Dexamethasone promotes IL-4-induced alternative activation at PPARγ point, instead of upstream STAT6 in BV2 microglial cells

Microglia are innate immune effector cells in the central nervous system and play an extremely important role in the physiological processes of the central nervous system. When microglia are activated, there are two polarization states, M1 and M2 phenotype. Dexamethasone is a glucocorticoid widely used in clinical practice, which pharmacological effects are mainly anti-inammatory, anti-toxic. However, whether Dexamethasone affects polarization state of microglia is unknown. In this study, we investigate the effect of Dexamethasone on IL-4-induced alternative activation in murine BV-2 microglial cells. to of markers found we the effects of Dexamethasone on induced of transducer and activators of transcription 6 (STAT6) and peroxisome proliferator-activated receptor-gamma (PPARγ).


Results
Dexamethasone promoted IL-4 induced microglia alternative activation by increasing the expression of Arg1 and FIZZ1. Dexamethasone also enhanced the expression of PPARγ. These effects were reversed by RU486 (a Dexamethasone antagonist). Further, the effects of Dexamethasone and IL-4 on Arg1 and FIZZ1 were blocked by the application of GW9662 (a PPARγ antagonist).

Conclusions
Our studies con rm that Dexamethasone promotes IL-4 induced alternative activation via STAT6/PPARγ signaling pathways in microglia. At the same time, it was con rmed that Dexamethasone acts on PPARγ instead of STAT6. These ndings support that Dexamethasone has a therapeutic potential for neuroin ammatory diseases via alternative activation.

Background
Microglia are considered to be the resident macrophages, which play a critical role in in ammatory reactions of the central nervous system (CNS) [1]. They are activated multiple states: M1 phenotype and M2 phenotype, during diverse conditions, such as trauma, in ammation, and infection. Generally, M1 microglia, also called classical phenotype, participate a pro-in ammatory role through secretion of proin ammatory factors, including TNF-α, IL-1, IL-6 [2][3][4][5][6]. M2 microglia, also called alternative phenotype, play an anti-in ammatory role, and promote tissue repair through phagocytosis of protein aggregates and cell debris [2][3][4][5][6][7]. Therefore, suppression of the M1 phenotype and promotion of the M2 phenotype has been considered as a potential strategy in trauma and in ammatory therapy in CNS.
Identifying the microglia phenotype is critical for studying the function of microglia. speci c M1 versus M2 markers have been examined in the context of determining the microglia phenotype. Arg1 is a typical marker for M2 microglia activation that participates in arginine metabolism [8]. FIZZ1 (also known as RELMα) belongs to the resistin-like molecule (RELM) family of secreted mammalian proteins [9]. Both Arg1 and FIZZ1 could be induced in IL-4 mediated M2 classical activation pathway.
Corticosteroids (GC) are widely used clinically for anti-in ammatory and immunosuppressive agents.
Dexamethasone (Dex), a synthetic glucocorticoid, has been demonstrated to suppress the LPS-induced ROS production and NO release, then suppress the LPS-induced M1 phenotype activation in BV2 microglia [10,11]. Microglia showed a reduction of phagocytosis function, pro-and anti-in ammatory cytokine production, and cell proliferation after microglia exposed persistently to glucocorticoid (GC) [12]. However, the effect of Dexamethasone on microglia M2 polarization and molecular mechanisms is still unknown.
Here, we investigated the impact of Dexamethasone on IL-4-induced expression of M2 markers in BV-2 microglial cells and the signaling pathways involving in these processes. Our data show that Dexamethasone up-regulates expression of Arg1 and FIZZ1 in IL-4-treated microglial cells depending on STAT6 and PPARγ signaling pathways.

Cell culture
The murine microglia cell line BV-2 was maintained at 25 cm 2 culture bottles in DMEM ( Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Beyotime, Shanghai, China) at 37℃ in a humidi ed atmosphere of 95% air and 5% CO 2 . BV2 cells were plated on 12-well culture plates at about a density of 1.0×10 6 per well for protein extraction, at 0.8×10 6 per well on a 12-well plate for immuno uorescence. After 12 hours of plating, BV2 cells were treated with different treatments.

Protein extraction
After handled, the cells were washed three times with PBS, and were directly lysed with SDS-PAGE Sample Loading Buffer (2×) on ice and denatured at 100℃ for 10 minutes. The samples were stored at -20℃ for later use.

Statistical analysis
Data are expressed as means ± SEM of the indicated number of independent experiments. Statistical signi cance between multiple groups was analyzed by one-way ANOVA. Statistical analysis was performed using the SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically signi cant.

