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Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury Maoxing Fei a, Handong Wang a, b, *, Mengliang Zhou b, **, Chulei Deng c, Li Zhang b, Yanling Han d a
Department of Neurosurgery, Jinling Hospital, Nanjing Medical University, Nanjing, 210002, PR China Department of Neurosurgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, 210002, PR China Department of Neurosurgery, Jinling Hospital, Southern Medical University, Guangzhou, 510000, PR China d Department of Neurosurgery, Jinling Hospital, Nanjing, 210002, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2019 Accepted 1 December 2019 Available online xxx
Traumatic brain injury (TBI) represents a major cause of death and disability worldwide. Exacerbated neuroinflammation following TBI causes secondary injury. Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that promotes the inflammatory response in different tissues and cells. However, the contribution of PDPN to neuroinflammation and microglial activation is unknown. Here, we found that PDPN was correlated with microglial activation after TBI in mice. Meanwhile, PDPN expression could be induced by trauma-related stimuli, such as lipopolysaccharide (LPS), ATP, H2O2 and hemoglobin (Hb), in primary microglia. Furthermore, with Hb treatment in vitro, knockdown of PDPN could decrease the proportion of M1-like microglia and increase the proportion of M2-like microglia via reduced secretion of IL-1b and TNF-a and increased secretion of IL-10 and TGF-b compared to the control microglia. Immunofluorescence also showed that CD86-positive microglia were decreased and CD206-positive microglia were elevated in the PDPN-KD group. Additionally, PDPN knockdown impaired microglial mobility and phagocytosis and decreased the expression of matrix metalloproteinases (mainly MMP2 and MMP9). In summary, PDPN plays an important role in microglia-mediated inflammation and may serve as a potential target for TBI treatment. © 2019 Elsevier Inc. All rights reserved.
Keywords: Podoplanin Traumatic brain injury Microglia Matrix metalloproteinases
1. Introduction Traumatic brain injury (TBI) can cause primary injury and secondary injury [1]. Since primary injury is characterized by severe neuronal damage and injured axons, which cannot be ameliorated, prevention of secondary injury is the main treatment strategy for TBI [2]. Secondary injury is inextricably linked with microglia/ macrophage activity, and M1 phenotype activation increases the release of proinflammatory cytokines, which cause irreversible neuronal apoptosis [3]. The M1 phenotype of microglia is considered neurotoxic after TBI, results in the release of proinflammatory
* Corresponding author. Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing, Jiangsu, 210002, PR China. ** Corresponding author. Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing, Jiangsu, 210002, PR China. E-mail addresses: [email protected] (H. Wang), mengliangzhou@yahoo. com (M. Zhou).
cytokines and causes deleterious effects in neurons, while the M2 phenotype results in the production of growth factors and antiinflammatory cytokines [4e6]. Although the M1/M2 classification of microglia is being debated [7], Kumar A et al. showed that microglia isolated following controlled cortical impact (CCI) injury displayed an increase in the M2-like phenotype within the first day but subsequently displayed the M1-like phenotype [8]. Therefore, targeting M1/M2 changes in microglia could be a strategy to inhibit secondary injury following TBI. Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that is widely expressed in different tissues and cell types, such as glomerular podocytes (hence its name), type I alveolar cells, osteocytes, mesothelial cells, choroid plexus and different types of fibroblasts [9]. Numerous studies have investigated the expression of PDPN in different tumors and the potential role of PDPN in different mechanisms promoting tumor cell metastasis, such as the EMT pathway and non-EMT pathway [10]. PDPN is generally enriched in the F-actin-rich invadopodia, and its role in
https://doi.org/10.1016/j.bbrc.2019.12.003 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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cell migration is likely mediated by its anchorage to the actin cytoskeleton through the ezrin/radixin/moesin (ERM) proteins and control of the cytoskeletal organization via regulating the activity of small Rho GTPases [9]. Additionally, PDPN was shown to participate in the dendritic cell antigen presenting process, keratinocyte migration during wound healing process and phagocytic macrophages in inflammatory conditions [11e13]. PDPN expression was highly correlated with several inflammatory diseases [12e14]. However, its role in neuroinflammation has rarely been examined. Previously, Kolar K et al. found that PDPN was highly associated with reactive astrocytes in tumor models and nonneoplastic brain injury models [15]. However, Song Y et al. found that PDPN was elevated in neurons rather than astrocytes and associated with neuronal apoptosis in a lipopolysaccharide (LPS)-induced neuroinflammatory model [16]. These differences could be attributed to the different models used in the two experiments but still cast doubts on the role of PDPN in the brain. Here, we found that PDPN was highly expressed in microglia rather than neurons or activated astrocytes in a TBI model in vivo. We extracted primary microglia from neonatal mice and transfected them with lentivirus targeting PDPN to study their role in the inflammatory process. Our results demonstrated that PDPN could affect the inflammatory states of the microglia and that knockdown of PDPN facilitated an anti-inflammatory phenotype (M2-like phenotype) in microglia following treatment with Hb. Meanwhile, PDPN was highly expressed in the invadopodia, and knockdown of PDPN impaired microglial mobility and phagocytosis by decreasing the expression of matrix metalloproteinases (MMP2 and MMP9). 