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Activation of P2X7R- NLRP3 pathway in Retinal microglia contribute to Retinal Ganglion Cells death in chronic ocular hypertension (COH)
Journal Pre-proof Activation of P2X7R- NLRP3 pathway in Retinal microglia contribute to Retinal Ganglion Cells death in chronic ocular hypertension (COH) Yujian Zhang, Yang Xu, Qing Sun, Shengding Xue, Huaijin Guan, Min Ji PII:
S0014-4835(19)30044-2
DOI:
https://doi.org/10.1016/j.exer.2019.107771
Reference:
YEXER 107771
To appear in:
Experimental Eye Research
Received Date: 17 January 2019 Revised Date:
25 July 2019
Accepted Date: 19 August 2019
Please cite this article as: Zhang, Y., Xu, Y., Sun, Q., Xue, S., Guan, H., Ji, M., Activation of P2X7R- NLRP3 pathway in Retinal microglia contribute to Retinal Ganglion Cells death in chronic ocular hypertension (COH), Experimental Eye Research (2019), doi: https://doi.org/10.1016/ j.exer.2019.107771. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Activation of P2X7R- NLRP3 pathway in Retinal microglia contribute to Retinal Ganglion Cells death in chronic ocular hypertension (COH)
Yujian Zhang1, Yang Xu2, Qing Sun1, Shengding Xue1, Huaijin Guan1**, Min Ji1* 1. Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001; China 2. Institute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, 221002; China.
* Corresponding author. ** Corresponding author. Dr. Min Ji and Professor. Huaijin Guan were the corresponding author of this article. E-mail addresses: Dr. Min Ji, [email protected]; Professor. Huaijin Guan, [email protected]
Abstract
Activation of P2X7R is linked to the occurrence and development of glaucoma. The present study concentrated on the activated P2X7R-NLRP3 pathway underlying the retinal microglia in retinal ganglion cells (RGCs) in chronic ocular hypertension (COH). Mouse COH model was set up to investigate the changes of P2X7R-NLRP3 inflammatory pathway in vivo. Primary microglia cells and primary RGCs were cultured and purified in vitro experiments. The expression of P2X7R, NLRP3, CASP-1, and ASC was detected and analyzed using Western blot, Quantitative polymerase chain reaction (qPCR) and immunofluorescence. Hoechst stains labeled nucleus to count microglia cells after experimental treatment. RGCs survival rate was examined utilizing LIVE/DEAD viability kit. The level of cytokines was measured by qPCR and enzyme-linked immunosorbent assay (ELISA). Consequently, the expression of P2X7R, NLRP3, CASP-1, and ASC was raised in COH mice retina. The number of microglia cells was increased after addition of BzATP, the agonist of P2X7R, to the culture medium of primary rat microglia cells. However, survival rates of RGCs decreased after addition of conditioned media to the RGC cultures. A438079 (100 µM), the inhibitor of P2X7R, and Mcc950 (1 µM), the inhibitor of NLRP3, blocked the effect of P2X7R activation in rat retinal microglia cells. Both inhibitors attenuated RGC death with the treatment of retina microglia cell conditioned medium (MCM). The production of some pro-inflammatory cytokines, such as TNF-α, CXCL-1, CSF-1, IL-6, IL-1β, and IL-18 was increased markedly with the activation of P2X7R in microglia.
However, the effect suffered as a result of A438079 and partially inhibited by Mcc950. These data suggested a role of P2X7R -NLRP3 pathway in activated retinal microglia cell-mediated RGC damages in COH.
Keywords: “microglia”; “glaucoma”; “chronic ocular hypertension”; “P2X7 receptor”; “NLRP3 inflammasome”; “cytokine”; “retinal ganglion cells”.
