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TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease
Biochemical and Biophysical Research Communications xxx (2018) 1e6
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TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease Youwen Zhang a, b, Shujun Feng a, Kun Nie a, Yan Li a, Yuyuan Gao a, Rong Gan a, Limin Wang a, Bing Li a, Xuegang Sun c, Lijuan Wang a, **, Yuhu Zhang a, * a b c
Department of Neurology, Guangdong Neuroscience Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China Department of Neurology, The People's Hospital of Gaozhou, Guangdong, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2018 Accepted 31 March 2018 Available online xxx
Neuroinflammation and overactivated microglia underlies the pathogenesis of Parkinson's disease (PD). Furthermore, microglia could polarize into classic inflammatory M1 and immunosuppressive M2 phenotype. Thus, inhibiting the overactivated inflammatory M1 microglia by promoting the transformation of microglia to the protective M2 phenotype provides potential therapy for PD, but the mechanism that modulates microglia polarization remains unknown. Triggering receptor expressed on myeloid cells-2 (TREM2) is a recently identified immune receptor expressed by the microglia in the brain. Emerging evidence indicates that TREM2 enhances the phagocytosis function of microglia and suppress inflammation. Based on these evidence, we hypothesized that TREM2 might play a protective role through regulating microglia polarization. Here, we employ a lentiviral strategy to overexpress or suppress TREM2 on microglia and found that TREM2 was essential for M2 microglia polarization. Knockdown of TREM2 in BV2 microglia inhibited M2 polarization and lead to exaggeration of M1 microglial inflammatory responses, whereas overexpression of TREM2 promoted M2 polarization and alleviated microglial inflammation. We also observed that the TREM2 level was higher in the midbrain of PD mice, which was accompanied by an elevated level of Arginase-1 and increased proinflammatory cytokines, suggesting that TREM2 is an important factor in switching the microglia phenotypes. Taken together, these findings indicate that TREM2 plays a crucial role in altering the proinflammatory M1 microglia to M2 phenotype and has beneficial effects in the immune pathogenesis of PD. © 2018 Elsevier Inc. All rights reserved.
Keywords: Triggering receptor expressed on myeloid cells 2 Parkinson's disease Microglia Polarization Neuroinflammation
1. Introduction Parkinson's disease (PD) is the second most common neurodegenerative disorder of unknown etiology [7]. Pathobiologically, PD is characterized by progressive degeneration of dopamine (DA) neurons, deposition of Lewy bodies and focal accumulation of activated microglia in the substantia nigra in the midbrain [3]. Increasing evidence implicate the chronic inflammation and microglia activation in the degeneration of dopaminergic neurons in PD patients [14].
* Corresponding author. 106 Zhongshan Er Road, Guangzhou 510080, Guangdong Province, PR China. ** Corresponding author. 106 Zhongshan Er Road, Guangzhou 510080, Guangdong Province, PR China. E-mail addresses: [email protected] (L. Wang), [email protected] (Y. Zhang).
Neuroinflammation could be a two-edged sword which has both neurotoxic and neuroprotective effects in PD [2,28]. Deactivated microglia are identified to be the resident immune cells in the brain. However, microglial activation can polarized into inflammatory M1 and immunosuppressive M2 microglia, depending on different types of stimuli [5,6]. The overactivation of M1 microglia and the lack of M2 response might be an important mechanism underlying the neuroinflammation and neurodegeneration in PD. It is therefore very important to uncover the mechanisms that regulate M2 polarization. Recent reports revealed that microglial activation states may change during the progression of PD, that is protective in the initial stages but later becomes detrimental [11,13,27]. As the disease progresses, excessive microglial activation may caused great damages to the dopaminergic neurons [23]. Therapeutic strategies of switching the inflammatory M1 polarization or promoting the beneficial M2 polarization may be an exciting approach for future
https://doi.org/10.1016/j.bbrc.2018.03.226 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Zhang, et al., TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.226
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neuroprotective therapy in PD. In recent years, the importance of regulating microglia M1/M2 balance in the chronic neuroinflammation including PD have drawn increasing attention. However, the understanding of molecular mechanism underlying microglia polarization in PD remains unclear. Significantly, recent studies showed that triggering receptor expressed on myeloid cells 2 (TREM2) is a novel risk gene for sporadic Parkinson's disease, as a rare coding variant (R47H) within exon 2 of this gene increases the susceptibility of sporadic PD in Americans [26] and Spanish [1]. TREM2 is a type I transmembrane receptor uniquely expressed on the microglia [9,29]. Soon afterwards, TREM2 was revealed to have a potential capacity for regulating the functions of microglia by modulating microglial activation [18,22,30,32]. In addition, recent evidence revealed a crucial role of TREM2 in maintaining the survival of microglia [24,31]. By inference, we hypothesize that changes in TREM2 function could regulate the polarization of microglia phenotype and therefore halt the inflammatory processes in PD. To prove this possibility we examined the polarization of BV2 microglia employing both loss-of-function and gain-of-function of TREM2 by lentivirus infection. And we further testified the role of TREM2 in M1/M2 differentiation in MPTP-intoxicated PD mice. In the present study, we show that TREM2 plays an important role in M2 microglia polarization. Suppression of TREM2 switches the protective M2 microglia into the inflammatory M1 phenotype, which finally accelerates neuroinflammation in PD. Taken together, our findings support the critical role of TREM2 in M2 microglia polarization in PD.
