Journal of Neuroimmunology 279 (2015) 50–63

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The activation of NG2 expressing cells is downstream to microglial reaction and mediated by the transforming growth factor beta 1 Ping Xiang a, Lie Zhu b, Hua Jiang b, Bei Ping He a,⁎ a b

Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Department of Plastic Surgery, Chang Zheng Hospital, Shanghai, China

a r t i c l e

i n f o

Article history: Received 13 October 2014 Received in revised form 25 December 2014 Accepted 14 January 2015 Keywords: NG2 NG2 expressing cells Microglia Lipopolysaccharide TGF-β1

a b s t r a c t In the present study, we investigated the mechanism of activation of NG2 expressing cells. Application of microglial inhibitors not only attenuated morphological changes but also significantly retarded increase in the number of NG2 expressing cells. Intracerebral injection of TGF-β1 led to a profound activation of NG2 glia as well as an earlier accumulation of NG2+-microglia, whilst inhibition of TGF-β1 Smad2/3 signalling pathway eventually attenuated their active responses. We conclude that the activation of NG2 expressing cells is an event downstream to microglial reaction and TGF-β1 secreted from microglia might play an important role in modulation of the function of NG2 expressing cells. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The constitutive NG2 glia, the fourth major type of glial cells in the CNS, express chondroitin sulphate proteoglycan NG2 (CSPG4) as their antigen marker. In the adult CNS, the constitutive NG2 glia, making up 5–10% of all glial cells and existing ubiquitously in the grey and white matter (Horner et al., 2002), are considered as a distinct cell type (Peters, 2004; Nishiyama et al., 2009) as they do not co-stain with specific markers for other three glial cells, namely microglia, astrocytes and oligodendrocytes, in the normal CNS. Although the function of constitutive NG2 glia is not clear at present, the cells have presented remarkable active responses in pathological conditions. The rapid increase in expression of NG2 is a common feature of damages to the CNS, such as cortical stab injury (Fitch and Silver, 1997) and spinal cord injury (Lemons et al., 1999). An interesting phenomenon in neuropathologies is that NG2 protein could be expressed in activated microglia and infiltrating macrophages in various CNS injury models (Bu et al., 2001; Jones et al., 2002; Matsumoto et al., 2008; Zhu et al., 2010). Therefore, the pool of activated NG2 expressing cells should include two portions: the constitutive NG2 glia (NG2+/OX42− cells in this study) and NG2-positive microglia (NG2+/OX42+ cells). For the latter, the induced expression of NG2 protein in microglia may suggest an alteration in their functional activities (Gao et al., 2010; Smirkin et al.,

⁎ Corresponding author at: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Singapore 117597. E-mail address: [email protected] (B.P. He).

http://dx.doi.org/10.1016/j.jneuroim.2015.01.006 0165-5728/© 2015 Elsevier B.V. All rights reserved.

2010; Zhu et al., 2012). NG2 has been reported to regulate the production of inflammatory cytokines in activated microglia (Gao et al., 2010). In addition, NG2 positive macrophages might play a beneficial role in the ischaemic brain, possibly through secretion of neuroprotective factors (Smirkin et al., 2010). Our lab showed that microglia lost phagocytic capacity after gaining NG2 expression in a LPS-induced neuroinflammation model (Zhu et al., 2012). However, how NG2 glia became activated and the signal molecules triggering NG2 expression in microglia in vivo remains unclear at present. NG2, a member of CSPG family, is composed of a core glycoprotein of 300 kDa and a single chondroitin sulphate chain (Nishiyama et al., 1991; Stallcup, 2002). It is a prominent component of glial scar (Tan et al., 2005). TGF-β1 was reported to increase the expression of CSPG by induction of a few genes encoding CSPG core proteins and enzymes regulating GAG chain synthesis (Smith and Strunz, 2005; Wang et al., 2008). Since NG2 belongs to CSPG families, it is reasonable to assume that TGFβ1 might be a potential stimulator to induce upregulation of NG2 protein expression in the lesions. We have investigated the possible mechanisms on activation of both constitutive NG2 glia and NG2-positive microglia in a LPS induced neuroinflammatory model. In the LPS intracerebral injection model, the integrity of the blood brain barrier has been disrupted. Monocytes may flow into the brain parenchyma and turn into macrophages. Therefore, the term of microglia used in this study may include both resident microglia and blood-borne macrophages. Our results showed that the activation of NG2 glia was an event downstream to microglial activation and the expression of NG2 proteoglycan in microglia was possibly induced by TGF-β1 released from cells in the injured brain through Smad2/3 signalling pathway.

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Table 1 Sequence of primers used for Quantitative Real-time PCR. Gene

Forward primer (5′–3′)

Reverse primer (5′–3′)

TNF-α IL-1β TGF-β1 IL-10 TβRI TβRII GAPDH

AGTCCGGGCAGGTCTACTTT TCGTTGCTTGTCTCTCCT ATACGCCTGAGTGGCTGTCT ATCCAGAGGGTCTTCAGCTTC TTTGAGGAGGGCAGCTTTTA AGTTTTGCGACGTGACACTG ACATGCCGCCTGGAGAAACCTGCC

GGCCACTACTTCAGCGTCTC TGATGACGACCTGCTAGTGTG GTTTGGGACTGATCCCATTG ACCAGCTGGACAACATACTGC CACCAGTGAGGAGACCCAAT GGCATCTTCCAGAGTGAAGC TGCCAGCCCCAGCATCAAAGGTGGA

2. Materials and methods 2.1. Animals Adult male Sprague–Dawley rats (250–350 g, 6–8 weeks) were used in this study. All animal work has been approved by Institutional Animal Care & Use Committee and National University of Singapore (IACUC 017/12). The animals were maintained in a 12-hour day/night cycle and provided with food and water. All efforts were made to relieve the pain and reduce the number of rats used in the current study. 2.2. Cortical injection Intracerebral injection of lipopolysaccharide (LPS) to Sprague– Dawley rats was performed as previously described (Zhu et al., 2012). LPS solution (1 μg/μl, 10 μg/rat; Escherichia coli serotype 0127:B8, Sigma, USA) was injected at the location of 3.0 mm posterior and 2.0 mm lateral to the bregma and 2.0 mm deep to the skull surface at the left hemisphere. The injection was completed in 5 min and the needle was kept there for another 5 min prior to withdrawal from the brain.