Results
Dexamethasone promotes IL-4-induced alternative activation of BV-2 microglial cells Firstly, we evaluated the effects of Dexamethasone on IL-4-induced microglia alternative activation; BV-2 cells were treated with 1 μM Dexamethasone for 60 minutes and stimulated with IL-4 (10 ng/ml) for 36 hours. The expression of Arg1 and FIZZ1 were analyzed to evaluate the alternative activation of BV2 cells. We tested the protein levels of Arg1 and FIZZ1 using Western blot analysis. As shown in Figure 1A and B, IL-4 stimulation signi cantly enhanced Arg1 and FIZZ1 protein levels in BV-2 cells (P < 0.01). Treatment with Dexamethasone and RU486 alone did not affect Arg1 and FIZZ1 protein levels (P > 0.05); while a combination of Dexamethasone and IL-4 increased expression of Arg1 and FIZZ1 in BV-2 cells compared to IL-4 treatment alone. Immuno uorescence staining demonstrated that Arg1 and FIZZ1 staining intensity increased after IL-4 treatment, pretreatment with Dexamethasone further increased the staining intensity ( Figure 1C). To evaluate the effects of Dexamethasone, a Dexamethasone antagonist RU486 (2 μM) was administrated 60 minutes before treatment with Dexamethasone. The effects in response to Dexamethasone were reversed by RU486 ( Figure 1A-B).
As shown in Figure 2A, IL-4 increased PPARγ protein levels. Dexamethasone alone failed to activate PPARγ (P > 0.05). However, the combination of Dexamethasone and IL-4 further increased PPARγ protein levels compared to treatment with IL-4 alone, the effects of Dexamethasone were abolished by pretreatment with RU486.
We also examined the effects of Dexamethasone on IL-4 stimulated phosphorylated-STAT6 in BV-2 cells. Treatment with Dexamethasone and RU486 alone did not in uence the IL-4-induced expression of STAT6 (P > 0.05, Figure 2B).
Immuno uorescence staining demonstrated that PPARγ staining intensity increased after IL-4 treatment, pretreatment with Dexamethasone further increased the staining intensity ( Figure 2C ).

PPARγ is essential for induction of Arg1 and FIZZ1 by IL-4 and Dexamethasone
Dexamethasone promotes IL-4-induced PPARγ activation, these facts suggest, but do not prove, that PPARγ is required for the effects of Dexamethasone and IL-4 on Arg1 and FIZZ1 induction. To directly test the effects of PPARγ, BV-2 cells were pretreated with or 2 μM GW9662 for 30 minutes to inhibit the PPARγ signaling pathway and stimulated with Dexamethasone and IL-4 for 36 hours.
Immuno uorescence and Western blot were performed to determine Arg1 and FIZZ1 expression. As expected, Arg1 and FIZZ1 induction by Dexamethasone, Arg and FIZZ1 were markedly blunted by inhibition of PPARγ ( Figure 4).