After that, real-time PCR was performed with SYBR Premix Ex Taq (Takara) and monitored by using the Real-time PCR System (ABI 7500). Amplification of GAPDH was used for sample normalization. 2.4. Western blot analysis Brain tissues or microglia after transduction and cytokines stimulation were homogenized by ultrasonication. Different samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were then blocked and then incubated overnight at 4 C with the primary antibody against TREM2 protein (1:750, Abcam) and Arginase-1 (1:10000, Abcam). After washes, membranes were incubated with appropriate HRP-coupled secondary antibody. After washes again, protein bands were visualized with a chemiluminescent HRP substrate (Millipore). The size and signal intensity were analyzed using Adobe Photoshop CS5. 2.5. Cell cultures and treatments Murine microglial BV2 cells were cultured with DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) at 37 C in a humidified, 5% CO2 incubator. After microglia were transduced with the TREM2-shRNA vector or the scramble TREM2 control vector, or transduced with the wtTREM2 vector or the GFP control vector, cells were then insulted with LPS (100 ng/ml, Sigma), IFN-g (10 ng/ml, Peprotech), IL-4 (10 ng/ml, Peprotech) and IL-13 (10 ng/ ml, Peprotech) for 24 h. 2.6. Lentiviral particles preparation
2. Materials and methods 2.1. Animals and surgery Male C57BL/6 mice (5e6 weeks, weight 18e22 g, Sun Yat-sen University Laboratory) were housed at constant temperature (22e25 C) and humidity (40e70%). All animals care and experiments were conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory Animals. The Ethics Committee of Guangdong General Hospital approved the study protocol. The mice of MPTP group were intraperitoneally injected with MPTP (Sigma, 25 mg/kg) and probenecid (Sigma, 250 mg/kg) for 10 times at 3.5 d intervals. Mice of probenecid group and control group received only probenecid or saline respectively. Mice were finally anesthetized and then perfused transcardially with cold 0.9% saline followed by 4% paraformaldehyde. Brains were post-fixed with 4% paraformaldehyde for 24 h. 2.2. Immunohistochemistry Paraffin-embedded brain sections were dewaxed, rehydrated, heat-induced antigen retrieval. Then sections were incubated overnight at 4 C with a rabbit anti mouse TH (1:500, Millipore) antibody. After washing, sections were incubated for 10 min with the biotinylated goat anti rabbit antibody (1:200, Proteintech group), washed again and immersed in streptavidin-peroxidase for 10 min. Immunoreactivity was visualized by using the diaminobenzidine solution. After dehydration, sections were photographed by microscope and total numbers of TH-positive dopaminergic neurons in midbrain was counted. 2.3. RT-PCR analysis Total RNA was isolated from brain samples or cultured microglia by Trizol reagent (Invitrogen). Two micrograms of RNA were used for reverse transcription using PrimeScript RT Master Mix (Takara).
For overexpression of TREM2, the mouse TREM2 cDNA (NM_031254) and GFP gene were inserted into the GV358 lentiviral vector. And TREM2 short hairpin RNA sequence tagged with the GFP gene was cloned into the GV248 lentiviral vector to knockdown TREM2. And scramble TREM2 short hairpin RNA sequence for control study and the GFP gene were inserted into the GV248 lentiviral vector. The shRNA sequences are as follows: GATGCTGGAGATCTCTGGG. Lentiviral vectors were purified and then transfected into 293 T cells. Lentiviral particles were collected after 48 h. BV2 microglia were seeded into 24-well plates. Lentiviral particles were added to the culture for 8e12 h. The efficiency of microglia transduction was at least 90% as determined by the number of microglia expressing GFP fluorescence. 2.7. Immunofluorescence for TREM2 BV2 microglia were fixed in 4% paraformaldehyde for 15 min, blocked by 5% BSA for 30 min, and then incubated at 37 C for 1 h with polyclonal rabbit anti-TREM2 (Santa Cruz) antibody and incubated at 37 C for 1 h in the dark with a secondary fluorescence FITC-conjugated goat anti-mouse IgG antibody (1:50; Cell signaling Technology). After washing, cells were stained with DAPI for 15 min at 37 C. Images were visualized using a fluorescence microscope (Leica). 2.8. Nitrate measurement Culture supernatants was collected after cell treatment. NO production was determined by measuring the accumulation of nitrate (an indicator of NO) in the culture supernatants using the Griess reagent kit (Beyotime). 2.9. Statistical analysis Results are presented as mean ± SD of at least three
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independent experiments. One sample t-test, independent samples t-test or one-way ANOVA were used to determine significant differences among groups. P < 0.05 was considered statistically significant. All experiments were performed in triplicate and each experiment was conducted three times.
3. Results 3.1. MPTP damage dopaminergic neuron in MPTP-intoxicated mouse model After MPTP administration, numbers of midbrain TH positive dopaminergic neurons decreased significantly by 39.7% in MPTP group compared to the probenecid group and control group (P < 0.001, Supplemental Fig. 1).
3.2. TREM2 and proinflammatory cytokines mRNA are upregulated in MPTP-intoxicated mice We found that MPTP injection induce microglial overactivation in midbrain (Fig. 1). The mRNA levels of IL-6, IL-1b and TNF-a in midbrain were elevated in MPTP-intoxicated mice (Fig. 1AeC). Interestingly, the mRNA level of TREM2 in the midbrain showed an upregulation in MPTP-intoxicated mice compared with controls (Fig. 1D), which can be interpreted as an attempt to prevent the neuroinflammation in MPTP-intoxicated mice.