Fig. 2. Decrease in protein expression of Iba1 after minocycline treatment for 3 days. A. Iba1, a marker for activated microglia, is expressed at a low level in the normal control group (N). At 3 days after LPS treatment (LPS), protein expression of Iba1 significantly increases. However, this effect is inhibited by minocycline (LPS + M) (S: saline treatment); B. Relative density of Iba1 compared with internal β actin control in different treatment groups. Study time point: 3 days post treatment (P3d) (*P b 0.05, **P b 0.01).

The survival time point was set as 1, 3 and 5 days post treatments. Some animals were sacrificed for mRNA and protein analysis at 6 and 12 h after LPS injection.

Fig. 1. Inhibition in expression of LPS-induced cytokines after minocycline treatment. Minocycline was administrated 12 h before and immediately after LPS microinjection and then followed by daily injection for 3 days. A. The mRNA expression of TNF-α, IL-1β, IL-10 and TGF-β1 at 3 days after LPS or LPS + M treatment. B. Protein expression of cytokines TNF-α, IL-1β, IL-10 and TGF-β1 at 3 days after LPS or LPS + M treatment. C. Significant downregulation of protein expression after LPS + M treatment for 3 days compared with LPS administration only shown by the relative density. N: normal control; LPS: LPS treatment; LPS + M: minocycline treatment following LPS injection; S: saline control. Study time point: 3 days post treatment (P3d) (*P b 0.05, **P b 0.01).

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Fig. 3. Double immunofluorescence labelling of NG2 and OX42 and the number of NG2+ or OX42+ cells at 1, 3 and 5 days after minocycline treatment. Expression of NG2 decreases after minocycline treatment at 1 (Ad), 3 (Bj) and 5 days (Cp) compared with LPS injection (Aa, Bg and Cm). Double labelling between NG2 (red) and OX42 (green) is observed at 3 (Bi) and 5 days (Co) after LPS injection. However, the colocalization decreases at both 3 (Bl) and 5 days (Cr) after minocycline treatment. (D) The number of NG2+ cells significantly reduces at different time points after minocycline treatment (LPS + M) compared with that of LPS injection. (E) The number of OX42+ cells also decreases at different time points after minocycline treatment. Study time point: 1, 3 and 5 days post treatment (P1d, P3d and P5d) (bar: 50 μm, average counting area: 0.1 mm2, **P b 0.01).

For anti-CD11b antibody blockage, anti-CD11b antibody solution (1 μg/μl, 1 μg/rat; Millipore, USA) was injected into the cerebral cortex. LPS was then injected into the same site 24 h later. The survival intervals were set as 1, 3, and 5 days. In order to observe the effect of TGF-β1 on activation of NG2 glia, exogenous TGF-β1 (5 ng/μl, 20 ng/rat, PeproTech, USA) was injected at the same location as LPS injection groups. The survival time point was set as 1, 3 and 5 days post treatments. On the other hand, neutralizing TGF-β1 antibody was injected to block the effect of endogenous secreted TGF-β1 immediately following LPS injection. Briefly, the LPS stimulation groups were randomly divided into two groups: one was injected with anti-TGF-β1 (2 μg/rat, 0.5 mg/ml, R&D system, USA) immediately following LPS injection and the other received PBS served as the control. The survival time point was set as 1, 3 and 5 days post treatments. In order to confirm the effective signalling pathway of activation of NG2 glia, anti-TβRII (2 μg/rat, 1 mg/ml, Millipore, USA) as the antagonist of endogenous secreted TGF-β1 was injected immediately after LPS injection. The study time point was set as 3 days after treatments. In summary, the control groups used for different treatments were made as follows: rats without any treatment served as the normal control; rats with saline injection served as LPS injection control; rats with saline in 0.1% BSA injection served as TGF-β1 injection control; rats with PBS injection served as anti-TGF-β1 control and rats with unrelated

antiserum injection served as anti-CD11b control as well as anti-TβRII control. 2.3. Minocycline treatment Minocycline was purchased from Sigma and dissolved in sterile water at 25 mg/ml. The solution was sonicated to ensure complete solubilization. The LPS-treated group was injected intraperitoneally (I.P.) with minocycline 12 h before and immediately after LPS intracerebral injection and then once a day until sacrifice. This treatment protocol was based on those studies in which inhibition of microglial activation has been achieved (Fan et al., 2005a, 2005b). The animals injected with 0.9% saline solution served as the control group. 2.4. Perfusion and tissue sampling At the designated time point, rats with different treatments were deeply anesthetized and then perfused intra-aortically with Ringer's solution (pH 7.4) and subsequent 2% paraformaldehyde (pH 7.4). Brains were removed, postfixed in the same fixative for 4 h and further equilibrated in 20% sucrose of 0.1 M phosphate-buffered solution (PB) overnight at 4 °C. Each frozen brain containing the injection site was sectioned into 30 μm coronal sections using a cryostat (Leica CM 3050, Germany).