2. Materials and methods 2.1. Animal ethical approval All animal experiments were approved by the animal ethical committee of Nanjing Medical University (Jiangsu, China). The experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health (NIH). Adult male mice weighing 28e32 g from the Institute of Cancer Research (ICR) were housed under controlled conditions with a 12-h light/dark cycle and were given free access to food and water. 2.2. Mouse model of TBI Marmarou’s weight-drop model, as previously described by Flierl et al. [17], was employed in our study. Briefly, mice were anesthetized via isoflurane inhalation (induced at 4%, maintained at 1.5%). Subsequently, a mouse was fastened on a platform, and a long longitudinal midline scalp incision was made. Then, a 200 g weight, released from a height of 2.5 cm, hit the selected region (1.5 mm left-lateral to the midline on the mid-coronal plane). Finally, the scalp was closed, and the mice were returned to the quondam cages. The sham group was subjected to the same procedures without brain injury. 2.3. Primary cell culture Primary microglial culture was obtained from postnatal (1e3 d) ICR mice. Briefly, neonatal mice were decapitated and sterilized in 75% alcohol. The cortex was separated in HBSS under a dissection microscope. Then, the cortex was digested in 0.125% trypsin for 5 min at 37 C in a water bath. Subsequently, microglia culture medium was added to stop the digestion. The suspension was stirred with a pipette, filtered through a screen with 70 mm pores
and then centrifuged at 1500 r/min for 5 min. The remaining was resuspended in microglia culture medium (DMEM with 10% FBS and 1% penicillin-streptomycin). 2.4. Cell transfection For establishment of PDPN knockdown microglia, lentiviruses carrying shRNA targeting mouse PDPN or scramble shRNA as a negative control were designed and generated by GeneChem Co., Ltd. (Shanghai, China). The primer sequence of PDPN shRNAs were listed as follows: shPDPN, 50 -GCTACTGGAGGGCTTAATGAATCAAGAGTTCATTAAGCCCTCCAGTAGC-3’. Primary microglia were transfected with the lentivirus according to the company’s instructions. The knockdown efficiency was detected via RT-PCR and western blotting. 2.5. Regents and antibodies For F-actin staining, Phalloidin-iFluor 488 (ab176753) was purchased from Abcam (Cambridge, MA, USA). Anti-PDPN(ab11936), anti-Iba1(ab178846), anti-CD86(ab119857), anti-CD206(ab64693) and anti-GAPDH(ab181602) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-MMP2(#87809) and antiMMP9(#15561) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-NeuN(MAB377X) antibodies were obtained from EMD Millipore (Billerica, MA, USA). Anti-GFAP (539892-82) antibodies were obtained from ThermoFisher (Massachusetts, USA). LPS, ATP, H2O2 and hemoglobin (Hb) were purchased from Sigma-Aldrich (Missouri, USA). 2.6. Quantitative real-time reverse transcriptase polymerase chain reaction RNA was extracted by TRIzol Reagent and then reversetranscribed into cDNA. Quantitative real-time PCR analysis was performed with UltraSYBR Mixture using the LightCycler 96 RealTime PCR System (Roche). The primers were as follows: IL-1b, 50 GCCTGTGTTTTCCTCCTTGC-30 (forward) and 50 -TGCTGCCTAAT GTCCCCTTG-30 (reverse); TNF-a 50 -CCCTCACACTCACAAACCACC-30 (forward) and 50 -CTTTGAGATCCATGCCGTTG-30 (reverse); IL-10, 50 TTTAAGGGTTACTTGGGTTGCC-30 (forward) and 50 - AATGCTCCTTGATTTCTGGGC-30 (reverse); TGF-b, 50 - CAACAATTCCTGG 0 CGTTACCT-3 (forward) and 50 - GCCCTGTATTCCGTCTCCTT-30 (reverse); PDPN, 50 - GGACCGTGCCAGTGTTGTTC-30 (forward) and 50 - AGAGGTGCCTTGCCAGTAGATT-30 (reverse); IL-6, 50 -GAGACTTCCATCCAGTTGCCT-30 (forward), 50 -TGGGAGTGGTATCCTCTGTGA-30 (reverse); MMP1, 50 - CTCTGGAGTAATGTCACACCTCT-30 (forward), 50 - TGTTGGTCCACCTTTCATCTTC-30 (reverse); MMP2, 50 -TCTCCCC CAAAACAGACAAAGAG-30 (forward), 50 -TCCTTCAGCACAAAGAGGTT GC-30 (reverse); MMP3, 50 - ACATGGAGACTTTGTCCCTTTTG-30 (forward), 50 - TTGGCTGAGTGGTAGAGTCCC-30 (reverse); MMP7, 50 CTGCCACTGTCCCAGGAAG-30 (forward), 50 - GGGAGAGTTTTCCAG TCATGG-30 (reverse); MMP9, 50 -CTGCCTGCACCACTAAAGG-30 (forward), 50 -GAAGACGAAGGGGAAGACG-30 (reverse); MMP12, 50 CTGGGCAACTGGACACCT-30 (forward), 50 -CTACATCCGCACGCTTCA30 (reverse); MMP13, 50 -CTTCTTCTTGTTGAGCTGGACTC-30 (forward), 50 - CTGTGGAGGTCACTGTAGACT-30 (reverse); MMP14, 50 GTTCTGGCGGGTGAGGAATAAC-30 (forward), 50 -TCATAGGCAGTG TTGATGGATGC-30 (reverse) and Rpl5, 50 -GGAAGCACATCATGGG TCAGA-30 (forward) and 50 -TACGCATCTTCATCTTCCTCCATT-30 (reverse). Rpl5 was used as a housekeeping gene. The PCR program was as follows: 95 C for 30 s and 40 cycles consisting of a denaturation step (95 C, 5 s) and an annealing step (60 C, 30 s).
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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2.7. Western blotting analysis
2.12. Statistical analysis
The cerebral cortex of the sham group and the injured cerebral cortex were collected at 3 h, 6 h, 12 h, 1 d, 2 d, 3 d and 7 d after TBI. Microglia from the different groups were treated as described above. Protein samples from tissues and cells were extracted according to the manufacturer’s instructions (Protein Extraction Kit, Beyotime Biotechnology). Protein samples at the same quantities were separated by electrophoresis and then transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then were separately immunoblotted with primary antibodies. Next, the secondary antibodies were added. The blots were visualized with a chemiluminescent detection kit (P90720, Millipore, MA, USA). ImageJ software was used to analyze the blots.
SPSS version 22.0 (IBM Corporation, Armonk, NY, USA) software was employed to analyze all the data. Data are expressed as the mean ± SEM and were evaluated by Student’s t-test and ANOVA for multiple comparisons. P < 0.05 was considered significant.
