PPAR-γ promotes the polarization of rat retinal microglia to M2 phenotype by regulating the expression of CD200-CD200R1 under hypoxia

Recent reports suggest that peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms that trigger PPAR-γ’s anti-inflammatory ability in microglia are yet to be expounded. Thus, in this study, we aimed to explore the molecular mechanisms behind the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat retinal microglial cells. We used shRNA expressing lentivirus to knock down PPAR-γ and CD200 genes, and we assessed hypoxia-induced polarization markers release – M1 (iNOS, IL-1β, IL-6, and TNF-α) and M2 (Arg-1, YM1, IL-4, and IL-10) by RT-PCR. We also monitored PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200, and CD200Rs) by Western blot and RT-PCR. Our results showed that hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. Moreover, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, whereas sh-PPAR-γ had the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype. Our findings demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via the CD200-CD200R1 pathway.


Introduction
Microglia are the resident macrophages in the central nervous system (CNS), including the retina.These cells possess properties that make them suitable for mediating cellular inflammatory responses, and their activation is one of the indications of neuroinflammation [1].In the retina, microglia exert scavenger, cytoprotective, or cytotoxic effects depending on the state of the micro-environment [2].Similar to peripheral macrophages, there exist two activated phenotypes, M1 and M2, in microglia.The classically activated M1 phenotype performs a pro-inflammatory function by liberating pro-inflammatory factors.On the contrary, as an anti-inflammatory phenotype, alternatively activated M2 microglia mediate inflammation suppression, immune regulation, parasite removal, and tissue remodeling [1].
Proper microglial activation can aid in microenvironmental recovery, while an over pro-inflammatory reaction can exacerbate damage [3].Furthermore, self-released inflammatory factors can activate microglia further, leading to the deterioration of the inflammatory cascade.CD200/ CD200R1 interaction provides a mechanism inhibiting the microglial inflammatory response.CD200 is a surface glycoprotein, while its receptor, CD200R1, is mainly located in microglia [4].In the retina, CD200 and CD200R promote microglia migration and prevent M1 phenotype activation of resident microglia [5].Previously, we demonstrated that recombinant CD200Fc protein inhibits LPS-induced microglial inflammation by suppressing the TLR4-mediated MyD88-TAK1-NF-κB pathway activation, resulting in decreased NF-κB expression and translocation and reduced levels of inflammatory mediators [6].
Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a ligand-activated nuclear transcription factor involved in energy metabolism, adipocyte differentiation, and neurodegenerative disorders [7].Notably, PPAR-γ has been found to be up-regulated in neurodegenerative disorders like multiple sclerosis and amyotrophic lateral sclerosis [8,9], with its activation reducing the inflammatory response by suppressing the NF-κB pathway [10].Primarily, PPAR-γ regulates macrophage polarization, playing a crucial anti-inflammatory role [11].However, the pathway underlying how PPAR-γ ameliorates inflammation remains unclear.Thus, this study aimed to elucidate the role of PPAR-γ in hypoxia-stimulated microglia.We identified that CD200-CD200R1 signaling pathway-modulated microglial M2 polarization plays a critical function in PPAR-γ amelioration of inflammation outcome.Our findings provide further evidence for the potential therapeutic value of PPAR-γ and its ligands in inflammation-related retinal diseases.

Primary retinal microglia culture and hypoxic exposure
Sprague-Dawley rats (1 week; Laboratory of Guangxi Medical University, Nanning, Guangxi, China) were used in this study.The project was approved by the Animal Ethics Committee from the People's Hospital of Guangxi Zhuang Autonomous Region.The primary microglial culture protocol followed the methods described by Roque [12] and Dong [13].Initially, retinas were digested with 0.25% trypsin at 37 ℃ for 30 min and then trypsin was inactivated using Dulbecco's modified Eagle's medium (DMEM)/F-12 (Life Technologies, Grand Island, NY).Subsequently, the tissue was passed through 200 µM filters, and re-suspended filtered cells in DMEM/F-12 culture medium containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.Next, for the normoxia group, cells were kept in a humidified atmosphere of 5% CO 2 and 95% air.Hypoxic culture conditions were achieved with a multi-gas incubator containing 94% N 2 , 5% CO 2 , and 1%O 2 .The medium was changed after 24 h of incubation, and then changed twice a week thereafter.After 2 weeks, the flask was shaken at 200 rpm for 1 h to harvest microglial cells, which were then employed in subsequent experiments.