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Fig. 4. Immunostaining of NG2 (red) and OX42 (green) after CD11b blockage. Hypertrophy of NG2+ cells and colocalization between NG2 (red) and OX42 (green) are evident after LPS treatment at 1 (D), 3 (E) and 5 days (F). However, administration of anti-CD11b antibody seems to attenuate the activation of NG2+ cells and inhibit the colocalization between NG2 and OX42 (A–C). Bar: 50 μm.

2.5. Immunohistochemistry The sections were washed with 1 × PBS plus 0.1% Triton X-100 for 10 min × 3 times and then blocked with 5% normal goat serum for 1 h at room temperature. Sections were further incubated with mouse anti-NG2 (Abcam, UK), rabbit anti-NG2 (Millipore, USA), mouse anti-OX42 (Santa Cruz, USA), rabbit anti-TGF-β1 (Abcam, UK), rabbit anti-TβRI (Santa Cruz, USA), rabbit anti-TβRII (Millipore, USA), rabbit anti-IL-1β (Millipore, USA) and rabbit anti-iNOS (Abcam, UK) overnight at room temperature. Next day, sections were washed with 1 × PBS for 3 times and incubated with goat anti-mouse Alexa Fluor 488 (Invitrogen, USA) and goat anti-rabbit Alexa Fluor 594 (Invitrogen, USA) for 1 h. DAPI solution was then added to the sections for 2 min to stain nuclei. Finally, sections on the slides were mounted with nonfluorescent mounting medium (Dako, Denmark), viewed and photographed using a Laser-Scanning Confocal Microscope (Olympus FluoView™ FV1000, Japan). 2.6. Western blot Total proteins were extracted from the brain tissues containing the injury site using M-PER tissue Protein Extraction Reagent (Pierce, USA), EDTA, and protease inhibitor cocktail (Pierce, USA). The concentration of total protein in the tissue was measured according to Bradford's method (Bradford, 1976). Samples containing 90 μg (for pSmad2/3) or 20 μg proteins (for other proteins) were separated by SDS gel electrophoresis and protein bands were electroblotted onto 0.2 μm PVDF membrane (Bio-Rad Laboratories, USA). After blocking 1 h with 5% milk in TBS-0.1% Tween buffer, membranes with the respective protein bands were then incubated with the primary antibodies: rabbit anti-TNF-α (Millipore, USA), rabbit anti-IL-1β (Millipore, USA), rabbit anti-IL-10 (Abcam, UK), rabbit anti-TGF-β1 (Abcam, UK), rabbit anti-Iba1 (Wako, Japan), mouse anti-GFAP (Millipore, USA), rabbit anti-Smad2/3 (Cell Signaling, USA) and mouse anti-β actin (Sigma, USA) overnight at 4 °C. The membrane staining for p-Smad2/3 was blocked 1 h with 5% BSA in TBS-0.1% Tween buffer before incubation with rabbit anti-p-Smad2/3 (Cell Signaling, USA). Next day, the

membranes were incubated with HRP-conjugated secondary antibodies (Pierce, USA) for 1 h at room temperature. After further washing, supersignal west dura extended duration substrate (Thermo Scientific, USA) was applied to the protein bands before X-ray exposure. The band density was then measured using the Quantity One software (Bio-Rad, USA). 2.7. RNA extraction, Reverse transcription and Quantitative Real time PCR Total RNA was extracted from the brain tissue containing the injury site using RNeasy Microarray Tissue Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. The quantity of total RNA was measured with a Nanodrop machine (Thermo Scientific, USA). Total 2 μg of RNA was converted to cDNA with the reverse transcriptase kit (Promega, USA), containing Oligo dT primers, dNTP, RNase inhibitor and M-MLV reverse transcriptase. The converted cDNA was then used as the template for qPCR with the ABI real-time PCR instrument (Invitrogen, USA). Briefly, a mixture was composed of 5 μl 2 × SYBR Green fast master mix (Invitrogen, USA), 0.5 μl of 5 μM forward primer, 0.5 μl of 5 μM reverse primer, 1 μl of 10 times diluted cDNA and top up to final volume of 10 μl with RNase/DNase free water. The programme was set as 95 °C for 15 min for the initial step followed by 40 cycles of amplification. Each cycle consisted of denaturation at 95 °C for 1 s and annealing at 60 °C for 20 s. The expression of target genes was normalised to GAPDH as an internal control. The forward and reverse primer sequences for each gene were listed in Table 1. Gene expression was quantified using the 2− ΔΔCT method as previously described (Livak and Schmittgen, 2001). 2.8. Cell counting The number of total NG2+ cells, NG2+/OX42+ cells and total OX42+ cells was determined in a confocal microscope (Olympus FluoView™ FV1000, Japan). Eight randomly selected microscopic fields (each measuring an area of 0.1 mm2) at 40× objective from each section were examined (n = 3). Cells with a NG2 (red) labelled cell body overlapping with OX42 (green) labelled cell body were NG2+/OX42+ cells (yellow

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Fig. 5. mRNA and protein expression of cytokines in the LPS focal injury model. A. The mRNA expression of proinflammatory cytokines like TNF-α and IL-1β as well as anti-inflammatory cytokines like TGF-β1 and IL-10 at 6 h, 12 h, 1 day, 3 days and 5 days after LPS treatment and normal/saline control group. B. Western blot of TNF-α, IL-1β, TGF-β1 and IL-10 at different time points. N: normal control; L: LPS injection; S: saline control. In this experiment, saline injection was used as a control for LPS injection up to 5 days (*P b 0.05, **P b 0.01).

colour). The cells were counted only where a cell body and nucleus could be identified.