Introduction
Glaucoma is a disease that damages retinal ganglion cells(RGCs) and the optic nerve, resulting in visual field loss and the leading cause of permanent blindness in the world(Crabb, 2015; Hong et al., 2011). Accumulating evidence suggests that glial cells play a critical role in the development of glaucoma (Feilchenfeld et al., 2008; Seitz et al., 2013; Tezel, 2009). A large number of studies have reported the presence of neuroinflammatory responses by microglia, astrocytes and other immune cells in the optic nerve head (ONH) at different stages of experimental glaucoma (Gramlich et al., 2015; Hernandez, 2000; Rita et al., 2002). P2X7 receptors (P2X7R) are a nonselective cation channel receptor in inflammation and degeneration of the central nervous system(Stokes et al., 2015). Interestingly, P2X7R operates at the intersection of metabolism, inflammation, and cellular response to stress. In previous study, the P2X7R was shown to be activated in Müller cells of rat chronic ocular hypertension (COH) models (Xue et al., 2016; Ying et al., 2016), which might damage the RGCs. However, the role of P2X7R in retinal microglia cells and during the COH process is not yet elucidated. Long-term exposure to P2X7R agonist such as Adenosine 5′-triphosphate (ATP) or 3`-O-(4-benzoylbenzoyl) ATP (BzATP) resulted in formation of larger aqueous pores and finally leads to cell death(Jiang et al., 2013). Constitutive expression of NLRP3 inflammasome has detected in several immune cells (Andersen et al., 2014; Makoto et al., 2012; Segovia et al., 2018). It expressed predominantly in macrophages and plays a central role in response to infections and in general in the
pathogenesis of inflammation(Olaf et al., 2011). Consequently, downstream inflammatory cytokines mature and are released following the activation of the NLRP3 inflammasome. Furthermore, extracellular ATP can activate P2X7R, which is one of the most potent activators of the NLRP3 inflammasome (Franceschini et al., 2015b). This inflammasome is an intracellular protein complex, which mainly includes NLRP3, caspase1 (CASP-1), and apoptosis-associated speck-like protein containing CARD (ASC). The present study aimed to construct and detect the involvement of NLRP3 inflammasome in P2X7R-activated retinal microglia cells and the subsequent effect on RGCs.
Materials and Methods Animals All experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines on the Care and Use of Laboratory Animals and the guidelines of Nantong University on the ethical use of the animal. About 60 C57BL/6J (B6) mice for our experiment, including 30 males and 30 females, were used for establishing the COH model. 1-day-old Sprague–Dawley (SD) rats were used for primary retinal cell culture. All the animals in this experiment were ordered from the Nantong Laboratory Animal Company. No. 20160302-007 Table of Animal Experimental Ethical Inspection was approved by the Experimental Animals Center of Nantong University. During this study, all possible efforts were made to minimize the sacrifice to animals and alleviate
their suffering.
COH model The mice COH model was reproduced following a previous research(Huihui et al., 2011), which is described in more detail. All the 8-week-old mice without gender preference were anesthetized by a mixture of ketamine (25 mg/kg) and xylazine (10 mg/kg). After general anesthesia, ocular surface was anesthetized by proxymetacaine hydrochloride eye drops. The intracellular pressure (IOP) was elevated unilaterally in adult mice C57BL/6J (B6) mice after the anesthetic was injected into the anterior chamber using a needle with 5 µm polystyrene microbeads (Invitrogen, Carlsbad, CA) into the right eye at 0 and 14 days. A Hamilton syringe connected to a glass micropipette was used for the injection of small volume (2 µL). Another group was injected with only 2 µL of 0.9% saline to the ipsilateral anterior chamber that served as the control. After COH operation, eyes with cataract, endophthalmia, or other damage after injection were excluded.
IOP measurement of COH mice Mice were anesthetized by 2%– 4% isoflurane inhalation delivered in 100% oxygen, depending on a previous experiment(Yang et al., 2012). After mice lost consciousness, the IOP was measured daily in both eyes with a TonoLab tonometer (Colonial Medical Supply, Franconia, NH) as described previously
(Tadashiro et al., 2008). The IOP was measured in the morning about 3 min after the animal lost consciousness. The IOP was calculated as the mean of six measurements.