2.8. Cell viability assay Cell viability was detected by Cell Counting Kit-8 (CCK-8) assays (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, cells were plated in 96-wells at a density of 5 103/well for 12 h. After treatment, the cells were incubated with 100 mL of DMEM containing 10% CCK-8 reagent in a 37 C incubator. Two hours later, the optical density values were detected with an enzyme-linked immunosorbent assay plate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). Then, the following formula was used to calculate the viability: cell viability¼ (OD450 of the treated groups/ OD450 of the control group) 100%. 2.9. Phagocytosis test Primary microglia were transferred with lentivirus targeting PDPN in advance. pHrodo™ Green E. coli BioParticles® Conjugate was purchased from ThermoFisher. The procedure was carried on according to the manufacturer’s instructions. The pictures of different groups were recorded under a Zeiss immunofluorescence microscope (Zeiss, German) and the fluorescence intensity was recorded by Cytofluor 4000 Fluorescence. 2.10. Transwell migration assay Transwells were employed to detect the microglial motility under different conditions. A total of 2 105 cells in 200 mL of DMEM were added into the upper chamber of 6.5 mm transwells with 8.0 mm pores (Corning Incorporated, Corning, NY, USA). After incubation at 37 C for 16 h, the cells in the interior of the chamber were removed. Then, the chambers were fixed with paraformaldehyde for 15 min and subsequently stained with 0.1% crystal violet for 10 min. The migrating cells were observed in six microscopic fields (200 ) under an inverted microscope (Carl Zeiss Meditec AG, Jena, Germany). 2.11. Immunofluorescence analysis of cells and tissues Cells were seeded onto microscope coverslips overnight and treated as indicated. Mouse brains were obtained, fixed, and embedded in paraformaldehyde for IHC and IF staining. Tissue sections (7 mm) were generated with a freezing microtome. Briefly, the slides were fixed with paraformaldehyde for 15 min and then treated with 0.3% Triton X-100 for 10 min. After fetal bovine serum was added for 30 min, specific primary antibodies and secondary antibodies were added to the slides. Nuclei were stained with DAPI (Sigma-Aldrich, D9542, 1/2000) for 10 min. All slides and tumor sections were observed using a Zeiss immunofluorescence microscope (Zeiss, German).
3. Results 3.1. PDPN expression patterns in healthy mouse brains and brains after TBI First, we generated TBI in mice by using a weight striking model. Then, immunofluorescence staining was used to determine the expression patterns of PDPN in normal mouse brains and brains after TBI (1, 3, and 7 d) (Fig. 1A). PDPN was rarely expressed in the cerebral cortex, hippocampus or corpus callosum in the sham group (Fig. 1A). However, strong immunofluorescence staining of PDPN was observed around the injured cortex and corpus callosum starting on the first day after TBI (Fig. 1A). Dual staining of PDPN and glial markers (NeuN, GFAP and Iba1) showed that PDPN strongly colocalized with Iba1, but PDPN did not show colocalization with neurons or astrocytes (Fig. 1B). PDPN and Iba1 staining in normal mouse brains showed that microglia expressed PDPN at a low level in the ‘rest state’ (Fig. 1C). Then, we traced the PDPNpositive microglia in the corpus callosum and capsula externa white matter. This result indicated that PDPN could be involved in the chemotaxis of microglia (Fig. 1D). 3.2. PDPN was substantially elevated in the early stages of TBI and was induced by inflammatory stimuli in vitro Samples around the lesion were collected at different time points after TBI for western blotting and PCR analyses (Fig. 2A). As shown in Fig. 2B and C, the PDPN mRNA levels rapidly increased at 6 h after induction of TBI, and the peak of PDPN appeared earlier than the peak of Iba1. Meanwhile, inflammatory factors such as IL1b, IL-6 and TNF-a were increased after the TBI (Fig. 2DeF). Western blotting also confirmed that PDPN was highly expressed at 1 d, and this level was maintained after 7 days (Fig. 2GeI). To determine whether TBI-related inflammatory stimuli induce the expression of PDPN in vitro, we used primary microglial cultures. As shown in Fig. 2J-L, the mRNA and protein levels of PDPN were elevated by ATP, LPS, H2O2 and Hb, which indicated an underlying role of PDPN in microglial inflammation. 3.3. The expression of PDPN was correlated with microglial inflammation To confirm the role of PDPN in microglia, we employed a lentivirus targeting PDPN to knock down PDPN in primary microglia and stimulated the microglia with Hb. As shown in Fig. 3AeC, PDPN knockdown was verified by western blotting and immunofluorescence staining. Though cell viability was not affected, as shown by the CCK-8 assay (Fig. 3D), microglial morphology was more ameba-like in the shCtrl þ Hb group than in the shPDPN þ Hb group (Fig. 3E). Interestingly, Hb significantly increased the expression of M1-like microglial inflammatory factors, such as IL1b and TNF-a, and downregulated M2-like microglial inflammatory factors, such as IL-10 and TGF-b, while no significant change of IL1b and TNF-a and significant elevation of IL-10 and TGF-b were observed after knockdown of PDPN (Fig. 3F and G). Furthermore, immunofluorescence staining showed a lower level of CD86positive microglia and a higher level of CD206-positive microglia in the shPDPN group than in the shCtrl group following exposure to
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Fig. 1. PDPN expression patterns in healthy mouse brains and brains after TBI. (A) Sections of normal mouse brains and traumatic brains at different times (1, 3, 7 d) were stained with PDPN antibody. PDPN was rarely expressed in the normal brain sections. However, strong immunofluorescence intensity of PDPN was recorded at the peri-injury cerebral cortex and corpus callosum. Scale bar, 400 mm. (B) Dual staining of PDPN and glial markers (NeuN, GFAP and Iba1) showed that PDPN strongly colocalized with Iba1 (Pearson’s Coefficient, 0.654; Overlap Coefficient, 0.961). Scale bar, 25 mm. (C) PDPN and Iba1 staining in normal mouse brains showed that microglia expressed PDPN in resting microglia. Scale bar, 25 mm. (D) PDPN-positive microglia were scattering at the capsula externa white matter, which indicated that PDPN could be involved in the microglial chemotaxis. Scale bar, 25 mm.