3. Results

2.9. Statistical analysis

3.1. Inhibition of microglial activation in LPS induced neuroinflammation model with minocycline

In this study, at least 3 animals for each group have been used. After LPS injection, a total of 30 coronal sections with 30 μm of sickness from each rat were taken from the area containing the lesion centre (about 900 μm from rostral to caudal) for the immunohistochemical study. This collected part of brain contained the most intense glial reactivity. For mRNA and protein analysis, around 3 × 3 × 3 mm3 tissue piece was dissected out from the operation side containing the lesion centre. All the immunostaining pictures were taken at the distance around 30 μm to 300 μm from the edge of lesion centre. The quantitative data were presented as the mean ± standard deviation. The statistical significance of differences between normal control, saline control, LPS injection and treatment groups was evaluated using Student's t-test or the one-way ANOVA. The level of significance was determined by *P b 0.05 and **P b 0.01.

3.1.1. Attenuation in expression of LPS-induced cytokines Minocycline, a tetracycline-type antibiotic, is a potent inhibitor of microglial activation with no direct effect on astrocytes and neurons. In this study, minocycline was employed to block the activation of microglia and then the responses of NG2 glia were investigated. The mRNA expression of IL-1β, IL-10 and TGF-β1 significantly decreased after minocycline treatment compared with LPS treatment (Fig. 1A). The protein expression of cytokines was subsequently detected by the western blot. At 3 days after LPS injection, protein expression of cytokines including TNF-α, IL-1β, IL-10 and TGF-β1 profoundly increased in comparison to the normal control group, but was attenuated after minocycline treatment (Fig. 1B and C). This result demonstrated that minocycline attenuated LPS-induced cytokine production, which was consistent with other studies that minocycline has the

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NG2+ cells also decreased. This may suggest that the activation of NG2+ cells was possibly under the downstream of microglial reaction. 3.3. Attenuation of activation of NG2+ cells after anti-CD11b treatment Since the minocycline may not selectively inhibit microglia, the antibody against CD11b, a subunit of complement receptor type 3 of microglia, was administrated. After LPS focal injection, NG2+ cells morphologically transformed from a typically small cell body with long and fine processes to a larger cell body with thicker processes (Fig. 4D–F). Administration of anti-CD11b antibody, however, attenuated the LPS-stimulated NG2 positive staining as well as the colocalization between NG2 and OX42 (Fig. 4A–C), suggesting that antibody specific blockage of microglial CD11b inhibited the reactive responses of NG2+ cells. 3.4. Correlation of the expression of TGF-β1 with the active responses of NG2+ cells

Fig. 6. mRNA expression of TβRI and TβRII in the LPS focal injury model. A. Fold change of TβRI in LPS treatment group, saline control group and normal control group across 1, 3, and 5 days. B. Fold change of TβRII in different treatments across 1, 3, and 5 days (*P b 0.05, **P b 0.01).

anti-inflammatory effects (Yrjanheikki et al., 1999; Sanchez Mejia et al., 2001; Tomas-Camardiel et al., 2004). 3.1.2. Decreases in protein expression of Iba1 after minocycline treatment To assess the effect of minocycline on the activation status of microglia, protein expression of Iba1, a marker of activated microglia was investigated. Western blot bands of Iba1 were shown in Fig. 2A and relative density to β actin was calculated in Fig. 2B. At 3 days post-LPS administration, the expression of Iba1 greatly increased compared with the normal control group. However, this LPS-induced increase in expression of Iba1 was significantly retarded after minocycline treatment for continuous three days. This observation confirmed the suppression of microglial activation by minocycline. 3.2. Downregulation in responses of NG2+ cells by minocycline treatment In order to investigate the responses of NG2+ cells after minocycline treatment, double immunofluorescence staining of antibodies against NG2 (red) and OX42 (green) was carried out at 1 (Fig. 3A), 3 (Fig. 3B) and 5 days (Fig. 3C) after minocycline treatment. The responses of OX42+ microglia (green) declined at different time intervals (Fig. 3Ae, Bk and Cq) compared with LPS treatment, suggesting the successful inhibition of microglial activation. Additionally, the staining of NG2 also became weaker at 1 (Fig. 3Ad), 3 (Fig. 3Bj) and 5 days (Fig. 3Cp). Furthermore, fewer NG2+/OX42+ cells after minocycline treatment (Fig. 3Bl and Cr) were observed compared with LPS treatment. In order to confirm this observation, the number of NG2+ cells as well as OX42+ cells was quantified through cell counting. The number of total NG2+ cells significantly decreased at different time points after minocycline treatment (Fig. 3D), as well as the number of OX42+ microglia (Fig. 3E). Taking together, the above results have shown that in parallel to the inhibition of microglial activation, the responses of