Primary rat retinal microglia cell culture 1-day-old SD rats were used to prepare the primary retinal microglia cell cultures. Briefly, fresh retina was aseptically removed from the rats in phosphate-buffered saline (PBS) at low temperature in advance. Then, the tissues were transferred to serum-free Dulbecco’s Modified Eagle’s Medium/Nutrient
Mixture
F-12
Ham
(DMEM/F12)
(Gibco
BRL
Life
Technologies, Rockville, MD, USA), minced to small fragments, and the mixture is filtered through Corning® 40 µm Cell Strainer after digested with 0.25% trypsin(Gibco BRL Life Technologies, Rockville, MD, USA) for about 15 min. Finally, the mixed cells were cultured at 37℃ in 5% CO2/95% air in DMEM/F12 with 10% fetal bovine serum (FBS) (Gibco BRL Life Technologies, Rockville, MD, USA). Mixed RGCs were grown in a humidified atmosphere for 5 days, and the growth medium was refreshed for the first time. Half a month after the implantation, microglia cells was separated from the others using a table concentrator. After 1 h agitation, the cells in the supernatant were collected and replated for the next experiment. Purity of the retina microglia cells was determined by the positive staining of Iba1, a microglia cell marker, in 10 random fields per dish, and the rate of Iba1-positive cells in each field was
calculated using a Leica microscope. A total of 60 fields were selected.
Primary rat retinal mixed neuron culture Primary rat retinal mixed neuron culture was prepared as described previously (Kerrison and Zack, 2006) with minor modification. The cell suspension was plated in poly-D-lysine-coated dishes and cultured in a Neurobasal-A medium (Gibco BRL Life Technologies, Rockville, MD, USA) supplemented with 2 mM glutamine, 25µM glutamic acid, 1% penicillin/streptomycin, B-27 supplement (1:50), insulin (5 µg/mL), 40 ng/mL of each Brain-derived neurotrophic factor (BDNF) (Miltenyi Biotec, Bergisch Gladbach, Germany) and Ciliary neurotrophic factor (CNTF) (Miltenyi Biotec, Bergisch Gladbach, Germany), and 5 mM forskolin (Sango Biotech, Shanghai, China). The RGCs were identified using an antibody against Brn3a, a RGC marker. Subsequently, primary neuron cells were cultured for at least 48 h before further use.
Western blot Rat retinas were collected in 4℃ PBS and lysed in lysis buffer (containing 1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet P-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After boiling for 15 min, samples were subject to centrifugation at 12,000 rpm for 5 min, and the supernatant was collected. Protein concentration was measured using a NanoDrop 2000
(Thermo Fisher Scientific). An equivalent of 30 µg protein sample was resolved by 8% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were estimated by the Bio-Rad protein assay (Bio-Rad Laboratories, Segrate, Milan, Italy). Also, cell lysates containing an equal amount of protein were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes that were blocked with EZ-Block B in 1× Tris-buffered saline (TBS), followed by probing with primary antibodies against P2X7R (1:1000), NLRP3 (1:800), caspase-1 (1:500), TMS1/ASC (1:200), and GAPDH (1:2000) at 4 ℃ overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5000) at room temperature for 2 h. The immunoreactive bands were detected using a chemifluorescent reagent (ECL) and exposure to X-ray film. The experiments were performed in triplicate, and the protein intensity was quantified by Image J software.
qPCR Total RNA was extracted from mouse retinas or primary rat retinal cells in modified TRIzol reagent (Invitrogen, Carlsbad, CA). Primary rat retina microglia (2 x 104 cells/well) was seeded into 96-well plates and allowed to adhere overnight. On the following day, microglia were primed 1 h before the experiments with serum-free DMEM-F12. Also, inhibitors of P2X7R or NLRP3
were added into the cells simultaneously. The content and purity of our total RNA were detected by Nano Drop 2000. The cDNA synthesis was carried out using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Run corn, Cheshire, UK). Primers involved in this experiment were designed and synthesized by Sangon Biotech. Amplification and qPCR measurements were performed by ABI 7500 real-time PCR systems. The analysis was performed by real-time qPCR using PowerUpTM SYBR® Green Master Mix (Thermo Fisher Scientific, Run corn, Cheshire, UK) according to the standard protocol. The amount of target mRNA in the test samples was normalized to that of Gapdh. 2-∆∆Ct was used to calculate the fold-change in gene expression. The primers are listed in Table 1.