Fig. 2. PDPN was substantially elevated in the early stages of TBI and was induced by inflammatory stimuli in vitro. (A) Samples for RT-PCR and western blotting were collected as illustrated. Scale bar, 5 mm. (BeF) The mRNA levels of PDPN, Iba1, IL-b,IL-6 and TNF-a at the different time points (sham, 1 d, 2 d, 3 d and 7 d) after TBI were detected by RT-PCR. (GeI) The protein levels of PDPN and Iba1 in sham group and in groups at different time points (1 d, 2 d, 3 d and 7 d) were detected by western blotting. Then, the intensity of the blots was quantified and analyzed. (JeL) Primary microglia were extracted and cultured as described. The mRNA and protein levels of PDPN were detected after different stimuli (ATP 500 mM, LPS 1 mg/ml, H2O2 0.1 mM, Hb 13 mg/ml) were separately added for 24 h in the cultures. Data were represented as the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Hb (Fig. 3H). This finding demonstrated that PDPN could influence the inflammatory phenotype of microglia. 3.4. PDPN was associated with microglial mobility by Regulating Matrix Metalloproteinases Since PDPN could influence the inflammatory phenotype of microglia, we further investigated whether PDPN influenced microglial movement. Firstly, to determine the subcellular localization of PDPN in primary microglia, dual staining of PDPN and Factin in the Hb-treated microglia was employed. As shown in Fig. 4A, PDPN was localized with F-actin-rich invadopodia. Next, we detected the migration ability via transwell assays and found that fewer cells penetrated through the micropore in the shPDPN þ Hb group than in the shCtrl þ Hb group (Fig. 4B and C). Interestingly, Phagocytosis of microglia was also impaired after PDPN knockdown (Fig. 4D and E). Because matrix metalloproteinases (MMPs) could facilitate cell mobility, we wondered whether MMPs were changed after PDPN was knocked down in microglia. RT-qPCR showed that MMP2 and MMP9 were significantly down-regulated in the shPDPN group
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(Fig. 4F). However, other MMPs were not significantly influenced after PDPN knockdown (Fig. 4F). Western blotting also confirmed that expression of MMP2 and MMP9 was decreased in the shPDPN and shPDPN þ Hb group compared with the shCtrl and shCtrl þ Hb group respectively (Fig. 4GeI). 4. Discussion In this study, we demonstrated that PDPN was highly expressed in the microglia of the mouse TBI groups. Although previous studies reported elevated expression of PDPN in astrocytes and hippocampal neurons in other neuroinflammatory models [15,16], we failed to detect colocalization of PDPN with neuron or astrocyte markers (NeuN or GFAP). In vitro experiments showed that elevated level of PDPN could be induced by LPS, ATP, H2O2 and Hb in microglia, indicating the possible functions of PDPN in the inflammatory states of the microglia. The roles of PDPN in inflammation have been reported in rheumatoid arthritis (RA), sepsis and wound healing processes [12e14]. The CLEC-2-PDPN pathway could facilitate PDPN-positive immune cell infiltration at the site of infection and promote
Fig. 3. The expression of PDPN was correlated with microglial inflammation. (A-C) Lentivirus targeting PDPN was transferred into microglia. The knockdown efficiency of PDPN was confirmed either in normal condition or in Hb exposed condition by western blotting and immunofluorescence staining. Scale bar, 10 mm. (D) CCK8 assay showed that PDPN knockdown did not affected microglia viability. (E) The morphology of microglia after PDPN knockdown was subtly changed. However, microglial morphology in the shCtrl þ Hb group was more ameba-like than in the shPDPN þ Hb group. Scale bar, 20 mm. (F, G) The mRNA levels of IL-b, TNF-a, IL-10 and TGF-b were detected by RT-PCR. (H) Immunostaining of CD86 and CD206 were employed to determine the inflammatory phenotypes of microglia. Scale bar, 20 mm. Data were represented as the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Fig. 4. PDPN was associated with microglial mobility by Regulating Matrix Metalloproteinases. (A) Primary microglia were exposed in culture medium with Hb for 24 h. Dual staining of PDPN and F-actin showed that PDPN was mainly localized with F-actin-rich invadopodia. Scale bar, 5 mm. (B, C) Transwell migration assays were conducted, pictured, calculated and analyzed. Scale bar, 50 mm. (D, E) Representative pictures of E.coli phagocytosis tests in shCtrl and shPDPN group were shown. Inflorescence intensity of two groups was detected by a fluorescence plate reader and then analyzed (D). Scale bar, 25 mm. (F) The mRNA levels of matrix metalloproteinases, (MMP1-3, MMP7, MMP9-14), were detected after PDPN knockdown. (GeI) The protein levels of MMP2 and MMP9 were significantly decreased in the shPDPN group and the shPDPN þ Hb group compared with in the shCtrl group and shCtrl þ Hb group respectively. Data were represented as the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.
macrophage recruitment. PDPN expression could contribute to chronic inflammation in RA by increasing cytokines, including IL-6, IL-8, and IL-17 [13,18]. Chronic microglial inflammation was also detected after TBI, and the activation phenotype of the microglia is strongly related to TBI outcomes [19,20]. The dual microglial phenotypes (M1-like and M2-like phenotype) in neuroinflammation have been widely reported [4e6]. The M1-like microglial phenotype is considered neurotoxic after TBI, promotes the release of proinflammatory cytokines and causes deleterious effects in neurons, while the M2-like phenotype results in the production of growth factors and anti-inflammatory cytokines [4e6]. Therefore, decreasing the neurotoxic phenotype of microglia could potentially improve the long-term prognosis of TBI patients. Here, we found that knockdown of PDPN in primary microglia prevented the induction of the M1 phenotype not only by simultaneously decreasing Hb-induced TNFa and IL-1b and increasing TGF-b and IL-10 expression but also by decreasing CD86-positive cells and elevating the proportion of CD206-positive cells after addition of Hb. These results indicated that PDPN might act as an inflammation sensor in microglia. In addition to the effects on the microglial phenotype, mobility and phagocytosis of microglia were affected after knockdown of PDPN under Hb exposure. Previously, other researchers reported that the intracellular domain of PDPN directly binds to the ERM proteins [21,22]. In addition, the PDPN-ERM complex regulates and cell motility and morphology not only by physically binding to the cytoskeleton but also by activating various cellular signaling pathways, such as Rho GTPases [21,23]. Consistently, in the microglia, we found that PDPN was highly expressed in the F-actinrich invadopodia and PDPN knockdown could significantly
decrease microglial mobility and phagocytosis. Interestingly, we also observed PDPN knockdown down-regulated expression of matrix metalloproteinases, MMP2 and MMP9 but not other matrix metalloproteinases. The influence of PDPN on the expression pattern of microglial matrix metalloproteinases needs further studies. Since high levels of MMPs can promote activated microglia to migrate through the white matter and aggravate the white matter injury after TBI [24], PDPN could be a potential target for TBI treatment. In conclusion, we found that PDPN was highly elevated in microglia after TBI. The elevated level of PDPN was related to the M1-like phenotype of microglia, and knockdown of PDPN increased the proportion of M2-like microglia under exposure to Hb in vitro, suggesting a potential treatment strategy for TBI. Additionally, knockdown of PDPN decreased microglial mobility and phagocytic activity partly by decreasing matrix metalloproteinases, MMP2 and MMP9. However, the mechanism of PDPN in microglial inflammation still needs to be elucidated. Meanwhile, the therapeutic and prognostic effects of targeting PDPN in post-TBI should be verified in further experiments in vivo.
Declaration of competing interest The authors declare no potential conflict of interest.
Acknowledgements This study was grant-supported by the National Natural Science Foundation of China (No.81672503 and No.81702484).