Since microglia secrete a lot of cytokines upon LPS stimulation, it may be possible that those cytokines lead to the activation of NG2+ cells. Therefore, in this study, the mRNA and protein expression of pro-inflammatory cytokines like TNF-α and IL-1β as well as antiinflammatory cytokines like IL-10 and TGF-β1 was investigated (Fig. 5). Considering TNF-α and IL-1β were usually produced and peaked at early time point after LPS induction, 6 h and 12 h-time points were chosen in this study. The mRNA expression of TNF-α significantly increased and peaked at 6 h after LPS injection. IL-1β had a similar pattern of expression, which significantly increased at 6 h and peaked at 12 h after LPS induction. However, the anti-inflammatory cytokines such as IL-10 and TGF-β1 were expressed at a high level during the late time points. IL-10 was expressed highly at 12 h, 1 day and 3 days, whilst TGF-β1 significantly increased at 1 day, 3 days and remained a high level at 5 days. Protein expression of these cytokines was shown in Fig. 5B. Our group previously reported the colocalization between NG2 and OX42 in the same LPS injection model and the gradual increase in the number of NG2+/OX42+ cells (Zhu et al., 2012). It seems that the appearance of NG2+/OX42+ cells was closely consistent with the increase in expression of TGF-β1. Therefore, it may be possible that the appearance of NG2+/OX42+ cells was related to the expression of TGF-β1. 3.5. Activation of TGF-β1 signalling pathway in LPS induced neuroinflammation model 3.5.1. Upregulation of TGF-β1 receptors In view of the above results, TGF-β1 seems to be more correlated with the progression of inflammation process. Therefore, the signalling pathway of TGF-β1 was observed in this study through detecting the cellular expression of its two receptors. The mRNA expression of TβRI in LPS treatment group significantly increased at 3 and 5 days compared with the normal group (Fig. 6A). The mRNA expression of TβRII also significantly increased at different time points compared with the normal control (Fig. 6B). In order to find out which cells responded to TGF-β1 activity, antiTβRI/anti-TβRII antibody was co-stained with either cell markers of NG2 glia or microglia. TβRI (Fig. 7A, B and C) and TβRII (Fig. 7G, H and I) were co-localized with anti-OX42, appearing in yellow colour indicated by the arrows. This result was consistent with another study that activated microglia expressed both two receptors in response to hypoxia exposure (Li et al., 2008). NG2+ cells also expressed TβRI (Fig. 7D, E and F) and TβRII (Fig. 7J, K and L). 3.5.2. Cellular sources of secreted TGF-β1 It has been reported that TGF-β1 was upregulated in astrocytes, microglia and neurons in response to injuries (Wu et al., 2007, 2008). In

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Fig. 7. Immunofluorescence staining of anti-TβRI and anti-TβRII with anti-OX42 or ant-NG2 antibodies in the LPS focal injury model. Expression of TβRI and TβRII is observed at 1, 3 and 5 days after LPS injection. TβRI (A, B and C) as well as TβRII (G, H and I) are co-localized with OX42 labelled microglia (yellow cells or green cell bodies encircling red). TβRI (D, E and F) and TβRII (J, K and L) are also expressed in NG2+ cells. Bar: 50 μm.

this LPS focal injury model, the cellular localization of TGF-β1 was investigated through double labelling between anti-TGF-β1 and OX42 or NG2. As shown in Fig. 8, TGF-β1 was expressed by OX42+ microglia at 1 (Fig. 8Ac), 3 (Fig. 8Af) and 5 days (Fig. 8Ai) post-LPS injection. It was consistent with other findings that activated microglia were the primary source of TGF-β1 in a middle cerebral artery occlusion animal model (Lehrmann et al., 1998; Doyle et al., 2010). However, NG2+ cells do not co-localize with TGF-β1 at different time intervals (Fig. 8Bl, Bo and Br), suggesting that TGF-β1 may influence NG2+ cells in a paracrine manner. 3.6. Activation of NG2+ cells by direct injection of exogenous TGF-β1 into the cerebral cortex 3.6.1. Active responses of NG2+ cells In order to find out the direct effect of TGF-β1 on NG2+ cells, exogenous TGF-β1 was injected at 5 ng/μl, total 20 ng into the rat brain cortex, as the same injection site as LPS treatment. NG2+ cells were more activated and expression of NG2 more intense in TGF-β1 injection

group (Fig. 9Ad, Ae and Af), compared with saline control (Fig. 9Aa, Ab and Ac). Additionally, it seems that TGF-β1 direct injection led to a significant increase in the colocalization between NG2 and OX42 (Fig. 9Bj, Bk and Bl) compared with the control group (Fig. 9Bg, Bh and Bi). Further through quantification of cell number, TGF-β1 treatment induced increase in the number of NG2+ cells (Fig. 9C) as well as NG2+/OX42+ cells (Fig. 9D) compared with the saline control. 3.6.2. More profound increase in the number of NG2+ cells and NG2+/ OX42+ cells compared with LPS injection In order to compare the effect between TGF-β1 injection and LPS injection, the number of NG2+/OX42+ cells (Fig. 10A) and total NG2+ cells (Fig. 10B) was quantified. As shown in Fig. 10A, there was no difference in the number of NG2+/OX42+ cells between two groups post 1 day injection. However, at 3 days after injection, TGF-β1 led to a significant increase in the number of NG2+/OX42+ cells compared with LPS. At 5 days after treatment, it seems no difference. Similar changes could also be observed in the number of total NG2+ cells (Fig. 10B). These results indicate that exogenous TGF-β1 may induce an increase in the

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Fig. 8. Immunostaining between anti-TGF-β1 and anti-OX42 or anti-NG2 antibodies in the LPS focal injury model. Expression of TGF-β1 (red) is observed at 1 (b, k), 3 (e, n) and 5 days (h, q) after LPS injection. It is expressed by OX42+ microglia at 1 (Ac), 3 (Af) and 5 days (Ai), appearing in yellow colour indicated by the arrows. However, it is not expressed in NG2+ cells at different time points (Bl, Bo and Br). Bar: 50 μm.