Immunofluorescence (IF) After treatment, mice from different groups were sacrificed, and eyeballs were enucleated and placed in 4% paraformaldehyde for almost 4 h. After overnight dehydration with 30% sucrose, the eyeballs were wetted with SAKURA Tissue-Tek® O.C.T. compound and frozen at -80 ℃ for the next sections. Primary retinal cells were fixed with fresh paraformaldehyde (4%) for about 1 h at room temperature with gentle agitation. Then, samples were blocked with 3% BSA in PBS for at least 1h at room temperature and incubated with Iba1 (1:200, Wako Pure Chemical Industries, Osaka, Japan), NLRP3 (1:200, Abcam, Cambridge, MA, USA), caspase-1 (1:200, Abcam, Cambridge, MA, USA), and
TMS1/ASC (1:200, ProteinTech Group, Chicago, IL, USA) overnight at 4 ℃. Subsequently, the samples were incubated with the corresponding secondary antibodies for 6 h at room temperature in the dark, attended by Hoechst 33342 (1:4000). In addition, equivalent primary rat retina microglia (1 x 104 cells/well) was seeded into 24-well plates and nucleus was marked by Hoechst 33342 (1:5000) after experimental treatment to detect cell proliferation. Fluorescence was detected by a confocal microscope (Leica, Germany) or a fluorescence microscope (Leica).
Co-culture technique We collected supernatant of primary microglia cells treated with 1 h 100 µM BzATP (Sigma-Aldrich, St. Louis, MO, USA). We centrifuge the supernatant for 10 min at 4℃ until 4000 g. Sediment would be deserted and supernatant will be kept for the follow-up experiment. We co-cultured RGCs with the different conditioned medium for 2 h.
LIVE/DEAD viability assay The survival rate of RGCs was determined by the LIVE/DEAD viability assay kit (Invitrogen, Carlsbad, CA), which can simultaneously distinguish between live and dead cells as described previously (Harvey and Chintala, 2007). Briefly, cells plated on sterile glass coverslips were incubated (without fixation) for 30 min with a mixture of a solution containing 1 µM green-fluorescent
calcein-AM and 0.5 µM red-fluorescent ethidium homodimer-1 prepared in 1 mL Dulbecco’s PBS (D-PBS). After incubation, the mixture was removed, a drop of a D-PBS was added, and a coverslip was placed on the slides to be captured by a fluorescence microscope. The live and dead cells were individually counted from at least 100 cells (from each condition) and expressed as mean ± SD.
Enzyme-linked immunosorbent assay（ （ELISA） ） ELISA was conducted on microglia cell culture media collected after experimental treatment. The samples were subjected to centrifugation for 5 min at 4000 ×g; the conditioned supernatant was collected and stored at -80 °C until further use. The secretion of inflamma tory cytokines such as rat TNF-α, IL-1β, IL-18, CXCL1/CINC-1/KC (IL-8), IL-6, and IL-11 using ELISA kits (NeoBioscience Technology Co. Shenzhen, China) according to manufacturer’s instructions.
Statistical analysis All data are presented as means ± SD. The differences among groups involved in this study were analyzed by ANOVA or Student’s t-test. The statistical analyses were carried out using SPSS 22.0 software packages and GraphPad Prism version 7.0. P < 0.05 indicated statistical significance.
Results 1. COH activated P2X7R-NLRP3 pathway in the mice retina The mouse COH model was successfully established, and the changes in the IOP of the operated eyes were similar to that reported previously (Qiang et al., 2012) (Figure 1A and 1B). The expression of Iba1 was used to evaluate the microglia cell activation in the full-thickness retina. The sustained expression of microglia could be observed during 3 days in the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL) during COH process (Fig. 1C). The expression level of P2X7R, NLRP3, cleaved caspase-1, and ASC proteins was elevated during the COH process (Fig. 1D). Levels of P2x7r and Nlrp3 mRNA rose gradually and peaked at 14 days during COH (Fig. 1E and 1F). Cleaved caspase-1, ASC, and the other two parts of NLRP3 inflammasome showed varying degrees of elevation at different stages of the COH process. Cleaved caspase-1 and ASC were enhanced in the early stages (Fig. 1G and 1H). Consistent results could be acquired in qPCR (Fig. 1 I–L) as assessed by Western blot.