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury Maoxing Fei a, Handong Wang a, b, *, Mengliang Zhou b, **, Chulei Deng c, Li Zhang b, Yanling Han d a
Department of Neurosurgery, Jinling Hospital, Nanjing Medical University, Nanjing, 210002, PR China Department of Neurosurgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, 210002, PR China Department of Neurosurgery, Jinling Hospital, Southern Medical University, Guangzhou, 510000, PR China d Department of Neurosurgery, Jinling Hospital, Nanjing, 210002, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2019 Accepted 1 December 2019 Available online xxx
Traumatic brain injury (TBI) represents a major cause of death and disability worldwide. Exacerbated neuroinflammation following TBI causes secondary injury. Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that promotes the inflammatory response in different tissues and cells. However, the contribution of PDPN to neuroinflammation and microglial activation is unknown. Here, we found that PDPN was correlated with microglial activation after TBI in mice. Meanwhile, PDPN expression could be induced by trauma-related stimuli, such as lipopolysaccharide (LPS), ATP, H2O2 and hemoglobin (Hb), in primary microglia. Furthermore, with Hb treatment in vitro, knockdown of PDPN could decrease the proportion of M1-like microglia and increase the proportion of M2-like microglia via reduced secretion of IL-1b and TNF-a and increased secretion of IL-10 and TGF-b compared to the control microglia. Immunofluorescence also showed that CD86-positive microglia were decreased and CD206-positive microglia were elevated in the PDPN-KD group. Additionally, PDPN knockdown impaired microglial mobility and phagocytosis and decreased the expression of matrix metalloproteinases (mainly MMP2 and MMP9). In summary, PDPN plays an important role in microglia-mediated inflammation and may serve as a potential target for TBI treatment. © 2019 Elsevier Inc. All rights reserved.
Keywords: Podoplanin Traumatic brain injury Microglia Matrix metalloproteinases
1. Introduction Traumatic brain injury (TBI) can cause primary injury and secondary injury [1]. Since primary injury is characterized by severe neuronal damage and injured axons, which cannot be ameliorated, prevention of secondary injury is the main treatment strategy for TBI [2]. Secondary injury is inextricably linked with microglia/ macrophage activity, and M1 phenotype activation increases the release of proinflammatory cytokines, which cause irreversible neuronal apoptosis [3]. The M1 phenotype of microglia is considered neurotoxic after TBI, results in the release of proinflammatory
* Corresponding author. Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing, Jiangsu, 210002, PR China. ** Corresponding author. Department of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing, Jiangsu, 210002, PR China. E-mail addresses: [email protected] (H. Wang), mengliangzhou@yahoo. com (M. Zhou).
cytokines and causes deleterious effects in neurons, while the M2 phenotype results in the production of growth factors and antiinflammatory cytokines [4e6]. Although the M1/M2 classification of microglia is being debated [7], Kumar A et al. showed that microglia isolated following controlled cortical impact (CCI) injury displayed an increase in the M2-like phenotype within the first day but subsequently displayed the M1-like phenotype [8]. Therefore, targeting M1/M2 changes in microglia could be a strategy to inhibit secondary injury following TBI. Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein that is widely expressed in different tissues and cell types, such as glomerular podocytes (hence its name), type I alveolar cells, osteocytes, mesothelial cells, choroid plexus and different types of fibroblasts [9]. Numerous studies have investigated the expression of PDPN in different tumors and the potential role of PDPN in different mechanisms promoting tumor cell metastasis, such as the EMT pathway and non-EMT pathway [10]. PDPN is generally enriched in the F-actin-rich invadopodia, and its role in
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Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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cell migration is likely mediated by its anchorage to the actin cytoskeleton through the ezrin/radixin/moesin (ERM) proteins and control of the cytoskeletal organization via regulating the activity of small Rho GTPases [9]. Additionally, PDPN was shown to participate in the dendritic cell antigen presenting process, keratinocyte migration during wound healing process and phagocytic macrophages in inflammatory conditions [11e13]. PDPN expression was highly correlated with several inflammatory diseases [12e14]. However, its role in neuroinflammation has rarely been examined. Previously, Kolar K et al. found that PDPN was highly associated with reactive astrocytes in tumor models and nonneoplastic brain injury models [15]. However, Song Y et al. found that PDPN was elevated in neurons rather than astrocytes and associated with neuronal apoptosis in a lipopolysaccharide (LPS)-induced neuroinflammatory model [16]. These differences could be attributed to the different models used in the two experiments but still cast doubts on the role of PDPN in the brain. Here, we found that PDPN was highly expressed in microglia rather than neurons or activated astrocytes in a TBI model in vivo. We extracted primary microglia from neonatal mice and transfected them with lentivirus targeting PDPN to study their role in the inflammatory process. Our results demonstrated that PDPN could affect the inflammatory states of the microglia and that knockdown of PDPN facilitated an anti-inflammatory phenotype (M2-like phenotype) in microglia following treatment with Hb. Meanwhile, PDPN was highly expressed in the invadopodia, and knockdown of PDPN impaired microglial mobility and phagocytosis by decreasing the expression of matrix metalloproteinases (MMP2 and MMP9). 2. Materials and methods 2.1. Animal ethical approval All animal experiments were approved by the animal ethical committee of Nanjing Medical University (Jiangsu, China). The experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health (NIH). Adult male mice weighing 28e32 g from the Institute of Cancer Research (ICR) were housed under controlled conditions with a 12-h light/dark cycle and were given free access to food and water. 2.2. Mouse model of TBI Marmarou’s weight-drop model, as previously described by Flierl et al. [17], was employed in our study. Briefly, mice were anesthetized via isoflurane inhalation (induced at 4%, maintained at 1.5%). Subsequently, a mouse was fastened on a platform, and a long longitudinal midline scalp incision was made. Then, a 200 g weight, released from a height of 2.5 cm, hit the selected region (1.5 mm left-lateral to the midline on the mid-coronal plane). Finally, the scalp was closed, and the mice were returned to the quondam cages. The sham group was subjected to the same procedures without brain injury. 2.3. Primary cell culture Primary microglial culture was obtained from postnatal (1e3 d) ICR mice. Briefly, neonatal mice were decapitated and sterilized in 75% alcohol. The cortex was separated in HBSS under a dissection microscope. Then, the cortex was digested in 0.125% trypsin for 5 min at 37 C in a water bath. Subsequently, microglia culture medium was added to stop the digestion. The suspension was stirred with a pipette, filtered through a screen with 70 mm pores