3.3. TREM2 and Arginase-1 protein are upregulated in MPTPintoxicated mice Consistent with RNA result, the levels of TREM2 and Arginase-1 proteins were also increased in the cortex and midbrain of MPTPintoxicated mice (Fig. 2). Arginase-1 is identified as a typical M2 marker [6] involved in the neuron survival and axon regeneration. Taken together, these results indicate that upregulation of TREM2 in vivo is possibly a failed compensatory attempt to keep inflammation in control.
3.4. Microglia can be polarized into different phenotypes By using immunofluorescence, we find that microglia can be polarized into detrimental M1 and beneficial M2 phenotypes. After applying LPS þ IFN-g as M1 activators and IL-4þIL-13 cytokines as M2 inducers to stimulate BV2 microglia, we found that LPS þ IFN-g stimulation downregulated the expression of TREM2 (Supplemental Fig. 2). On the contrary, treatment with IL-4þIL-13 enhanced the expression of TREM2 (Supplemental Fig. 2).
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3.5. Transduction of the lentivirus effectively influence TREM2 and Arginase-1 expression in BV2 microglia To further investigate the role of TREM2 in M2 microglia polarization, we employed both loss-of-function and gain-of-function strategies by lentivirus infection to investigate whether TREM2 was involved in regulating the M1/M2 differentiation. At 96 h after transduced with the TREM2-shRNA vector, the mRNA and protein levels of TREM2 and Arginase-1 were downregulated (Supplemental Fig. 3). On the contrary, after transduced with the wtTREM2 vector, we found the mRNA and protein levels of TREM2 and Arginase-1 were significantly increased (Supplemental Fig. 4). Overall, these results show that TREM2 is implicated in M2 microglia polarization. 3.6. TREM2 is required for M2 microglia polarization After transduced with the lentivirus, BV2 cells were stimulated with IL-4 and IL-13 or LPS and IFN-g. Interestingly, we found that when TREM2 was knocked down, the M2-related Arginase-1 were greatly decreased even without IL-4þIL-13 administration, whereas it was subsequently upregulated by IL-4þIL-13 treatment but decreased by LPS þ IFN-g treatment (Fig. 3A and C). What's more, the upregulation of Arginase-1 upon IL-4þIL-13 treatment was significantly suppressed in the knockdown BV2 microglia (Fig. 3A and C). Nitric oxide (NO), which can cause neuron damage, has been found to play a role in the pathogenesis of PD [19]. We found that NO was significantly enhanced in M1 microglia but was suppressed by M2 cytokines (Fig. 3B). Furthermore, we found that NO was increased in TREM2-knockdown BV2 cells but was decreased after IL-4þIL-13 treatment (Fig. 3B). These results suggest that TREM2 is essential for M2 polarization. We then overexpressed the TREM2 in BV2 cells to confirm the role of TREM2 in M2 microglia polarization and found that it upregulated TREM2 expression independent of LPS þ IFN-g or IL-4þIL-13 treatment (Fig. 4A and C). What's more, TREM2 overexpression lead to a reduction of NO (Fig. 4B). 4. Discussion Our study present the evidence that TREM2 plays a crucial role in regulating M2 microglia polarization. Overexpression of TREM2 was able to upregulate the M2 associated marker in response to IL4 and IL-13 stimuli, whereas suppression of TREM2 could shift microglia from the immunosuppressive M2 phenotype into the detrimental inflammatory M1 microglia, suggesting a critical role for TREM2 in the polarization of M2 microglia. TREM2 was recently discovered as a protective factor involved in the neuroinflammation of neurodegenerative diseases [15e17,30,31]. Furthermore, mutations in TREM2 are associated with increased risk of PD [1,26] and Alzheimer's disease [4,10,26].
Fig. 1. MPTP contributes to an elevated level of TREM2 and a mix of M1/M2 markers in the midbrain. The mRNA expression of M1 markers including TNF-a(A), IL-6 (B) and IL-1b (C), M2 marker TREM2 (D) in the midbrain was determined using real-time PCR. Saline control group, Probenecid group, MPTP group. n ¼ 5e9, *P < 0.05.
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Fig. 2. MPTP upregulated the protein level of TREM2 and Arginase-1 in the midbrain and cortex. Immunoblotting was performed to assess the protein level of TREM2 and Arginase-1 in the midbrain (A) and cortex (B). Saline control group, Probenecid group, MPTP group. n ¼ 6, *P < 0.05.
Fig. 3. Knockdown of TREM2 enhances M1 microglia polarization. The TREM2-knockdown BV2 cells were stimulated with M1 (LPS þ IFN-g) and M2 (IL4þIL13) triggers, respectively, for 24 h. (A) Proteins were then isolated to detect the expression of M2 markers TREM2 and Arginase-1 using immunoblotting. (B) The concentration of nitrate in the culture supernatant collected was measured using Griess reagent. (C) The mRNA expression of related M2 markers TREM2 and Arginase-1 was then determined using real-time PCR. n ¼ 3.