Discussion
In this study, we rst found that Dexamethasone enhances the IL-4 induced M2 polarization in BV-2 microglial cells. The expression of Arg1 and FIZZ1, IL-4-induced expression of two major alternative activation markers, increase when pretreated with Dexamethasone before IL-4 treated. Furthermore, Dexamethasone enhanced the effects of IL-4 on PPARγ signaling pathways, administering GW9662 before Dexamethasone and IL-4 blunted the induction of Arg1 and FIZZ1. These results verify that Dexamethasone can promote IL-4-induced M2 polarization in BV2 cells via STAT6/PPARγ signaling pathways. Also, Dexamethasone plays a role at PPARγ point, instead of upstream STAT6.
After spinal cord injury, early treatment with steroids can promote the recovery of nerve function and reduce the scope of permanent paralysis [17][18][19]. High-dose methylprednisolone steroid therapy is the only pharmacologic therapy shown to have e cacy in phase three randomized trials when administered within eight hours of injury [20]. However, an aqueous solution of methylprednisolone is unstable [21], and in this study, we chose a more stable and 7 times more powerful glucocorticoid analog, Dexamethasone.
In the central nervous system, microglia are usually maintained in a resting state. When activated, they have been de ned as two speci c activation states: M1 (classical activation) and M2 (alternative activation) in vitro [22]. In this study, we chose Arg1 and FIZZ1 as markers for microglia M2 state. Arg1 expression in macrophages is silent until signals from Th2 cytokines such as IL-4 and IL-13 [23]. In the cytoplasm, Arginase catalyzes the hydrolysis of L-arginine to L-ornithine and urea, which comprises the nal step of the urea cycle. This cycle provides the principal route for the disposal of nitrogenous waste from protein catabolism. Apart from Arg1, FIZZ1 is another marker for alternative activation [24]. Found in in ammatory zone 1 (FIZZ1), also known as resistin-like moleculeα (RELMα), a member of the newly described cysteine-rich secreted family of FIZZ/RELM.7-9. FIZZ1 is considered to be relevant to probrosis [25] and regulating in ammation [26]. First, we examined the effect of IL-4 and Dexamethasone on Arg1 and FIZZ1 expression. In our study, treatment with Dexamethasone alone had no effect on Arg1 and FIZZ1 expression in microglia, while pretreatment with Dexamethasone enhanced the effect of IL-4 on Arg1 and FIZZ1 expression. This indicated that Dexamethasone may be possible to promote the resolution of neuroin ammation by regulating microglia polarization. The effect of Dexamethasone was reversed by the use of Dexamethasone antagonist RU486.
The synergistic effect of Dexamethasone and IL-4 on Arg1 and FIZZ1 expression may be explained by the enhancement of IL-4 induced STAT6 / PPARγ activation by Dexamethasone. IL4 induces the expression of Arg1 and FIZZ1, depending on the activation of STAT6/PPARγ. After IL4 stimulated microglia, STAT6 was phosphorylated, transferred to the nucleus and bound to the Jmjd3 promoter, and activated the activation of the M2 marker gene [27]. STAT6 can also regulate the activity of the nuclear receptor PPARγ [28]. PPARγ plays an important role in the replacement activation of macrophages and microglia; PPARγ agonists can directly stimulate the expression of Arg1 and FIZZ1 [29][30][31][32]. In this study, Dexamethasone could not enhance IL4-induced phosphorylation of STAT6 but could increase IL4-induced PPARγ activity. ABC et al. Reported that pioglitazone can increase the expression of Arg1 and Ym1 by activating PPARγ, thereby promoting the M2 polarization of microglia [30]. In this study, the PPARγ antagonist GW9662 can reduce the synergistic effect of Dexamethasone and IL-4 on Arg1 and FIZZ1 expression, proving that PPARγ is necessary for Dexamethasone and IL-4-induced microglial M2 polarization. These results indicate that Dexamethasone relies on the STAT6 / PPARγ signaling pathway to enhance IL-4 induced activation of microglia M2 subtypes (Fig. 5).

Conclusions
The results showed that Dexamethasone promoted IL-4 induced microglia replacement activation by activating the STAT6 / PPARγ signaling pathway. The promoting effect of Dexamethasone on the replacement activation of microglia may be one of the possible mechanisms of Dexamethasone treatment of spinal cord injury. Dex promotes the IL-4-induced alternative activation of BV-2 cells. BV-2 cells were respectively treated with 1 μM Dex for 60 minutes, 2 μM RU486 and stimulated with IL-4 (20 ng/ml) for 36 hours. The expression of Arg1 and FIZZ1 were analyzed to evaluate the alternative activation of BV2 cells. We tested the protein levels of Arg1 and FIZZ1 using Western blot analysis (A, B). A representative result from three independent experiments is shown. Quanti cation for Arg1 and FIZZ1 was normalized by β-Actin and Grapdh respectively. Data are presented as mean ± SEM for three independent experiments. Asterisks indicate statistically signi cant difference (*P < 0.01, **P < 0.05). Representative images of BV-2 cells with positive immuno uorescence staining for Arg1 and FIZZ1 (C). Scale bars indicate 20 μm.  Dex enhances IL-4-induced PPARγ activation. BV-2 cells were pretreated with Dex (1 μM) or GW9662 (2 μM). Nuclear extracts were prepared 24 hours after IL-4 treatment, and PPAR γ protein levels were detected by Western blot. The representative results of three independent experiments are given. Grapdh was used to regulate the quanti cation of PPARγ. The data showed the mean value of three independent experiments ± SEM. The asterisk showed statistically signi cant difference (* P < 0.01, * * P < 0.05).

Figure 4
Inhibition of PPARγ impacts microglia alternative activation induced by IL-4 and Dex. BV-2 cells were treated with 1 μM Dex, 2 μM GW9662, IL-4 (20 ng/ml) or combination of them. Levels of Arg1 and FIZZ1 were detected by Western blot 24 hours after IL-4 stimulation. A representative result from three independent experiments is shown. Quanti cation of Arg1 and FIZZ1 was normalized by β-actin and Grapdh respectively. Data are presented as mean ± SEM for three independent experiments. Asterisks indicate signi cant difference (*P < 0.01, ** P 0.05).