2. P2X7R activation enhanced expressions of NLRP3 inflammasome in microglia cells The purity of retinal microglia cells was deduced by immunofluorescent staining with the antibodies against Iba1, the representative cell marker for retinal microglia cells. The percentage of purified microglia cells was 89.2 ± 4.9%
(Fig. 2A). Consequently, the cell number was altered during BzATP treatment (Fig. 2B). After treatment with 100 µM BzATP for 1 h, microglia cells increased obviously detected by immunofluorescence (Fig. 2C). The expression of P2x7r, Nlrp3, Casp-1, and Asc mRNA in rat retinal microglia cells was measured by qPCR. The average mRNA level of P2x7r, Nlrp3, Casp-1, and Asc mRNA elevated after treatment with 100 µM BzATP for 1 h as compared to the control (Fig. 2D–2G).
3. Inhibiting P2X7R-NLRP3 pathway reduced NLRP3 inflammasome expression in microglia cells Compared to the BzATP groups (100 µM, 1 h), microglia cell number decreased significantly after addition of A438079 (100 µM, 2 h,Sigma-Aldrich, St. Louis, MO, USA) or Mcc950 (1 µM, 2 h, Sigma-Aldrich, St. Louis, MO, USA)) (Fig. 3A-3C). Each group of cells was subjected to serum starvation for 2 h before the use of inhibitors against P2X7R or NLRP3. 100 µM A438079 and 1 µM Mcc950 were selected as the appropriate dose for this experiment. After pre-addition of A438079 or Mcc950, the fluorescence intensity of NLRP3, CASP-1, and ASC was decreased as compared to BzATP groups (100 µM, 1 h) as assessed by immunofluorescence staining (Fig. 3D). A similar result was obtained by qPCR (Fig. 3E–3G).
4. Inhibiting P2X7R-NLRP3 pathway in microglia cells increased the
survival rate of RGCs in MCM The ratio of RGCs was identified by immunofluorescence staining with the antibodies against Brn3a, the representative cell marker for RGCs. The percentage of Brn3a-positive cells was about 67.9 ± 8.9% of the total cells (Fig. 4A). Compared to BzATP group, MCM group significantly reduced the survival rate of RGCs (Fig. 4B). Correspondingly, a sharp decrease was noted in the Bcl2 mRNA of the RGCs with MCM as compared to the BzATP groups (Fig. 4D). However, the Bax and Casp-3 mRNAs of the RGCs showed opposite results (Fig. 4E and 4F). These phenomena could be inhibited after pre-addition of A438079 or Mcc950 (Fig. 4B–4F).
5. P2X7R-NLRP3 pathway was involved in the production of cytokines of microglia cells The production of some cytokines, associated with microglia cells, was examined using qPCR and ELISA. Strikingly, A438079 significantly inhibited the transcription of these cytokines after treatment with 100 µM BzATP (Fig. 5A–5G). Nevertheless, Mcc950 could not effectively decrease the mRNA level of Il-6, Il-1β, Il-18, and Il-11 (Fig. 5D–5G). The extracellular cytokines in the supernatant post-treatment showed a trend consistent to that of the intracellular mRNA (Fig. 5H–5M).
Discussion
In recent years, an increasing number of studies have been dedicated to understanding the role of neuroinflammation in glaucoma. Some studies even pointed out that therapies modulating the immune and cell response might represent a promising approach for reducing the loss of retinal ganglion cells associated with glaucoma (Adornetto et al., 2019).
The upregulation of P2X7R has been demonstrated to contribute to the neuroinflammatory and neurodegenerative disease progression(Savio et al., 2018). Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X7R expression in the neuroinflammatory foci also containing activated microglia(Monif et al., 2009). Also, P2X7R expresses ubiquitously in RGCs, thereby increase the mortality of the cells (Xue et al., 2016). Accumulating evidence demonstrated that antagonists of the P2X7R prevented the ATP-induced neuronal apoptosis in the retina (Jacobson and Civan, 2016). Reportedly, P2X7R interacts directly with the NLRP3 inflammasome scaffold protein
(Franceschini
et
al.,
2015a).