There is substantial evidence suggesting that TREM2 functions as a negative regulator of inflammation [15e18,30]. Recent studies have proved that knockdown of TREM2 on microglia deteriorated the neuroinflammation [15], on the contrary, overexpression of TREM2 could suppressed neuroinflammation in AD [17]. Previously, Wang and colleagues reported that TREM2 deficiency upregulated the microglial proinflammatory cytokines [31]. To date, there has been no direct evidence supporting a role for TREM2 on microglia polarization. On consideration of these evidences, we hypothesize that alleviation of neuroinflammation in PD might be achieved by
inhibition of TREM2 on microglia-mediated inflammatory response. In recent years, microglial activation states have been increasingly investigated. Activation of microglia in the central nervous system can be categorized into two opposite types: proinflammatory M1 phenotype and neuroprotective M2 phenotype [6]. When there is proper transition from the M1 to M2 microglia, the inflammation can be efficiently downregulated, so we hypothesized that TREM2 overexpression suppressed neuroinflammation through shifting M1 microglia to M2 activation. Treatment with M2
Please cite this article in press as: Y. Zhang, et al., TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.226
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Fig. 4. TREM2 is essential for M2 microglia polarization. The TREM2 gene was introduced into BV2 cells by wtTREM2 lentivirus followed by treatment with M1 and M2 triggers, respectively, for 24 h. (A) The level of TREM2 and Arginase-1 protein in the infected BV2 cells was determined using the immunoblotting assay. (B) The mRNA expression of related M2 markers TREM2 and Arginase-1 was then determined using real-time PCR. (C) Overexpress of TREM2 diminished the upregulation of NO in response to LPS and IFN-g. n ¼ 3.
polarizing stimuli resulted in enhanced expression of TREM2 and Arginase-1, whereas TREM2 and Arginase-1 was downregulated by M1 polarizing stimuli. In keeping with the role of M2 polarizing stimuli in promoting M2 polarization, we found that TREM2 overexpression elevated microglial M2 phenotype maker Arginase-1 in BV2 microglia, whereas knockdown of TREM2 could impair the increase in Arginase-1 expression. Not surprisingly given the previously identified role for TREM2 in neuroprotection [15,17,30,31], we observed that TREM2 is implicated in regulating M2 microglia polarization which may potentially alleviate neuroinflammation in PD. As M2 microglia are able to antagonize the M1 activation, it is interesting to assess the effect of TREM2 suppression on M1 polarization. We found that the level of pro-inflammatory M1associated marker NO in the culture supernatant was accordingly elevated when TREM2 was knocked down. Moreover, knockdown of TREM2 could immediately compromised the expression of M2related Arginase-1 even without any treatment. The phenotype of TREM2-knockdown microglia was similar to those treated with M1 activating stimuli, suggesting that the M1 effect of TREM2 suppression could be independent of M1 activator. Importantly whilst exposure of TREM2-deficient microglia to M2 polarizing stimuli did not result in increased Arginase-1 expression, however, we did observe that the elevated Arginase-1 expression induced by IL-4 and IL-13 was dismissed in the TREM2-deficient microglia, suggesting a switch from M2 to M1. Collectively, suppression of TREM2 compromises M2 microglia polarization and exaggerates M1 microglial inflammatory responses even in the presence of M2 polarizing stimuli, indicating that TREM2 is required for M2 microglia polarization. Microglia-mediated neuroinflammation has also been proved to play an important role in PD, it is tempting to propose that an M1/ M2 imbalance may be driving the inflammation process, so we investigated the role of TREM2 in M1/M2 differentiation in MPTP-
intoxicated PD mice. In our study, evident dopaminergic neurons loss and increased inflammatory cytokines were found in the brain of MPTP-intoxicate mice. This is in consistent with several other studies, where M1 cell induction resulted in brain injury pathology [8,12]. Theoretically, the brain inflammatory environment promotes M1 polarization and also inhibits the M2 switch. On the contrary, M2 response was observed instead that we detected an upregulation of TREM2 and immunosuppressive M2-associated marker Arginase-1 in MPTP-intoxicated PD mice. Coincidently, TREM2 is found highly expressed in microglia of the AD brain or in AD model mice [9,17,20,21,31]. One explanation for this contradiction is that there may be mixed populations of microglia in response to divergent stimuli in-vivo [25]. Promoting the M2 response could theoretically contribute to neuroprotection. However, in our study, TREM2 induces the expression of M2-related anti-inflammatory factor Arginase-1 but finally could not inhibit the release of inflammatory cytokines. There does seem to be a compensatory attempt of TREM2 to attenuate the excessive microglial overactivation and inflammation response initiated by MPTP treatment but finally failed to provide neuroprotection in PD. Together, our data suggests that TREM2 not only is an inducer of M2 microglia but also is a negative regulator of M1 activation. Overall, this study showed a modulatory effect of TREM2 on polarizing M1 microglia to an M2 phenotype, which further supports the idea that shifting proinflammatory M1 microglia to an M2 state may be a promising therapy for PD. However, microglial polarization is a complex process, the underlying downstream targets regulated by TREM2 should be further specified. Acknowledgements This work was supported by grants of Key Program of Natural Science Foundation of Guangdong Province, China (grant number 2017B030311015), Natural Science Foundation of Guangdong
Please cite this article in press as: Y. Zhang, et al., TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.226
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Province, China (grant number 2015A030313536), Medical Research Fund of Guangdong Province, China (grant number A2017317), National Key Research and Development Plan of China (grant number 2017YFC1310200), National Natural Science Foundation of China (grant number 81671275, 81501112). Thanks to Professor Sun Xuegang from Southern Medical University and his students for my experiment help and experimental design guidance. The authors declare that they have no conflict of interest.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.03.226. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.03.226.