number of NG2+ cells and trigger an earlier accumulation of NG2+/ OX42+ cells. 3.7. Reduced activation of NG2+ cells after blockage of TGF-β1 signalling pathway in LPS induced neuroinflammation model 3.7.1. Inhibition of active responses of NG2+ cells after neutralizing endogenously secreted TGF-β1 As shown in the above results, the activation of NG2+ cells was possibly downstream of microglial reaction. In addition, direct injection of TGF-β1 induced the activation of NG2+ cells and led to the increase in the number of NG2+/OX42+ cells compared with the LPS injection group. In order to further confirm this view, neutralizing TGF-β1 antibody was used to block the effect of TGF-β1 and then the responses of NG2+ cells were investigated through immunofluorescence study (Fig. 11). The responses of NG2+ cells decreased after co-injection of LPS and anti-TGF-β1 (Fig. 11D, E and F) compared with the LPS + PBS treatment group (Fig. 11A, B and C). In addition, fewer NG2+/OX42+ cells were observed at different time intervals after inhibition of the TGF-β1 effect. 3.7.2. Abolishment of increase in the number of NG2+ cells after neutralizing endogenously secreted TGF-β1 As shown in Fig. 11, the activation of NG2+ cells decreased after neutralizing secreted TGF-β1. In order to quantify this data, counting of the number of NG2+ cells, NG2+/OX42+ cells as well as percentage of NG2+/OX42+ cells was made at different time points. The number of total NG2+ cells significantly decreased at 1, 3 and 5 days compared with the LPS + PBS treatment (Fig. 12A). Furthermore, the number of NG2+/OX42+ cells also declined at different time points after injection of neutralizing anti-TGF-β1 antibody (Fig. 12B). The percentage of NG2+/OX42+ cells was calculated through the number of NG2+/ OX42+ cells divided by the number of total OX42+ cells. The percentage of NG2+/OX42+ cells also decreased a little at both 1 and 3 days, whilst significantly downregulated from 16.2% to 4.6% at 5 days after anti-TGFβ1 injection. Therefore, these results suggest that the NG2+ cells may be possibly regulated by the secreted TGF-β1 in this LPS induced neuroinflammation model.

3.7.3. Reduced activation of NG2+ cells with neutralizing anti-TβRII through Smad2/3 signalling pathway In this study, anti-TβRII, an antagonist of TGF-β1, was injected to the brain cortex immediately after LPS injection to block the TGF-β1 effective signalling pathway. As shown in Fig. 13A and B, protein expression of total Smad2/3 was not changed at 3 days after LPS + anti-TβRII treatment compared with either LPS + vehicle injection or normal control. However, p-Smad2/3 significantly increased after LPS + vehicle injection compared with the normal control group (Fig. 13A and C), suggesting that the TGF-β1 signalling pathway was activated upon LPS induced neuroinflammation, whilst this effect could be inhibited by the antiTβRII treatment. After inhibition of TGF-β1 signalling pathway with anti-TβRII injection, the responses of NG2+ cells were detected by the immunofluorescence staining (Fig. 14). At 3 days after co-injection of LPS and vehicle, colocalization between NG2 and OX42 was observed (Fig. 14A). However, this effect was inhibited after anti-TβRII treatment (Fig. 14C). In addition, it seems that the responses of NG2+ cells became weaker (Fig. 14D), showing by more processes, compared with LPS + vehicle treatment (Fig. 14B). Further quantification of cell numbers showed that the number of NG2+ cells significantly decreased in the antiTβRII treatment group as well as the number of NG2+/OX42+ cells (Fig. 14E). The percentage of NG2+/OX42+ cells to total OX42+ cells also decreased (Fig. 14F). Taken together, it may suggest that the activation of NG2+ cells was mediated through Smad2/3 signalling pathway.

4. Discussion We have observed activation of NG2 cells in the current neuroinflammatory model including two portions, i.e., constitutive NG2 glia (NG2+/OX42−) and NG2-positive microglia (NG2+/OX42+). In current immunohistochemistrical study, microglia became positive to the anti-NG2 antibody at different time points after focal LPS injection in the brain cortex, which is consistent with many studies describing NG2 expression in microglia (Gao et al., 2010; Zhu et al., 2010). But one possibility should be considered that the proteolysis-sensitive ectodomain of NG2 protein may be easily cleaved off from constitutive

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Fig. 9. Double labelling between anti-NG2 and anti-OX42 antibodies as well as quantification of cell number at 1, 3 and 5 days after TGF-β1 treatment. The responses of NG2+ cells become more intense after TGF-β1 treatment for 1 (Ad), 3 (Ae) and 5 days (Af) compared with saline control for 1 (Aa), 3 (Ab) and 5 days (Ac). In addition, the colocalization between NG2 (red) and OX42 (green) is more obvious in TGF-β1 treatment for 1 (Bj), 3 (Bk) and 5 days (Bl) compared with saline control for 1 (Bg), 3 (Bh) and 5 days (Bi). The number of NG2+ cells (C) and NG2+/OX42+ cells (D) increased after TGF-β1 injection at different time points compared with saline control (bar: 50 μm, average counting area: 0.1 mm2, *P b 0.05, **P b 0.01).

NG2 glia (Nishiyama et al., 1995; Asher et al., 2005) and then tightly stick to the surface of other cells in the brain parenchyma. However, if such non-specific binding could be true, we should be able to observe colocalization between NG2 and markers for other cell types.

