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(autophagy-related gene 16 like 1) mice, produced excessive amounts of IL-1β and

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IL-18 in response to endotoxin stimulation, and interestingly, the enhanced levels of

24

pro-inflammatory factors were found to be associated with higher level of caspase-1 11.

25

These

26

pharmacological inhibition or genetic deletion

27

mainly focused on the autophagy-mediated anti-inflammatory effect in macrophages.

28

In the central nervous system (CNS), microglia is the resident macrophage along with

29

primary immune cells exhibiting a key role in neuroinflammation

30

regulatory role of microglial autophagy in inflammasome has been the focus of

findings

have

been

confirmed

by

6, 7

. One of the major

manipulating

autophagy

8

. The

with

12, 13

. However, most of these studies

4


14

. Recently, the

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1

intense research. Increased inflammation was observed following the disruption of

2

microglial autophagy with Atg7 small interfering RNA (siRNA), while promoting

3

autophagy reduced the IL-1β release in Aβ-treated microglial cells

4

molecular machinery remains to be determined, these data postulate that promoting

5

microglial

6

neuroinflammation-related diseases.

7

autophagy

might

be

a

potential

15

. Although the

therapeutic

strategy

for

On the other hand, intracellular adenosine 3',5'-cyclic monophosphate (cAMP) 16-18

8

increase the autophagic activities reported in various cells in the past few years

.

9

cAMP is a well-established ubiquitous secondary messenger that plays a major role in

10

the regulation of various cellular processes. It is synthesized from ATP catalyzed by

11

adenylate cyclase and decomposed into AMP catalyzed by phosphodiesterases (PDEs).

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Ugland et al. reported that cAMP-mediated induction of autophagy in mesenchymal

13

stem cells might provide new targets for the intervention of neurological disorders 17.

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PDE type 4 (PDE4) inhibitor resveratrol could induce autophagy in endothelial cells

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by upregulating the cAMP signaling pathway

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Resveratrol induced autophagy through cAMP in HepG2 cells

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suggested that cAMP might mediate the induction of autophagy in certain types of

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cell. However, whether cAMP or PDE4 inhibitor exert similar roles on microglia

19

autophagy was unknown.

16

. A recent study also reported that 18

. These studies

20

PDEs are a ubiquitous superfamily of enzymes involved in the regulation of cAMP

21

and/or cyclic guanosine monophosphate (cGMP) by degrading these secondary

22

messengers to inactive nucleotides 19. Their vital role in cell signaling renders them as

23

therapeutic targets for many diseases, and the development of PDE inhibitors is under

24

study

25

enzyme, whose expression was detected in various cells including immune cells,

26

proinflammatory cells, and airway smooth muscle cells

27

inhibitors are considered as promising novel drugs for the treatment of chronic

28

inflammatory diseases based on the anti-inflammatory effect 22. Our previous studies

29

showed that inhibition of PDE4 can adequately attenuate neuroinflammation in

30

neurodegenerative and mental disorders 23-25. Roflupram (ROF, also known as Zl-n-91

20

. Among the 11 identified isoenzymes, PDE4 is a major cAMP-specific

5


21

. Remarkably, PDE4


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or FFPM), a selective PDE4 inhibitor, was designed based on the structures of

2

PDE4D2 and rolipram

3

anti-inflammatory effect and ameliorated the behavior of AD model in APP/PS1

4

transgenic

5

anti-neuroinflammatory effect are yet to be elucidated. Coupled with autophagy in the

6

regulation of inflammatory response, the role of ROF in microglial autophagy and the

7

putative correlation between the anti-inflammatory effect and autophagy is yet an

8

enigma. Therefore, the present study aimed to investigate the regulatory effect of ROF

9

on autophagic activities, as well as, the relevant contribution of autophagy to the

10

anti-inflammatory effect by emphasizing on the NLRP3 inflammasome pathway in

11

microglial cells.

mice27.