and then centrifuged at 1500 r/min for 5 min. The remaining was resuspended in microglia culture medium (DMEM with 10% FBS and 1% penicillin-streptomycin). 2.4. Cell transfection For establishment of PDPN knockdown microglia, lentiviruses carrying shRNA targeting mouse PDPN or scramble shRNA as a negative control were designed and generated by GeneChem Co., Ltd. (Shanghai, China). The primer sequence of PDPN shRNAs were listed as follows: shPDPN, 50 -GCTACTGGAGGGCTTAATGAATCAAGAGTTCATTAAGCCCTCCAGTAGC-3’. Primary microglia were transfected with the lentivirus according to the company’s instructions. The knockdown efficiency was detected via RT-PCR and western blotting. 2.5. Regents and antibodies For F-actin staining, Phalloidin-iFluor 488 (ab176753) was purchased from Abcam (Cambridge, MA, USA). Anti-PDPN(ab11936), anti-Iba1(ab178846), anti-CD86(ab119857), anti-CD206(ab64693) and anti-GAPDH(ab181602) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-MMP2(#87809) and antiMMP9(#15561) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-NeuN(MAB377X) antibodies were obtained from EMD Millipore (Billerica, MA, USA). Anti-GFAP (539892-82) antibodies were obtained from ThermoFisher (Massachusetts, USA). LPS, ATP, H2O2 and hemoglobin (Hb) were purchased from Sigma-Aldrich (Missouri, USA). 2.6. Quantitative real-time reverse transcriptase polymerase chain reaction RNA was extracted by TRIzol Reagent and then reversetranscribed into cDNA. Quantitative real-time PCR analysis was performed with UltraSYBR Mixture using the LightCycler 96 RealTime PCR System (Roche). The primers were as follows: IL-1b, 50 GCCTGTGTTTTCCTCCTTGC-30 (forward) and 50 -TGCTGCCTAAT GTCCCCTTG-30 (reverse); TNF-a 50 -CCCTCACACTCACAAACCACC-30 (forward) and 50 -CTTTGAGATCCATGCCGTTG-30 (reverse); IL-10, 50 TTTAAGGGTTACTTGGGTTGCC-30 (forward) and 50 - AATGCTCCTTGATTTCTGGGC-30 (reverse); TGF-b, 50 - CAACAATTCCTGG 0 CGTTACCT-3 (forward) and 50 - GCCCTGTATTCCGTCTCCTT-30 (reverse); PDPN, 50 - GGACCGTGCCAGTGTTGTTC-30 (forward) and 50 - AGAGGTGCCTTGCCAGTAGATT-30 (reverse); IL-6, 50 -GAGACTTCCATCCAGTTGCCT-30 (forward), 50 -TGGGAGTGGTATCCTCTGTGA-30 (reverse); MMP1, 50 - CTCTGGAGTAATGTCACACCTCT-30 (forward), 50 - TGTTGGTCCACCTTTCATCTTC-30 (reverse); MMP2, 50 -TCTCCCC CAAAACAGACAAAGAG-30 (forward), 50 -TCCTTCAGCACAAAGAGGTT GC-30 (reverse); MMP3, 50 - ACATGGAGACTTTGTCCCTTTTG-30 (forward), 50 - TTGGCTGAGTGGTAGAGTCCC-30 (reverse); MMP7, 50 CTGCCACTGTCCCAGGAAG-30 (forward), 50 - GGGAGAGTTTTCCAG TCATGG-30 (reverse); MMP9, 50 -CTGCCTGCACCACTAAAGG-30 (forward), 50 -GAAGACGAAGGGGAAGACG-30 (reverse); MMP12, 50 CTGGGCAACTGGACACCT-30 (forward), 50 -CTACATCCGCACGCTTCA30 (reverse); MMP13, 50 -CTTCTTCTTGTTGAGCTGGACTC-30 (forward), 50 - CTGTGGAGGTCACTGTAGACT-30 (reverse); MMP14, 50 GTTCTGGCGGGTGAGGAATAAC-30 (forward), 50 -TCATAGGCAGTG TTGATGGATGC-30 (reverse) and Rpl5, 50 -GGAAGCACATCATGGG TCAGA-30 (forward) and 50 -TACGCATCTTCATCTTCCTCCATT-30 (reverse). Rpl5 was used as a housekeeping gene. The PCR program was as follows: 95 C for 30 s and 40 cycles consisting of a denaturation step (95 C, 5 s) and an annealing step (60 C, 30 s).
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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2.7. Western blotting analysis
2.12. Statistical analysis
The cerebral cortex of the sham group and the injured cerebral cortex were collected at 3 h, 6 h, 12 h, 1 d, 2 d, 3 d and 7 d after TBI. Microglia from the different groups were treated as described above. Protein samples from tissues and cells were extracted according to the manufacturer’s instructions (Protein Extraction Kit, Beyotime Biotechnology). Protein samples at the same quantities were separated by electrophoresis and then transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then were separately immunoblotted with primary antibodies. Next, the secondary antibodies were added. The blots were visualized with a chemiluminescent detection kit (P90720, Millipore, MA, USA). ImageJ software was used to analyze the blots.
SPSS version 22.0 (IBM Corporation, Armonk, NY, USA) software was employed to analyze all the data. Data are expressed as the mean ± SEM and were evaluated by Student’s t-test and ANOVA for multiple comparisons. P < 0.05 was considered significant.
2.8. Cell viability assay Cell viability was detected by Cell Counting Kit-8 (CCK-8) assays (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, cells were plated in 96-wells at a density of 5 103/well for 12 h. After treatment, the cells were incubated with 100 mL of DMEM containing 10% CCK-8 reagent in a 37 C incubator. Two hours later, the optical density values were detected with an enzyme-linked immunosorbent assay plate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). Then, the following formula was used to calculate the viability: cell viability¼ (OD450 of the treated groups/ OD450 of the control group) 100%. 2.9. Phagocytosis test Primary microglia were transferred with lentivirus targeting PDPN in advance. pHrodo™ Green E. coli BioParticles® Conjugate was purchased from ThermoFisher. The procedure was carried on according to the manufacturer’s instructions. The pictures of different groups were recorded under a Zeiss immunofluorescence microscope (Zeiss, German) and the fluorescence intensity was recorded by Cytofluor 4000 Fluorescence. 2.10. Transwell migration assay Transwells were employed to detect the microglial motility under different conditions. A total of 2 105 cells in 200 mL of DMEM were added into the upper chamber of 6.5 mm transwells with 8.0 mm pores (Corning Incorporated, Corning, NY, USA). After incubation at 37 C for 16 h, the cells in the interior of the chamber were removed. Then, the chambers were fixed with paraformaldehyde for 15 min and subsequently stained with 0.1% crystal violet for 10 min. The migrating cells were observed in six microscopic fields (200 ) under an inverted microscope (Carl Zeiss Meditec AG, Jena, Germany). 2.11. Immunofluorescence analysis of cells and tissues Cells were seeded onto microscope coverslips overnight and treated as indicated. Mouse brains were obtained, fixed, and embedded in paraformaldehyde for IHC and IF staining. Tissue sections (7 mm) were generated with a freezing microtome. Briefly, the slides were fixed with paraformaldehyde for 15 min and then treated with 0.3% Triton X-100 for 10 min. After fetal bovine serum was added for 30 min, specific primary antibodies and secondary antibodies were added to the slides. Nuclei were stained with DAPI (Sigma-Aldrich, D9542, 1/2000) for 10 min. All slides and tumor sections were observed using a Zeiss immunofluorescence microscope (Zeiss, German).