But our current and previous observations discovered only selective co-staining of NG2 and microglial markers in the pathological condition. It is therefore unlikely that the co-labelling of NG2 and the markers of microglia/macrophages was due to non-specific

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Fig. 10. Comparison of number of NG2+/OX42+ cells and total NG2+ cells at 1, 3 and 5 days between LPS injection and TGF-β1 treatment. Number of NG2+/OX42+ cells (A) and total NG2+ cells (B) significantly increases at 3 days after TGF-β1 treatment compared with the LPS group. There are no significant changes in the number of total NG2+ cells and NG2+/OX42+ cells at 1 and 5 days in both groups (average counting area: 0.1 mm2, *P b 0.05, **P b 0.01).

binding of cleaved segment of NG2 protein to the surface of other cells. Our findings further suggest that activation of NG2+ cells may be an event downstream to microglial reaction. In current study, the responses of NG2+ cells decreased following minocycline treatment

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shown by a decline in staining intensity of NG2+ cells, in colocalization between NG2 and OX42 as well as in the cell number of NG2+ cells. Minocycline, a tetracycline derived antibiotic, has anti-inflammatory properties in the CNS. It blocks secretion of inflammatory cytokines through inhibition of p38 MAPK pathway in microglia (Tikka and Koistinaho, 2001; Tikka et al., 2001; Zhu et al., 2002). In our LPSinduced neuroinflammation model, the activation of microglia was attenuated after minocycline treatment. The expression of cytokines, including TNF-α, IL-1β, TGF-β1 and IL-10, decreased. In addition, the protein expression of Iba1, an antigen marker for activated microglia, was also downregulated. These observation is in agreement with other in vitro and in vivo findings, in which the anti-inflammatory effects of minocycline towards microglia were shown by attenuation in expression of cytokines and antigen marker OX42 (Yrjanheikki et al., 1999; Tikka and Koistinaho, 2001; Tikka et al., 2001; Krady et al., 2005; Bye et al., 2007; Guasti et al., 2009). Our current results showed that LPS-induced increase in GFAP expression in astrocytes was not affected by minocycline treatment (Supp. Fig. 1). Other studies have also reported that minocycline did not show direct effects on astrocytes and neurons (Amin et al., 1996; Tikka and Koistinaho, 2001; Tikka et al., 2001). Up-to-date no study has reported a direct effect of minocycline on the constitutive NG2 glia. It is reasonable to assume that the inhibition of microglial reaction with minocycline could result in blockage of constitutive NG2+ glial reaction. This notion is supported by our second evidence. We observed that the anti-CD11b antibody, which binds to α subunit of complement receptor type 3 of microglia/macrophages to specifically inhibit microglial functions (Bruck and Friede, 1990; Garcia et al., 1996), could eventually block NG2 cell activation. This direct evidence further suggests that the activation of NG2+ cells may be in a chain of cell reaction downstream to microglial reaction. As well known, after CNS injuries, the earliest cell responses come from infiltration of macrophages and activation of microglia. Microglia could express a variety of cytokines to influence the activities of oligodendrocytes (Takahashi et al., 2003) and astrocytes (Balasingam and Yong, 1996) in the CNS. After intracerebral injection of LPS into neonatal rat brain, IL-1β was mainly expressed by microglia/macrophages but not astrocytes (Pang et al., 2003). Another study also showed the similar finding that activated microglia were stained with TNF-α and iNOS after injection of LPS into rat cortex (Shin et al., 2004). The current result also showed that microglia were the major source to expression IL-1β and

Fig. 11. Double labelling between anti-NG2 (red) and anti-OX42 (green) antibodies at 1, 3 and 5 days after co-injection of LPS and neutralizing anti-TGF-β1 antibody. LPS triggers the colocalization between NG2 (red) and OX42 (green) at 1 (A), 3 (B) and 5 days (C). However, it decreases after blocking the effect of TGF-β1 with neutralizing antibody at different time intervals (D, E and F). Bar: 50 μm.

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Fig. 12. Decrease in the number of NG2+ cells, NG2+/OX42+ cells as well as percentage of NG2+/OX42+ cells after LPS plus anti-TGF-β1 injection. A. The number of NG2+ cells compared between LPS + anti-TGF-β1 treatment and LPS + PBS control at 1, 3 and 5 days. B. The number of NG2+/OX42+ cells compared between LPS + anti-TGF-β1 treatment and LPS + PBS control at different time points. C. The percentage of NG2+/OX42+ cells to total OX42 cells (average counting area: 0.1 mm2, *P b 0.05, **P b 0.01).

iNOS (Supp. Fig. 3). Since the expression of cytokines decreased following the downregulation of microglial responses in this study, the activation of NG2+ cells may be possibly assumed to mediate through the secreted cytokines from microglia. Therefore, cytokine expression was investigated in this LPS induced neuroinflammation model. Results have shown that pro-inflammatory cytokines as TNF-α and IL-1β were expressed at a high level at the early time points as 6 h and 12 h,

whilst anti-inflammatory cytokines such as TGF-β1 and IL-10 at late time points. The expression of TGF-β1 significantly increased at 1 day, 3 days and still remained a high level at 5 days, which was more closely consistent with the gradual increase in the number of NG2+/OX42+ cells. TGF-β1 may exert effects on constitutive NG2 glia. TGF-β1 was able to increase the chondroitin sulphate proteoglycan (CSPG) and related to

Fig. 13. Western blot analyses of the expression of Smad2/3 and p-Smad2/3 at 3 days after LPS plus anti-TβRII treatment. Immunoblotting with antibodies at 3 days post-injection. A. Smad2/3 shows no significant change between different groups. P-Smad2/3 significantly increases after LPS + vehicle treatment compared with the normal control group, whilst decreases after inhibition of TGF-β1 signalling pathway with anti-TβRII. B. Statistical analyses of the protein bands were done by normalising the target protein band densities to the β actin internal control. N: normal control (*P b 0.05).