26

. Our recent studies also showed that ROF exerted an

Nevertheless,

the

mechanisms

underlying

the

12 13

RESULTS AND DISCUSSION

14

Effect of ROF on cell viability in microglial BV-2 cells. The chemical structure of

15

ROF was shown in Figure 1A. This compound was kindly provided by Dr. Hengming

16

Ke (University of North Carolina, Chapel Hill, USA). Methyl thiazolyl tetrazolium

17

(MTT) assay was performed to determine the cellular compatibility of ROF with

18

BV-2 cells. As shown in Figure 1B, ROF (0.625–20 µM) did not exhibit toxicity on

19

BV-2 cells after 24 h incubation. ROF also showed adequate compatibility with

20

activated microglial BV-2 cells triggered by lipopolysaccharide (LPS) plus ATP, as

21

well as, Aβ25-35 exposure for 6 h (Figure 5A).

22

ROF induced autophagy in microglial BV-2 cells. Autophagy is a highly 28

23

conserved process for degradation in all types of eukaryotic cells

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altered LC3 expression in microglial BV-2 cells in two isoforms, LC3-I and LC3-II.

25

During

26

phospholipid-conjugated LC3-II, which is a valuable indicator of altered autophagic

27

activity29. The present study focused on whether PDE4 inhibitor ROF could induce

28

microglial autophagy. Thus, we firstly examined the expression of LC3 in BV-2 cells

29

treated with ROF. As shown in Figure 1C–G, ROF increased the protein level of

30

LC3-II in BV-2 cells treated with ROF while the that of p62/sequestosome 1 (p62), an

the

formation

of

autophagosome,

cytosolic

6


LC3-I

. and found an

converts

into

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autophagy substrate, was significantly decreased in a concentration- and

2

time-dependent manner. These results indicated that ROF had an active role in the

3

autophagic activity.

4

However, it is notable that both induction and inhibition of autophagy will lead 30

5

to an increase in LC3-II

6

reflects the entire process of autophagy, was used to determine the source of enhanced

7

LC3-II level. As autophagic flux can be evaluated based on the LC3-II turnover

8

through the use of lysosomal inhibitors

9

presence or absence of lysosomal inhibitor CQ. CQ suppresses the fusion of

10

autophagosome and lysosome at the late stage of autophagy, thereby resulting in the

11

impairment of LC3-II autophagic degradation and subsequent accumulation(29). As

12

shown in Figure 1H-I, compared to the cells treated with ROF alone, co-treatment

13

with CQ and ROF resulted in increased LC3-II accumulation, suggesting that

14

autophagic flux was enhanced after ROF treatment. Collectively, these data indicate

15

that ROF enhances the autophagic activities and stimulates the autophagic flux in

16

BV-2 cells.

17

. Therefore, further assessment of autophagic flux, which

31

, BV-2 cells were treated with ROF in the

In addition to LC3-II and p62, the quantity of autophagy-lysosome, formed at the 32

18

late stage of autophagy, is also an indicator of the autophagic activity

. Hence, we

19

labeled the lysosomes in BV-2 cells with acridine orange (AO) and lysotraker red

20

(LYT) stain to monitor the effect of ROF on the late stages of autophagic activity. As

21

shown in Figure 2A and 2B, compared to the cells in the vehicle control group, the

22

enhanced fluorescence signal was observed in cells treated with ROF. The typical

23

autophagy inducer rapamycin (RAPA, as a positive control) also induced fluorescence

24

signal of lysosome (Figure 2A-B), which further confirmed the induction effect of

25

ROF on autophagy. Notably, the classic PDE4 inhibitor rolipram (ROLI) and an

26

adenosine cyclase activator forskolin (FORS) could also induce high fluorescence

27

signal of lysosome (Figure 2A-B). These parallel experiments suggested that cAMP

28

contributes to microglial autophagy.

29

Furthermore, immunofluorescence staining showed that the increased LC3

30

punctuate staining in response to ROF was inhibited by class III PI3K inhibitor, 7



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Page 8 of 37

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3-methyladenine (3-MA), which is known to block autophagy at the early stage

2

(Figure 3A). The immunofluorescence images were consistent with results of Western

3

blot analysis for LC3-II (Figure 3B). Together, these results suggested that ROF

4

induces microglial autophagy involving the class III PI3K-dependent autophagy

5

signaling pathway. Although the underlying mechanisms require further study, it was

6

interesting to find that PDE4 inhibitor ROF could induce microglial autophagy.