Viral production and gene knock-down
Lentiviral shRNA clones targeting rat PPAR-γ, CD200 or green fluorescent protein (GFP) were procured from Shanghai HanBio Corporation.Lentivirus production was initiated by transfecting 293T cells with 10 µg of lentivirus vector followed by harvesting of supernatant after a 48-hour incubation period.Rat retinal microglial cells were then infected with said supernatant to induce PPAR-γ or CD200 knockdown, and the multiplicity of infection was set at 50.To confirm lentivirus infection efficiencies, the amount of GFP-positive cells were counted at 72 h post-infection.

Preparation of cytosolic and nuclear extracts
Microglial cells were centrifugated at 800 rpm and 4 ℃ for 5 min, followed by washing with ice-cold PBS.Utilizing a nuclear/cytosol fractionation kit (Biovision Inc., Mountain View, California) in accordance with the manufacturer's instructions, cell fractions were prepared for both nuclear and cytoplasmic components.After performing a final centrifugation step, resultant supernatants were collected for Western blot analysis.

Immunoprecipitation
To examine protein-protein interactions, microglial cells were lysed in 1 mL buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 10 mM NaF, 1 mM Na 3 VO 4 , 10 g/mL leupeptin, 10 g/mL aprotinin, and 20 mM PMSF after being harvested.Incubating aliquots of cellular lysates with primary antiTLR4 antibodies and shaking the complexes overnight at 4 ℃.Binding the complexes to 40 µL of recombinant Protein G Agarose beads (Invitrogen, USA) at 4 ℃ for 2 h, and washing the beads three times with lysis buffer.Then re-suspending the washed beads in electrophoresis sample buffer and boiling for 10 min.Finally, subjecting immunoprecipitated proteins to SDS-PAGE protein separation.

Western blot analysis
First, homogenizing microglial cells collected above in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na 3 VO 4 , 1 mM PMSF, 1 mM EDTA, 1% NP40, 50 mM NaF) for 30 min.Centrifuging cell lysates at 13,000 rpm, 4 ℃ for 15 min and then separated by 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.After electrophoresis, samples were transferred to PVDF membranes (Millipore, Billerica, MA), blocked with 5% BSA and 0.5% Tween 20 in PBS for 1 h, washed with PBS/Tween (TBST), and incubated with primary antibodies, as shown in Table 1.Then, after washing with TBST, HRP-conjugated secondary antibodies (goat anti-rabbit IgG and donkey anti-goat IgG, Santa Cruz Biotechnology) were applied.Using ECL Western blot Detection Reagents (Amersham Pharmacia Biotech) to develop blots, and Image Master TM 2D Elite software (Amersham Pharmacia Biotech) to perform densitometry analysis of bands.

RNA extraction and real-time PCR
Using the Trizol reagent (Life Technologies) to extract total mRNA of microglial cells.According to the manufacturer's instructions, using reverse transcription kit (Fermentas) to synthesize complementary DNA from 1 µg total RNA in 20 µL.TaqMan primers and probes were from Applied Biosystems, UK.Probes were labeled at the 5' end with a 60-carboxyfluorescein (FAM) reporter dye and at the 3' end with a 60-carboxy-tetramethyl rhodamine (TAMRA) quencher dye.Primers and probes for b-actin were supplied by Applied Biosystems (Warrington, UK).TaqMan PCR was performed from 1 µL of cDNA (corresponding to 50 ng RNA input) using Universal TaqMan Mastermix with 100 nM primers and a 50 nM probe.Cycling conditions were: 50 ℃ for 2 min, 95 ℃ for 10 min, followed by 40 cycles of amplification (95 ℃ for 15 s, 60 ℃ for 1 min).Samples were assayed on the Applied Biosystems PRISM 7000 Sequence detection system.Each sample was assayed in triplicate and a six-point standard curve run in parallel.To ensure the absence of genomic DNA contamination, a control sample of nonreverse-transcribed RNA was run for each set of RNA extractions.Relative quantification was obtained by calculating the ratio between the values obtained for each gene of interest and the housekeeping gene β-actin.The results are expressed as a percentage of the normal group.The primers of target genes are listed in Table 2.