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TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease Youwen Zhang a, b, Shujun Feng a, Kun Nie a, Yan Li a, Yuyuan Gao a, Rong Gan a, Limin Wang a, Bing Li a, Xuegang Sun c, Lijuan Wang a, **, Yuhu Zhang a, * a b c
Department of Neurology, Guangdong Neuroscience Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China Department of Neurology, The People's Hospital of Gaozhou, Guangdong, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2018 Accepted 31 March 2018 Available online xxx
Neuroinflammation and overactivated microglia underlies the pathogenesis of Parkinson's disease (PD). Furthermore, microglia could polarize into classic inflammatory M1 and immunosuppressive M2 phenotype. Thus, inhibiting the overactivated inflammatory M1 microglia by promoting the transformation of microglia to the protective M2 phenotype provides potential therapy for PD, but the mechanism that modulates microglia polarization remains unknown. Triggering receptor expressed on myeloid cells-2 (TREM2) is a recently identified immune receptor expressed by the microglia in the brain. Emerging evidence indicates that TREM2 enhances the phagocytosis function of microglia and suppress inflammation. Based on these evidence, we hypothesized that TREM2 might play a protective role through regulating microglia polarization. Here, we employ a lentiviral strategy to overexpress or suppress TREM2 on microglia and found that TREM2 was essential for M2 microglia polarization. Knockdown of TREM2 in BV2 microglia inhibited M2 polarization and lead to exaggeration of M1 microglial inflammatory responses, whereas overexpression of TREM2 promoted M2 polarization and alleviated microglial inflammation. We also observed that the TREM2 level was higher in the midbrain of PD mice, which was accompanied by an elevated level of Arginase-1 and increased proinflammatory cytokines, suggesting that TREM2 is an important factor in switching the microglia phenotypes. Taken together, these findings indicate that TREM2 plays a crucial role in altering the proinflammatory M1 microglia to M2 phenotype and has beneficial effects in the immune pathogenesis of PD. © 2018 Elsevier Inc. All rights reserved.
Keywords: Triggering receptor expressed on myeloid cells 2 Parkinson's disease Microglia Polarization Neuroinflammation
1. Introduction Parkinson's disease (PD) is the second most common neurodegenerative disorder of unknown etiology [7]. Pathobiologically, PD is characterized by progressive degeneration of dopamine (DA) neurons, deposition of Lewy bodies and focal accumulation of activated microglia in the substantia nigra in the midbrain [3]. Increasing evidence implicate the chronic inflammation and microglia activation in the degeneration of dopaminergic neurons in PD patients [14].
* Corresponding author. 106 Zhongshan Er Road, Guangzhou 510080, Guangdong Province, PR China. ** Corresponding author. 106 Zhongshan Er Road, Guangzhou 510080, Guangdong Province, PR China. E-mail addresses: [email protected] (L. Wang), [email protected] (Y. Zhang).
Neuroinflammation could be a two-edged sword which has both neurotoxic and neuroprotective effects in PD [2,28]. Deactivated microglia are identified to be the resident immune cells in the brain. However, microglial activation can polarized into inflammatory M1 and immunosuppressive M2 microglia, depending on different types of stimuli [5,6]. The overactivation of M1 microglia and the lack of M2 response might be an important mechanism underlying the neuroinflammation and neurodegeneration in PD. It is therefore very important to uncover the mechanisms that regulate M2 polarization. Recent reports revealed that microglial activation states may change during the progression of PD, that is protective in the initial stages but later becomes detrimental [11,13,27]. As the disease progresses, excessive microglial activation may caused great damages to the dopaminergic neurons [23]. Therapeutic strategies of switching the inflammatory M1 polarization or promoting the beneficial M2 polarization may be an exciting approach for future
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neuroprotective therapy in PD. In recent years, the importance of regulating microglia M1/M2 balance in the chronic neuroinflammation including PD have drawn increasing attention. However, the understanding of molecular mechanism underlying microglia polarization in PD remains unclear. Significantly, recent studies showed that triggering receptor expressed on myeloid cells 2 (TREM2) is a novel risk gene for sporadic Parkinson's disease, as a rare coding variant (R47H) within exon 2 of this gene increases the susceptibility of sporadic PD in Americans [26] and Spanish [1]. TREM2 is a type I transmembrane receptor uniquely expressed on the microglia [9,29]. Soon afterwards, TREM2 was revealed to have a potential capacity for regulating the functions of microglia by modulating microglial activation [18,22,30,32]. In addition, recent evidence revealed a crucial role of TREM2 in maintaining the survival of microglia [24,31]. By inference, we hypothesize that changes in TREM2 function could regulate the polarization of microglia phenotype and therefore halt the inflammatory processes in PD. To prove this possibility we examined the polarization of BV2 microglia employing both loss-of-function and gain-of-function of TREM2 by lentivirus infection. And we further testified the role of TREM2 in M1/M2 differentiation in MPTP-intoxicated PD mice. In the present study, we show that TREM2 plays an important role in M2 microglia polarization. Suppression of TREM2 switches the protective M2 microglia into the inflammatory M1 phenotype, which finally accelerates neuroinflammation in PD. Taken together, our findings support the critical role of TREM2 in M2 microglia polarization in PD.