Supposedly,
the
COH-activated
P2X7R-NLRP3 pathway led to a series of neuroinflammatory responses, which finally contributed to RGC death.
In another study, 300 µM BzATP was directly injected into the eyeballs of the rat to establish the animal COH model. The level of Iba-1, the characteristic marker of microglia, was increased significantly after P2X7R activation in retina
implied that the P2X7R stimulation could drive the microglia cells into a reactive form. We speculated that COH activated the P2X7R on the microglia cells and increased their viability. Also, several studies demonstrated that microglia activation could reduce number of RGCs(Bosco et al., 2012; Ke et al., 2015; Wu et al., 2014).
Microglia
are
the
resident
macrophages
that
maintain
retinal
homeostasis(Lloyd et al., 2017). Microglia are distributed in the outer plexiform layer (OPL), outer nuclear layer (ONL), IPL, GCL, and nerve fiber layer (NFL) of the primate retina (Ling et al., 2002). The microglia cells were extended and retracted in all directions randomly and repeatedly and monitored the whole retina homeostasis continuously. Herein, we found that the microglia could be activated in the INL, IPL, and GCL under a COH condition (Fig. 1C). In addition, the P2X7R-NLRP3 pathway was activated in the whole retina due to the COH. The role of microglia-mediated neuroinflammation in COH needs further investigation. Previous studies have shown that selective A2A receptor antagonist prevents microglia-mediated neuroinflammation and protects RGCs (Liu et al., 2016; Yang and Zhong, 2015). The current study showed that the P2X7R-NLPR3
pathway
was
involved
in
the
microglia-mediated
neuroinflammation (Fig 1. D–L).
Whether inhibiting the microglia activation could exert a protective effect on
RGCs by inhibiting the P2X7R-NLPR3 pathway is yet to be elucidated. Next, the primary rat retina microglia were cultured in a nutritive medium containing 10% FBS like in the in vitro model. We observed a sharp increase in cell number after treated with 100µM BzATP for 1 h (Fig. 2B). The Herve´Le Corronc group found proliferation of microglia in mammalian spinal cord depended almost entirely on P2X7R(Rigato et al., 2012). In addition, some other research also proved P2X7R played an important role in microglia cell proliferation(Monif et al., 2010; Reichenbach and Bringmann, 2016), which is consistent with our study.
The P2X7Rs effectuate the RGCs to lead to cytokine release, including IL-3. The RGCs also have IL-3Rs, and hence, the P2X7R agonism leads to auto-stimulation and changes in the Ca2+ levels (Lu et al., 2007). The mechanosensitive release of ATP stimulates the P2X7Rs on RGCs and ONH astrocytes. Moreover, the P2X7R stimulation on astrocytes or RGCs leads to IL-6 release; the stretch also leads to IL-6 release, the latter of which can be blocked by the P2X7R antagonists(Lu et al., 2017). Albalawi Farraj and et al (Albalawi et al., 2017) established acute ocular hypertension to evaluate the NLRP3 inflammasome and P2X7R interaction with respect to glaucoma. The increased expression of in vitro NLRP3 inflammasome and IL-1β was measured from ONH astrocytes following stretch or swelling. Although the glaucoma animal model is different, the P2X7R antagonist could effectively
decrease the expression of IL-1β in the study by Albalawi et al., which was consistent with the current results. In the central nervous system (CNS), the microglia responded to the damage by activating the ATP receptor and then extend to the injury area(Stefka and Traynelis, 2013). In the retina, the microglia cells were driven by extracellular ATP that was released from astrocytes and Müller cells (Wang and Wong, 2014). The cell interactions appear to be a mode of bi-directional communications that shape the overall injury response in the retina(Wang and Wong, 2014). Consequently, the ocular hypertension can drive the astrocytes or Müller cells to release the ATP to activate the microglia cells in glaucoma. However, additional experiments are essential to designate the cells found predominantly in vivo ocular hypertension.