3. Results 3.1. PDPN expression patterns in healthy mouse brains and brains after TBI First, we generated TBI in mice by using a weight striking model. Then, immunofluorescence staining was used to determine the expression patterns of PDPN in normal mouse brains and brains after TBI (1, 3, and 7 d) (Fig. 1A). PDPN was rarely expressed in the cerebral cortex, hippocampus or corpus callosum in the sham group (Fig. 1A). However, strong immunofluorescence staining of PDPN was observed around the injured cortex and corpus callosum starting on the first day after TBI (Fig. 1A). Dual staining of PDPN and glial markers (NeuN, GFAP and Iba1) showed that PDPN strongly colocalized with Iba1, but PDPN did not show colocalization with neurons or astrocytes (Fig. 1B). PDPN and Iba1 staining in normal mouse brains showed that microglia expressed PDPN at a low level in the ‘rest state’ (Fig. 1C). Then, we traced the PDPNpositive microglia in the corpus callosum and capsula externa white matter. This result indicated that PDPN could be involved in the chemotaxis of microglia (Fig. 1D). 3.2. PDPN was substantially elevated in the early stages of TBI and was induced by inflammatory stimuli in vitro Samples around the lesion were collected at different time points after TBI for western blotting and PCR analyses (Fig. 2A). As shown in Fig. 2B and C, the PDPN mRNA levels rapidly increased at 6 h after induction of TBI, and the peak of PDPN appeared earlier than the peak of Iba1. Meanwhile, inflammatory factors such as IL1b, IL-6 and TNF-a were increased after the TBI (Fig. 2DeF). Western blotting also confirmed that PDPN was highly expressed at 1 d, and this level was maintained after 7 days (Fig. 2GeI). To determine whether TBI-related inflammatory stimuli induce the expression of PDPN in vitro, we used primary microglial cultures. As shown in Fig. 2J-L, the mRNA and protein levels of PDPN were elevated by ATP, LPS, H2O2 and Hb, which indicated an underlying role of PDPN in microglial inflammation. 3.3. The expression of PDPN was correlated with microglial inflammation To confirm the role of PDPN in microglia, we employed a lentivirus targeting PDPN to knock down PDPN in primary microglia and stimulated the microglia with Hb. As shown in Fig. 3AeC, PDPN knockdown was verified by western blotting and immunofluorescence staining. Though cell viability was not affected, as shown by the CCK-8 assay (Fig. 3D), microglial morphology was more ameba-like in the shCtrl þ Hb group than in the shPDPN þ Hb group (Fig. 3E). Interestingly, Hb significantly increased the expression of M1-like microglial inflammatory factors, such as IL1b and TNF-a, and downregulated M2-like microglial inflammatory factors, such as IL-10 and TGF-b, while no significant change of IL1b and TNF-a and significant elevation of IL-10 and TGF-b were observed after knockdown of PDPN (Fig. 3F and G). Furthermore, immunofluorescence staining showed a lower level of CD86positive microglia and a higher level of CD206-positive microglia in the shPDPN group than in the shCtrl group following exposure to
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Fig. 1. PDPN expression patterns in healthy mouse brains and brains after TBI. (A) Sections of normal mouse brains and traumatic brains at different times (1, 3, 7 d) were stained with PDPN antibody. PDPN was rarely expressed in the normal brain sections. However, strong immunofluorescence intensity of PDPN was recorded at the peri-injury cerebral cortex and corpus callosum. Scale bar, 400 mm. (B) Dual staining of PDPN and glial markers (NeuN, GFAP and Iba1) showed that PDPN strongly colocalized with Iba1 (Pearson’s Coefficient, 0.654; Overlap Coefficient, 0.961). Scale bar, 25 mm. (C) PDPN and Iba1 staining in normal mouse brains showed that microglia expressed PDPN in resting microglia. Scale bar, 25 mm. (D) PDPN-positive microglia were scattering at the capsula externa white matter, which indicated that PDPN could be involved in the microglial chemotaxis. Scale bar, 25 mm.
Fig. 2. PDPN was substantially elevated in the early stages of TBI and was induced by inflammatory stimuli in vitro. (A) Samples for RT-PCR and western blotting were collected as illustrated. Scale bar, 5 mm. (BeF) The mRNA levels of PDPN, Iba1, IL-b,IL-6 and TNF-a at the different time points (sham, 1 d, 2 d, 3 d and 7 d) after TBI were detected by RT-PCR. (GeI) The protein levels of PDPN and Iba1 in sham group and in groups at different time points (1 d, 2 d, 3 d and 7 d) were detected by western blotting. Then, the intensity of the blots was quantified and analyzed. (JeL) Primary microglia were extracted and cultured as described. The mRNA and protein levels of PDPN were detected after different stimuli (ATP 500 mM, LPS 1 mg/ml, H2O2 0.1 mM, Hb 13 mg/ml) were separately added for 24 h in the cultures. Data were represented as the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.