After that, real-time PCR was performed with SYBR Premix Ex Taq (Takara) and monitored by using the Real-time PCR System (ABI 7500). Amplification of GAPDH was used for sample normalization. 2.4. Western blot analysis Brain tissues or microglia after transduction and cytokines stimulation were homogenized by ultrasonication. Different samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were then blocked and then incubated overnight at 4 C with the primary antibody against TREM2 protein (1:750, Abcam) and Arginase-1 (1:10000, Abcam). After washes, membranes were incubated with appropriate HRP-coupled secondary antibody. After washes again, protein bands were visualized with a chemiluminescent HRP substrate (Millipore). The size and signal intensity were analyzed using Adobe Photoshop CS5. 2.5. Cell cultures and treatments Murine microglial BV2 cells were cultured with DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) at 37 C in a humidified, 5% CO2 incubator. After microglia were transduced with the TREM2-shRNA vector or the scramble TREM2 control vector, or transduced with the wtTREM2 vector or the GFP control vector, cells were then insulted with LPS (100 ng/ml, Sigma), IFN-g (10 ng/ml, Peprotech), IL-4 (10 ng/ml, Peprotech) and IL-13 (10 ng/ ml, Peprotech) for 24 h. 2.6. Lentiviral particles preparation
2. Materials and methods 2.1. Animals and surgery Male C57BL/6 mice (5e6 weeks, weight 18e22 g, Sun Yat-sen University Laboratory) were housed at constant temperature (22e25 C) and humidity (40e70%). All animals care and experiments were conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory Animals. The Ethics Committee of Guangdong General Hospital approved the study protocol. The mice of MPTP group were intraperitoneally injected with MPTP (Sigma, 25 mg/kg) and probenecid (Sigma, 250 mg/kg) for 10 times at 3.5 d intervals. Mice of probenecid group and control group received only probenecid or saline respectively. Mice were finally anesthetized and then perfused transcardially with cold 0.9% saline followed by 4% paraformaldehyde. Brains were post-fixed with 4% paraformaldehyde for 24 h. 2.2. Immunohistochemistry Paraffin-embedded brain sections were dewaxed, rehydrated, heat-induced antigen retrieval. Then sections were incubated overnight at 4 C with a rabbit anti mouse TH (1:500, Millipore) antibody. After washing, sections were incubated for 10 min with the biotinylated goat anti rabbit antibody (1:200, Proteintech group), washed again and immersed in streptavidin-peroxidase for 10 min. Immunoreactivity was visualized by using the diaminobenzidine solution. After dehydration, sections were photographed by microscope and total numbers of TH-positive dopaminergic neurons in midbrain was counted. 2.3. RT-PCR analysis Total RNA was isolated from brain samples or cultured microglia by Trizol reagent (Invitrogen). Two micrograms of RNA were used for reverse transcription using PrimeScript RT Master Mix (Takara).
For overexpression of TREM2, the mouse TREM2 cDNA (NM_031254) and GFP gene were inserted into the GV358 lentiviral vector. And TREM2 short hairpin RNA sequence tagged with the GFP gene was cloned into the GV248 lentiviral vector to knockdown TREM2. And scramble TREM2 short hairpin RNA sequence for control study and the GFP gene were inserted into the GV248 lentiviral vector. The shRNA sequences are as follows: GATGCTGGAGATCTCTGGG. Lentiviral vectors were purified and then transfected into 293 T cells. Lentiviral particles were collected after 48 h. BV2 microglia were seeded into 24-well plates. Lentiviral particles were added to the culture for 8e12 h. The efficiency of microglia transduction was at least 90% as determined by the number of microglia expressing GFP fluorescence. 2.7. Immunofluorescence for TREM2 BV2 microglia were fixed in 4% paraformaldehyde for 15 min, blocked by 5% BSA for 30 min, and then incubated at 37 C for 1 h with polyclonal rabbit anti-TREM2 (Santa Cruz) antibody and incubated at 37 C for 1 h in the dark with a secondary fluorescence FITC-conjugated goat anti-mouse IgG antibody (1:50; Cell signaling Technology). After washing, cells were stained with DAPI for 15 min at 37 C. Images were visualized using a fluorescence microscope (Leica). 2.8. Nitrate measurement Culture supernatants was collected after cell treatment. NO production was determined by measuring the accumulation of nitrate (an indicator of NO) in the culture supernatants using the Griess reagent kit (Beyotime). 2.9. Statistical analysis Results are presented as mean ± SD of at least three
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independent experiments. One sample t-test, independent samples t-test or one-way ANOVA were used to determine significant differences among groups. P < 0.05 was considered statistically significant. All experiments were performed in triplicate and each experiment was conducted three times.
3. Results 3.1. MPTP damage dopaminergic neuron in MPTP-intoxicated mouse model After MPTP administration, numbers of midbrain TH positive dopaminergic neurons decreased significantly by 39.7% in MPTP group compared to the probenecid group and control group (P < 0.001, Supplemental Fig. 1).
3.2. TREM2 and proinflammatory cytokines mRNA are upregulated in MPTP-intoxicated mice We found that MPTP injection induce microglial overactivation in midbrain (Fig. 1). The mRNA levels of IL-6, IL-1b and TNF-a in midbrain were elevated in MPTP-intoxicated mice (Fig. 1AeC). Interestingly, the mRNA level of TREM2 in the midbrain showed an upregulation in MPTP-intoxicated mice compared with controls (Fig. 1D), which can be interpreted as an attempt to prevent the neuroinflammation in MPTP-intoxicated mice.