Data analysis
The statistical analysis involved a t-test when comparing two groups and a one-way ANOVA followed by Tukey's multiple comparison test for comparisons involving three or more groups.In all analyses, significance was defined as P < 0.05.
protein and mRNA levels were measured using Western blot and RT-PCR, respectively.Results revealed a significant increase in protein levels of CD200 and CD200R1 in the 1%O 2 group compared to that in the normoxia group.However, no significant differences were observed in CD200R2, R3, and R4 between the two groups.A consistent trend was also detected by RT-PCR analysis for mRNA levels of CD200 and CD200Rs (Fig. 2).These findings align with a previous study indicating that CD200 exclusively binds to CD200R1, whereas other CD200R isoforms are not its ligands [14].

Effect of hypoxia on PPAR-γ expression in microglial cells
To examine whether hypoxia affects PPAR-γ signaling, primary retinal microglial cells from rats were cultured in vitro and divided into two groups based on different oxygen concentrations: the 20% O 2 group (normoxia) and the 1% O 2 group (hypoxia).Western blot analysis showed a significant increase in protein levels of PPAR-γ in the 1% O 2 group compared to that in the 20% O 2 group.This result was further confirmed by RT-PCR analysis (Fig. 1a-c).As PPAR-γ is mainly localized in the nucleus, we extracted proteins separately from nuclei and cytoplasm of microglial cells before performing the immunoblot to examine their respective expression levels of PPAR-γ.The results revealed higher PPAR-γ protein levels in both the nuclei and cytoplasm of the hypoxia group than those of the normoxia group.(Fig. 1d-f).

Effect of hypoxia on CD200-CD200R1 expression in microglial cells
To investigate if hypoxia affects the expression of CD200 and its receptors (CD200Rs) in retinal microglial cells,

Discussion
The retina is highly metabolically active and requires more oxygen than other tissues [16].Consequently, the retina is especially susceptible to hypoxia, which can be linked to the development of various retinopathies such as glaucoma, diabetic retinopathy, retinal vessel occlusion, and age-related macular degeneration [17].CD200 is highly expressed within the retina, specifically on retinal neurons, RPE, and vascular endothelium, and is recognized for its ability to regulate inflammation in multiple retinal diseases such as autoimmune uveoretinitis and glaucoma [18][19][20].CD200 and PPAR-γ have been shown to reduce inflammation by preventing the activation of the NF-κB pathway [6,21], while the PPAR-γ pathway is crucial in the control of microglial polarization [10].These findings lead us to posit that CD200 could be a substantial target through which the PPAR-γ pathway operates in retinal microglia, ultimately contributing to the pathophysiological implications of retinal microglial polarization in hypoxia.Microglial cells are the third glial cell type besides Müller cells and astrocytes of the retina.In general, microglial