7

Blocking autophagy enhanced the activation of NLRP3 inflammasome in BV-2

8

cells. Next, we investigated the effect of ROF-induced microglial autophagy on

9

neuroinflammation focusing on NLRP3 inflammasome/caspase-1/IL-1β axis, which is 33, 34

10

critical in inflammatory response

, and recently, this axis was found to be

11

regulated by autophagic activities

12

NLRP3 inflammasome activity and microglia-mediated inflammation, NLRP3

13

inflammasome activators including LPS (1 µg/ml) plus ATP (1 mM) as well as Aβ25-35

14

(5 µM) were used to trigger BV-2 cells. As shown in Figure 4A, BV-2 cells were in

15

the ‘resting’ state in the normal culture medium. After stimulation for 4–6 h, BV-2

16

cells were in the ‘activated’ state with morphological changes, including bigger cell

17

body and more branches. To initiate and amplify the neuroinflammatory response,

18

activated microglia not only displayed a characteristic change in cell morphology but

19

also showed functional alterations including secretion of proinflammatory cytokines 36.

20

IL-1β is one of the main factors mediating inflammatory responses 37. In the present

21

study, mature-IL-1β (IL-1β p17) was detectable by in the supernatant of BV-2

22

microglial cell 4 h after LPS plus ATP as well as Aβ25-35 simulation (Figure 4B and

23

4C). Intriguingly, IL-1β production requires caspase-1 (CASP1) activation by

24

inflammasome, which is a molecular platform responsible for the processing of

25

caspase-1 and subsequent maturation and secretion of IL-1β and IL-18

26

blot analysis of caspase-1 showed a gradual increase in cleaved-caspase-1 (CASP1

27

p10) after stimulation for 4–6 h and the changes in CASP1 p10 were similar to that of

28

IL-1β p17 (Figure 4B and 4C). Together, these data show that inflammasome was

29

distinctly activated 6 h after LPS plus ATP as well as Aβ25-35 stimulation in BV-2 cells.

30

Based on the above cell model, we further investigated the interaction between

35

. In order to study the effect of autophagy on

8


38

. Western

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1

autophagy and microglial inflammation focusing on the modified NLRP3

2

inflammasome activities and LC3-II expression in BV-2 cells. As shown in Figure 4D,

3

4G, 4J, and 4K, the level of LC3-II was significantly increased in response to LPS

4

plus ATP as well as Aβ25-35 stimulation as compared to the control. These observations

5

were consistent with the previous reports that inflammatory stimuli could also

6

enhance the autophagic activities

7

in the negative feedback regulation thereby, restricting the inflammatory response 6.

8

Strikingly, as a response to 3-MA, a significant increase in CASP1 p10 and IL-1β p17

9

was observed in activated BV-2 cells as compared to cells treated without 3-MA

10

(Figure 4D–K). These results demonstrated that the blockage of microglial autophagy

11

amplified NLRP3 inflammasome activation and neuroinflammation. Moreover, the

12

protein levels of NLRP3 and precursor of interleukin-1β (pro-IL-1β) were increased

13

as a result of 3-MA treatment (Figure 4D–K). These data suggested that blockage of

14

autophagy promotes IL-1β processing that is associated with reduced autophagic

15

degradation of NLRP3 inflammasome component and IL-1β precursor.

15, 39

. The enhanced autophagy, in turn, participates

16

ROF-induced autophagy suppressed the activation of NLRP3 inflammasome

17

in BV-2 cells. In our previous in vivo studies, ROF showed an anti-inflammatory

18

effect in APP/PS1 transgenic AD mice27; however, the underlying mechanism was not

19

clarified. Although ROF-induced microglial autophagy was found in the present study,

20

the link between its anti-inflammatory effect and autophagy was yet to be determined.

21

Here, we found that ROF significantly increased the level of LC3-II in activated BV-2

22

cells (p

Roflupram, a Phosphodiesterase 4 Inhibitior, Suppresses

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