3.3. TREM2 and Arginase-1 protein are upregulated in MPTPintoxicated mice Consistent with RNA result, the levels of TREM2 and Arginase-1 proteins were also increased in the cortex and midbrain of MPTPintoxicated mice (Fig. 2). Arginase-1 is identified as a typical M2 marker [6] involved in the neuron survival and axon regeneration. Taken together, these results indicate that upregulation of TREM2 in vivo is possibly a failed compensatory attempt to keep inflammation in control.
3.4. Microglia can be polarized into different phenotypes By using immunofluorescence, we find that microglia can be polarized into detrimental M1 and beneficial M2 phenotypes. After applying LPS þ IFN-g as M1 activators and IL-4þIL-13 cytokines as M2 inducers to stimulate BV2 microglia, we found that LPS þ IFN-g stimulation downregulated the expression of TREM2 (Supplemental Fig. 2). On the contrary, treatment with IL-4þIL-13 enhanced the expression of TREM2 (Supplemental Fig. 2).
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3.5. Transduction of the lentivirus effectively influence TREM2 and Arginase-1 expression in BV2 microglia To further investigate the role of TREM2 in M2 microglia polarization, we employed both loss-of-function and gain-of-function strategies by lentivirus infection to investigate whether TREM2 was involved in regulating the M1/M2 differentiation. At 96 h after transduced with the TREM2-shRNA vector, the mRNA and protein levels of TREM2 and Arginase-1 were downregulated (Supplemental Fig. 3). On the contrary, after transduced with the wtTREM2 vector, we found the mRNA and protein levels of TREM2 and Arginase-1 were significantly increased (Supplemental Fig. 4). Overall, these results show that TREM2 is implicated in M2 microglia polarization. 3.6. TREM2 is required for M2 microglia polarization After transduced with the lentivirus, BV2 cells were stimulated with IL-4 and IL-13 or LPS and IFN-g. Interestingly, we found that when TREM2 was knocked down, the M2-related Arginase-1 were greatly decreased even without IL-4þIL-13 administration, whereas it was subsequently upregulated by IL-4þIL-13 treatment but decreased by LPS þ IFN-g treatment (Fig. 3A and C). What's more, the upregulation of Arginase-1 upon IL-4þIL-13 treatment was significantly suppressed in the knockdown BV2 microglia (Fig. 3A and C). Nitric oxide (NO), which can cause neuron damage, has been found to play a role in the pathogenesis of PD [19]. We found that NO was significantly enhanced in M1 microglia but was suppressed by M2 cytokines (Fig. 3B). Furthermore, we found that NO was increased in TREM2-knockdown BV2 cells but was decreased after IL-4þIL-13 treatment (Fig. 3B). These results suggest that TREM2 is essential for M2 polarization. We then overexpressed the TREM2 in BV2 cells to confirm the role of TREM2 in M2 microglia polarization and found that it upregulated TREM2 expression independent of LPS þ IFN-g or IL-4þIL-13 treatment (Fig. 4A and C). What's more, TREM2 overexpression lead to a reduction of NO (Fig. 4B). 4. Discussion Our study present the evidence that TREM2 plays a crucial role in regulating M2 microglia polarization. Overexpression of TREM2 was able to upregulate the M2 associated marker in response to IL4 and IL-13 stimuli, whereas suppression of TREM2 could shift microglia from the immunosuppressive M2 phenotype into the detrimental inflammatory M1 microglia, suggesting a critical role for TREM2 in the polarization of M2 microglia. TREM2 was recently discovered as a protective factor involved in the neuroinflammation of neurodegenerative diseases [15e17,30,31]. Furthermore, mutations in TREM2 are associated with increased risk of PD [1,26] and Alzheimer's disease [4,10,26].
Fig. 1. MPTP contributes to an elevated level of TREM2 and a mix of M1/M2 markers in the midbrain. The mRNA expression of M1 markers including TNF-a(A), IL-6 (B) and IL-1b (C), M2 marker TREM2 (D) in the midbrain was determined using real-time PCR. Saline control group, Probenecid group, MPTP group. n ¼ 5e9, *P < 0.05.
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Fig. 2. MPTP upregulated the protein level of TREM2 and Arginase-1 in the midbrain and cortex. Immunoblotting was performed to assess the protein level of TREM2 and Arginase-1 in the midbrain (A) and cortex (B). Saline control group, Probenecid group, MPTP group. n ¼ 6, *P < 0.05.
Fig. 3. Knockdown of TREM2 enhances M1 microglia polarization. The TREM2-knockdown BV2 cells were stimulated with M1 (LPS þ IFN-g) and M2 (IL4þIL13) triggers, respectively, for 24 h. (A) Proteins were then isolated to detect the expression of M2 markers TREM2 and Arginase-1 using immunoblotting. (B) The concentration of nitrate in the culture supernatant collected was measured using Griess reagent. (C) The mRNA expression of related M2 markers TREM2 and Arginase-1 was then determined using real-time PCR. n ¼ 3.