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Hb (Fig. 3H). This finding demonstrated that PDPN could influence the inflammatory phenotype of microglia. 3.4. PDPN was associated with microglial mobility by Regulating Matrix Metalloproteinases Since PDPN could influence the inflammatory phenotype of microglia, we further investigated whether PDPN influenced microglial movement. Firstly, to determine the subcellular localization of PDPN in primary microglia, dual staining of PDPN and Factin in the Hb-treated microglia was employed. As shown in Fig. 4A, PDPN was localized with F-actin-rich invadopodia. Next, we detected the migration ability via transwell assays and found that fewer cells penetrated through the micropore in the shPDPN þ Hb group than in the shCtrl þ Hb group (Fig. 4B and C). Interestingly, Phagocytosis of microglia was also impaired after PDPN knockdown (Fig. 4D and E). Because matrix metalloproteinases (MMPs) could facilitate cell mobility, we wondered whether MMPs were changed after PDPN was knocked down in microglia. RT-qPCR showed that MMP2 and MMP9 were significantly down-regulated in the shPDPN group
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(Fig. 4F). However, other MMPs were not significantly influenced after PDPN knockdown (Fig. 4F). Western blotting also confirmed that expression of MMP2 and MMP9 was decreased in the shPDPN and shPDPN þ Hb group compared with the shCtrl and shCtrl þ Hb group respectively (Fig. 4GeI). 4. Discussion In this study, we demonstrated that PDPN was highly expressed in the microglia of the mouse TBI groups. Although previous studies reported elevated expression of PDPN in astrocytes and hippocampal neurons in other neuroinflammatory models [15,16], we failed to detect colocalization of PDPN with neuron or astrocyte markers (NeuN or GFAP). In vitro experiments showed that elevated level of PDPN could be induced by LPS, ATP, H2O2 and Hb in microglia, indicating the possible functions of PDPN in the inflammatory states of the microglia. The roles of PDPN in inflammation have been reported in rheumatoid arthritis (RA), sepsis and wound healing processes [12e14]. The CLEC-2-PDPN pathway could facilitate PDPN-positive immune cell infiltration at the site of infection and promote
Fig. 3. The expression of PDPN was correlated with microglial inflammation. (A-C) Lentivirus targeting PDPN was transferred into microglia. The knockdown efficiency of PDPN was confirmed either in normal condition or in Hb exposed condition by western blotting and immunofluorescence staining. Scale bar, 10 mm. (D) CCK8 assay showed that PDPN knockdown did not affected microglia viability. (E) The morphology of microglia after PDPN knockdown was subtly changed. However, microglial morphology in the shCtrl þ Hb group was more ameba-like than in the shPDPN þ Hb group. Scale bar, 20 mm. (F, G) The mRNA levels of IL-b, TNF-a, IL-10 and TGF-b were detected by RT-PCR. (H) Immunostaining of CD86 and CD206 were employed to determine the inflammatory phenotypes of microglia. Scale bar, 20 mm. Data were represented as the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Fig. 4. PDPN was associated with microglial mobility by Regulating Matrix Metalloproteinases. (A) Primary microglia were exposed in culture medium with Hb for 24 h. Dual staining of PDPN and F-actin showed that PDPN was mainly localized with F-actin-rich invadopodia. Scale bar, 5 mm. (B, C) Transwell migration assays were conducted, pictured, calculated and analyzed. Scale bar, 50 mm. (D, E) Representative pictures of E.coli phagocytosis tests in shCtrl and shPDPN group were shown. Inflorescence intensity of two groups was detected by a fluorescence plate reader and then analyzed (D). Scale bar, 25 mm. (F) The mRNA levels of matrix metalloproteinases, (MMP1-3, MMP7, MMP9-14), were detected after PDPN knockdown. (GeI) The protein levels of MMP2 and MMP9 were significantly decreased in the shPDPN group and the shPDPN þ Hb group compared with in the shCtrl group and shCtrl þ Hb group respectively. Data were represented as the means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group.
macrophage recruitment. PDPN expression could contribute to chronic inflammation in RA by increasing cytokines, including IL-6, IL-8, and IL-17 [13,18]. Chronic microglial inflammation was also detected after TBI, and the activation phenotype of the microglia is strongly related to TBI outcomes [19,20]. The dual microglial phenotypes (M1-like and M2-like phenotype) in neuroinflammation have been widely reported [4e6]. The M1-like microglial phenotype is considered neurotoxic after TBI, promotes the release of proinflammatory cytokines and causes deleterious effects in neurons, while the M2-like phenotype results in the production of growth factors and anti-inflammatory cytokines [4e6]. Therefore, decreasing the neurotoxic phenotype of microglia could potentially improve the long-term prognosis of TBI patients. Here, we found that knockdown of PDPN in primary microglia prevented the induction of the M1 phenotype not only by simultaneously decreasing Hb-induced TNFa and IL-1b and increasing TGF-b and IL-10 expression but also by decreasing CD86-positive cells and elevating the proportion of CD206-positive cells after addition of Hb. These results indicated that PDPN might act as an inflammation sensor in microglia. In addition to the effects on the microglial phenotype, mobility and phagocytosis of microglia were affected after knockdown of PDPN under Hb exposure. Previously, other researchers reported that the intracellular domain of PDPN directly binds to the ERM proteins [21,22]. In addition, the PDPN-ERM complex regulates and cell motility and morphology not only by physically binding to the cytoskeleton but also by activating various cellular signaling pathways, such as Rho GTPases [21,23]. Consistently, in the microglia, we found that PDPN was highly expressed in the F-actinrich invadopodia and PDPN knockdown could significantly
decrease microglial mobility and phagocytosis. Interestingly, we also observed PDPN knockdown down-regulated expression of matrix metalloproteinases, MMP2 and MMP9 but not other matrix metalloproteinases. The influence of PDPN on the expression pattern of microglial matrix metalloproteinases needs further studies. Since high levels of MMPs can promote activated microglia to migrate through the white matter and aggravate the white matter injury after TBI [24], PDPN could be a potential target for TBI treatment. In conclusion, we found that PDPN was highly elevated in microglia after TBI. The elevated level of PDPN was related to the M1-like phenotype of microglia, and knockdown of PDPN increased the proportion of M2-like microglia under exposure to Hb in vitro, suggesting a potential treatment strategy for TBI. Additionally, knockdown of PDPN decreased microglial mobility and phagocytic activity partly by decreasing matrix metalloproteinases, MMP2 and MMP9. However, the mechanism of PDPN in microglial inflammation still needs to be elucidated. Meanwhile, the therapeutic and prognostic effects of targeting PDPN in post-TBI should be verified in further experiments in vivo.
Declaration of competing interest The authors declare no potential conflict of interest.
Acknowledgements This study was grant-supported by the National Natural Science Foundation of China (No.81672503 and No.81702484).
Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003
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Please cite this article as: M. Fei et al., Podoplanin influences the inflammatory phenotypes and mobility of microglia in traumatic brain injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.003