Strikingly, the RGCs suffered deep damage after co-culturing with MCM, implying that the damage was not caused by P2X7R activation of RGCs. In addition, the inhibition of P2X7R-NLPR3 pathway protected the RGCs (Fig. 4). Also, it has been proved that P2X7R antagonist exerts a protective effect on the RGCs by inhibiting the microglia activation in a rat COH model(Dong et al., 2018), which was consistent with the current results. Herein, MCM aggravated the RGC damage, which indicated that the amount of pro-inflammatory cytokines might be greater than that of the anti-inflammatory cytokines. Moreover, the pro-inflammatory effect played a dominant pattern. Hence, we evaluated some pro-inflammatory cytokines, including TNF-α, CXCL-1, CSF1,
IL-6, IL-1β, IL-18, and IL-11, related to P2X7R and NLRP3 in the microglia cells. Among these cytokines, TNF-α, CXCL-1, and CSF1 were correlated to the P2X7R-NLRP3 pathway. The inhibition of both P2X7R and NLRP3 can decrease the production of these three cytokines. Moreover, the production and process of IL-6, IL-1β, IL-18, and IL-11 after P2X7R activation occurs in an NLRP3-independent manner.
In summary, COH contributes to the activation of P2X7R-NLRP3 inflammatory pathway in vivo. The inhibition of P2X7R-NLRP3 pathway is an effective method to protect and support RGCs. However, the underlying mechanism needs to be addressed in future investigations. This study might provide an in-depth understanding of retinal immunity and provide a new perspective for targeted therapy of glaucoma.
Acknowledgements This study was supported by Natural Science Foundation of China (NSFC, No.81670852); China Postdoctoral Science Foundation (No. 2017M610343); Postdoctoral Science Foundation of Jiangsu Province, China (No. 1701009A). This is my first SCI paper. I would like to thank my mother and my dad for their selfless love during my life. I would like to thank Dr. Min Ji for her patient guidance and encouragement to me during my graduate student period.
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Segovia, J.A., Chang, T.H., Winter, V.T., Coalson, J.J., Cagle, M.P., Pandranki, L., Bose, S., Baseman, J.B., Kannan, T.R., 2018. NLRP3 is a Critical Regulator of Inflammation and Innate Immune Cell Response during Mycoplasma pneumoniae Infection. Infection & Immunity 86, IAI.00548-00517. Seitz, Roswitha, Ohlmann, Andreas, Tamm, R, E., 2013. The role of Muller glia and microglia in glaucoma. Cell and Tissue Research 353, 339-345. Stefka, G., Traynelis, S.F., 2013. Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors. Journal of Biological Chemistry 288, 15291-15302. Stokes, L., Spencer, S.J., Jenkins, T.A., 2015. Understanding the role of P2X7 in affective disorders—are glial cells the major players? Frontiers in Cellular Neuroscience 9, 258. Tadashiro, S., Makoto, A., Masaaki, O., Makoto, A., 2008. The efficacy of TonoLab in detecting
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pressure--comparison with TonoPen and microneedle manometery. Current Eye Research 33, 247-252. Tezel, G., 2009. The role of glia, mitochondria, and the immune system in glaucoma. Invest Ophthalmol Vis Sci 50, 1001-1012. Wang, M., Wong, W.T., 2014. Microglia-Muller cell interactions in the retina. Adv Exp Med Biol 801, 333-338. Wu, N., Yu, J., Chen, S., Xu, J., Ying, X., Ye, M., Li, Y., Wang, Y., 2014. α-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush. Life Sciences 94, 17-23. Xue, B., Xie, Y., Xue, Y., Hu, N., Zhang, G., Guan, H., Ji, M., 2016. Involvement of P2X 7
receptors in retinal ganglion cell apoptosis induced by activated Müller cells. Experimental Eye Research 153, 42-50. Yang, Q., Cho, K.S., Chen, H., Yu, D., Wang, W.H., Luo, G., Pang, I.H., Guo, W., Chen, D.F., 2012. Microbead-induced ocular hypertensive mouse model for screening and testing of aqueous production suppressants for glaucoma. Invest Ophthalmol Vis Sci 53, 3733-3741. Yang, Z., Zhong, Y., 2015. Effect of adenosine on GLAST expression in the retina of a chronic ocular hypertension rat model. Experimental & Therapeutic Medicine 10, 991. Ying, X., Xie, Y., Bo, X., Nan, H., Zhang, G., Guan, H., Min, J., 2016. Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7Receptor Expression and Retinal Ganglion Cell Death. BioMed Research International,2016,(2016-9-21) 2016.