There is substantial evidence suggesting that TREM2 functions as a negative regulator of inflammation [15e18,30]. Recent studies have proved that knockdown of TREM2 on microglia deteriorated the neuroinflammation [15], on the contrary, overexpression of TREM2 could suppressed neuroinflammation in AD [17]. Previously, Wang and colleagues reported that TREM2 deficiency upregulated the microglial proinflammatory cytokines [31]. To date, there has been no direct evidence supporting a role for TREM2 on microglia polarization. On consideration of these evidences, we hypothesize that alleviation of neuroinflammation in PD might be achieved by
inhibition of TREM2 on microglia-mediated inflammatory response. In recent years, microglial activation states have been increasingly investigated. Activation of microglia in the central nervous system can be categorized into two opposite types: proinflammatory M1 phenotype and neuroprotective M2 phenotype [6]. When there is proper transition from the M1 to M2 microglia, the inflammation can be efficiently downregulated, so we hypothesized that TREM2 overexpression suppressed neuroinflammation through shifting M1 microglia to M2 activation. Treatment with M2
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Fig. 4. TREM2 is essential for M2 microglia polarization. The TREM2 gene was introduced into BV2 cells by wtTREM2 lentivirus followed by treatment with M1 and M2 triggers, respectively, for 24 h. (A) The level of TREM2 and Arginase-1 protein in the infected BV2 cells was determined using the immunoblotting assay. (B) The mRNA expression of related M2 markers TREM2 and Arginase-1 was then determined using real-time PCR. (C) Overexpress of TREM2 diminished the upregulation of NO in response to LPS and IFN-g. n ¼ 3.
polarizing stimuli resulted in enhanced expression of TREM2 and Arginase-1, whereas TREM2 and Arginase-1 was downregulated by M1 polarizing stimuli. In keeping with the role of M2 polarizing stimuli in promoting M2 polarization, we found that TREM2 overexpression elevated microglial M2 phenotype maker Arginase-1 in BV2 microglia, whereas knockdown of TREM2 could impair the increase in Arginase-1 expression. Not surprisingly given the previously identified role for TREM2 in neuroprotection [15,17,30,31], we observed that TREM2 is implicated in regulating M2 microglia polarization which may potentially alleviate neuroinflammation in PD. As M2 microglia are able to antagonize the M1 activation, it is interesting to assess the effect of TREM2 suppression on M1 polarization. We found that the level of pro-inflammatory M1associated marker NO in the culture supernatant was accordingly elevated when TREM2 was knocked down. Moreover, knockdown of TREM2 could immediately compromised the expression of M2related Arginase-1 even without any treatment. The phenotype of TREM2-knockdown microglia was similar to those treated with M1 activating stimuli, suggesting that the M1 effect of TREM2 suppression could be independent of M1 activator. Importantly whilst exposure of TREM2-deficient microglia to M2 polarizing stimuli did not result in increased Arginase-1 expression, however, we did observe that the elevated Arginase-1 expression induced by IL-4 and IL-13 was dismissed in the TREM2-deficient microglia, suggesting a switch from M2 to M1. Collectively, suppression of TREM2 compromises M2 microglia polarization and exaggerates M1 microglial inflammatory responses even in the presence of M2 polarizing stimuli, indicating that TREM2 is required for M2 microglia polarization. Microglia-mediated neuroinflammation has also been proved to play an important role in PD, it is tempting to propose that an M1/ M2 imbalance may be driving the inflammation process, so we investigated the role of TREM2 in M1/M2 differentiation in MPTP-
intoxicated PD mice. In our study, evident dopaminergic neurons loss and increased inflammatory cytokines were found in the brain of MPTP-intoxicate mice. This is in consistent with several other studies, where M1 cell induction resulted in brain injury pathology [8,12]. Theoretically, the brain inflammatory environment promotes M1 polarization and also inhibits the M2 switch. On the contrary, M2 response was observed instead that we detected an upregulation of TREM2 and immunosuppressive M2-associated marker Arginase-1 in MPTP-intoxicated PD mice. Coincidently, TREM2 is found highly expressed in microglia of the AD brain or in AD model mice [9,17,20,21,31]. One explanation for this contradiction is that there may be mixed populations of microglia in response to divergent stimuli in-vivo [25]. Promoting the M2 response could theoretically contribute to neuroprotection. However, in our study, TREM2 induces the expression of M2-related anti-inflammatory factor Arginase-1 but finally could not inhibit the release of inflammatory cytokines. There does seem to be a compensatory attempt of TREM2 to attenuate the excessive microglial overactivation and inflammation response initiated by MPTP treatment but finally failed to provide neuroprotection in PD. Together, our data suggests that TREM2 not only is an inducer of M2 microglia but also is a negative regulator of M1 activation. Overall, this study showed a modulatory effect of TREM2 on polarizing M1 microglia to an M2 phenotype, which further supports the idea that shifting proinflammatory M1 microglia to an M2 state may be a promising therapy for PD. However, microglial polarization is a complex process, the underlying downstream targets regulated by TREM2 should be further specified. Acknowledgements This work was supported by grants of Key Program of Natural Science Foundation of Guangdong Province, China (grant number 2017B030311015), Natural Science Foundation of Guangdong
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Province, China (grant number 2015A030313536), Medical Research Fund of Guangdong Province, China (grant number A2017317), National Key Research and Development Plan of China (grant number 2017YFC1310200), National Natural Science Foundation of China (grant number 81671275, 81501112). Thanks to Professor Sun Xuegang from Southern Medical University and his students for my experiment help and experimental design guidance. The authors declare that they have no conflict of interest.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.03.226. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.03.226.
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Please cite this article in press as: Y. Zhang, et al., TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.226