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Fig. 14. Decrease in the colocalization between NG2 and OX42 at 3 days after injection of anti-TβRII antibody. Confocal images showing the colocalization between NG2 (red) and OX42 (green) at 3 days after co-injection of LPS and vehicle (A), whilst this colocalization is inhibited after anti-TβRII treatment (C). Cell counting of the number of total NG2+ cells, NG2+/OX42+ cells (E) as well as the percentage of NG2+/OX42+ cells to total OX42+ cells (F) (bar: 50 μm, average counting area: 0.1 mm2, **P b 0.01).

the formation of glial scar (Logan et al., 1994; Asher et al., 2000). It has been reported found that intracerebral injection of TGF-β1 could induce the increase in NG2 protein level and hypertrophy of NG2 glia (Rhodes et al., 2006). On the other hand, inhibition of endogenous TGF-β1 with its neutralizing antibody was shown to attenuate the scar formation (Logan et al., 1994). Application of decorin, potentially inhibiting TGFβ1 activities, reduced the expression of NG2 protein in the lesion site (Davies et al., 2004). In concert with these reports, we also found that exogenous TGF-β1 injection led to the activation and increase in the number of NG2+ cells. In comparison with the LPS treatment group, increases in the number of both NG2+ cells were more significant at

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3 days after TGF-β1 injection. These results indicate that exogenous TGF-β1 triggered an earlier activation of NG2+ cells. TGF-β1 might exert its effect on NG2 cells through microglia. In various injury models including stab wound injury (Lindholm et al., 1992), facial nerve axotomy (Kiefer et al., 1993) and ischemic injury model (Lehrmann et al., 1998; Doyle et al., 2010), activated microglia/macrophages were the primary cellular source to express TGF-β1 and expression of TGF-β1 is closely linked to microglial activation (Kiefer et al., 1995). As shown by a knife-cut injury model, microglia were colocalized with chondroitin sulphate (a side chain of NG2 protein). Further in vitro study demonstrated that the expression of above proteins on microglia was induced by TGF-β1 which upregulated the main enzymes required for biosynthesis of chondroitin sulphate (Yin et al., 2009). Moransard et al. (2011) demonstrated that TGF-β induced NG2 expression in macrophages by in vitro study. In the current study, the onset of NG2+/OX42+ microglia was induced by direct injection of TGF-β1 in the brain but attenuated after application of neutralizing TGF-β1 antibody. It therefore may be concluded that the phenomenon of microglia becoming NG2 positive may be induced by TGF-β1. A recent published paper based on an in vitro study also proved this view and reported that primary cultured microglia enhanced the expression of NG2 protein after stimulation with TGF-β1 (Sugimoto et al., 2014). However, the specific signalling pathway that TGF-β1 induced NG2 protein expression on microglia is still not elucidated. In our study, we present evidence to suggest that the NG2 protein expressed in microglia might be involved in the Smad2/3 signalling pathway. A previous in vitro study on astrocytes confirmed that the production of CSPG is Smad dependent (Susarla et al., 2011). In their study, knocking down Smad2 or Smad3 led to a differential change of CSPG core proteins as well as the enzymes for synthesis and modification of GAG side chains. The role of Smad proteins in regulation of CSPG was also confirmed in a stab wound in vivo model (Wang et al., 2007). The Smad3 null mice exhibited fewer NG2 glia and microglial cells near the injury site compared with the wild type, whilst the level of TGF-β1 was not influenced. Therefore, these studies suggest that TGF-β1 might induce the production of CSPG through Smad2/3 signalling pathway. In our current study, the number of NG2+/OX42+ cells decreased after treatment of anti-TβRII antibody, which specifically blocked the TGF-β1 Smad2/3 signalling pathway shown by a decrease in phosphorylation of Smad2/3. The NG2 protein expressed in microglia could therefore be induced through the Smad2/3 signalling pathway. In conclusion, our results suggest that the activation of NG2 glia, including constitutive NG2 glia and NG2-positive microglia, might be an event downstream to microglial activation which may be involved in TGF-β1 mediated Smad2/3 signalling pathway. In our previous study, NG2 positive microglia (NG2+/OX42+ cells) lost their phagocytic capacity in the same LPS injection model (Zhu et al., 2012). Since TGFβ1 could play a role in mediation of microglial activation and cytotoxicity (Kiefer et al., 1995) and induce NG2 protein expression in microglia in this study, it is reasonable to assume that TGF-β1 may regulate intensity (strong or weak) and property (destructive or protective) of microglial activation through regulation of NG2 protein expression in response to brain tissue damage. Further studies are required in order to fully understand this view. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2015.01.006. Abbreviations BSA bovine serum albumin CNS central nervous system CSPG 4 chondroitin sulphate proteoglycan NG2 GAG glycosaminoglycan IL-1β interleukin-1 beta IL-10 interleukin-10 iNOS inducible nitric oxide synthase I.P. intraperitoneally

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LPS MAPK PBS PCR TBS TGF-β1 TβRI TβRII TNF-α

lipopolysaccharide mitogen-activated protein kinases phosphate buffered saline polymerase chain reaction Tris-buffered saline transforming growth factor beta 1 transforming growth factor receptor I transforming growth factor receptor II tumour necrosis factor-alpha

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The activation of NG2 expressing cells is downstream to microglial reaction and mediated by the transforming growth factor beta 1.

In the present study, we investigated the mechanism of activation of NG2 expressing cells. Application of microglial inhibitors not only attenuated mo...
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