Effects of PPAR-γ and CD200 on microglial polarization under hypoxia
In this part, microglia were divided into several groups to explore the effects of PPAR-γ and CD200 on microglial polarization.M1 markers, including iNOS, IL-1β, IL-6, and TNF-α, were used to evaluate pro-inflammatory polarization; while M2 markers, including Arg-1, YM1, IL-4, and IL-10, were used to measure anti-inflammatory polarization.
Results indicated that treatment with 1%O 2 induced mRNA levels of M1 markers and reduced mRNA levels of M2 markers, which is consistent with earlier reports [15].However, the effects of hypoxia on M1 and M2 markers expressions were almost completely reversed by 15d-PGJ 2 .Interestingly, the influence of 15d-PGJ 2 on hypoxia-stimulated microglial cells was partially offset by knocking down CD200 using shRNA.In addition, whether it was knocking down PPAR-γ or CD200 in hypoxia-stimulated microglial cells, the mRNA levels of M1 markers were higher, and M2 markers were lower than that seen in the 1%O 2 group (Figs. 4 and 5).Previous studies have demonstrated the modulation of M2-activated microglia by PPAR-γ and its association with neuroprotective and anti-inflammatory functions [23][24][25], and we have similar findings with these studies in hypoxiainduced microglia.To prevent any non-specific effects from using a PPAR-γ antagonist, we employed shRNA to knock down PPAR-γ and examined subsequent microglial activation markers.Same as expected, knocking down PPAR-γ activation can be classified into two states, and hypoxia has been shown to shift microglial cells towards the M1 phenotype [22], highlighting the importance of regulating retinal microglial polarization in treating hypoxia-induced damage.In our experiments, we observed that hypoxia increased expression levels of PPAR-γ and CD200-CD200R1 in retinal microglial cells, while it also induced the activation of microglia into the M1 phenotype, which could be prevented by activating PPAR-γ.Furthermore, we showed that the PPAR-γ-CD200-CD200R1 pathway played a role in the comprises multiple members, with only CD200R1 containing a lengthy intracellular fragment capable of recruiting downstream molecules for physiological functions [27].Similar to the PPAR-γ pathway, the CD200-CD200R signaling pathway has been implicated in regulating microglial polarization and neuroinflammation [28,29].The study by Dentesano et al. [30] found that 15d-PGJ 2 (PPAR-γ agonist) can modulate the expression of CD200-CD200R1 and has anti-inflammatory effects on activated microglial cells.Our reversed the PPAR-γ-mediated M2 activation, which then promoted hypoxia-induced microglial M1 polarization.This result confirms the role of PPAR-γ in promoting microglial M2 polarization.
CD200 is a member of the immunoglobulin supergene family that is predominantly expressed in astrocytes, oligodendrocytes, and neural stem cells within the CNS [26].As the receptors of CD200, CD200Rs are primarily present on microglia in the CNS.The CD200Rs family research supports this finding by demonstrating that PPAR-γ upregulates CD200 expression, and knocking down CD200 reduces the inhibitory effects of 15d-PGJ 2 on pro-inflammatory factor expression, while knocking down CD200 alone causes microglial cells to polarize further towards the M1 phenotype, similar to knocking down PPAR-γ.
In brief, the findings of this study suggest that PPAR-γ promotes the shift from M1 to M2 phenotype in hypoxiainduced retinal microglia by upregulating CD200-CD200R1, pointing towards its potential therapeutic role in retinal diseases associated with neuroinflammation.Nonetheless, as our data does not fully replicate the highly intricate microenvironment of hypoxic retina, further comprehensive experiments are required to gain insight into the function of PPAR-γ in microglial polarization and the molecular mechanisms involved in PPAR-γ-mediated regulation of the CD200-CD200R1 pathway.

Fig. 1
Fig. 1 Effects of hypoxia on the expressions of PPAR-γ in microglial cells.Cells of 20%O 2 (normoxia) group were cultured in a humidified atmosphere with 5% CO 2 and 95% air.Cells of 1%O 2 (hypoxia) group were cultured in a humidified atmosphere with 94% N 2 , 5% CO 2 , and 1%O 2 .a Protein expressions of PPAR-γ were detected by Western blot.b Densitometric analysis of the protein levels of PPAR-γ between

Fig. 3 Fig. 2
Fig. 3 Effects of PPAR-γ pathway on the expressions of CD200 and CD200R1 in hypoxia-stimulated microglial cells.Before Western blot measurement, the cells were exposed to 15d-PGJ 2 (5µM) or knocked down mRNA of PPAR-γ by shRNA.a Protein expressions of CD200

Table 1
Primary antibodies used in western blot analysis of this study