Figure Legends Figure. 1 Changes of P2X7R and NLRP3 inflammasome in mice COH retina. (A) IOP measurements in mice COH eyes as compared to the 0 d group. *P < 0.05 vs. 0 d. The data represented the mean ± SD of three independent experiments. (B) Anterior segment photography of the mouse after COH surgery. (C) Immunofluorescence labeling showed changes in Iba1 protein expression in COH retina extracts at different time points. The microglia identified in the Iba1 panel are marked by white arrows. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (D) Representative immunoblots show the changes in P2X7R, NLRP3, cleaved-caspase-1, and ASC protein in mice COH retina extracts at different time points. A representative of three experiments. (E–H) Bar charts summarized the average densitometric quantification of
immunoreactive bands of P2X7R, NLRP3, cleaved-caspase-1, and ASC expression at different time points as shown in (D). *P < 0.05 vs. 0 d. The data represent the mean ± SD of three independent experiments. (I–L) Bar charts show the changes in P2x7r, Nlrp3, Casp-1, and Asc mRNA in COH retina for different time points. *P < 0.05 vs. 0 d. The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments. IOP, intraocular pressure; COH, chronic ocular hypertension.
Figure. 2 Primary rat microglia cell identification and cell response after treatment with BzATP. (A) Microphotographs of primary cultured rat retinal microglia cells (a3), stained with the antibody against Iba1 (red, a1). a2 is Hoechst image. (B) Microphotographs of retinal microglia cell nucleus marked by Hoechst before and after 100 µM BzATP treatment. (C) Bar chart summarizes the average cell numbers under different conditions as shown in (B). *P < 0.05 vs. Control. (D– G) Bar charts showing the changes in P2x7r, Nlrp3, Casp-1, and Asc mRNA with 100 µM BzATP with different time points. *P < 0.05 vs. Control. The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments.
Figure. 3 Effect of the P2X7R-NLRP3 pathway inhibition on rat retinal
microglia cells. (A) Microphotographs of retinal microglia cell nucleus marked by Hoechst in control, BzATP (100 µM, 1 h), and pre-added A438079 (10, 50, 100 µM, 2 h) before BzATP (100 µM, 1 h) groups. (B-C) Bar chart summarizes the average cell numbers under different conditions as shown in (A). *P < 0.05 vs. Control. &
P < 0.05 vs. BzATP (100 µM, 1 h). The data represent the mean ± SD of three
independent experiments. (D) Immunofluorescence labeling shows the changes in NLRP3, CASP-1, and ASC protein expression in the retina microglia cells with different treatments. (E–G) Bar charts show the changes in Nlrp3, Casp-1, and Asc mRNA with different treatments. *P < 0.05 vs. Control; &
P < 0.05 vs. BzATP (100 µM, 1 h). The mRNA expression of genes was
normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments.
Figure. 4 Effect of retina microglia cell conditioned medium (MCM) on RGCs. (A) Microphotographs of primary mixed cultured rat retinal cells (a3) stained with the antibody against Brn3a, an RGC marker (red, a1). a2 is a Hoechst image. (B) Microphotographs show representative confocal images of Live signals (green) and Dead signals (red) in the retina microglia cells with different conditions in the control group. Control group: RGCs was cultured with normal microglia cell medium; BzATP group: RGCs was cultured with
microglia cell medium containing 100 µM BzATP; MCM group: RGCs was cultured with collected supernatant; MCM+A438079: After treated with 2 h 10 µM A438079, RGCs was cultured with collected supernatant; MCM+Mcc950: After treated with 2 h 1 nM Mcc950, RGCs was cultured with collected supernatant. (C) Bar chart summarizes the average ratio of Live/Dead cells under different conditions as shown in (B). *P < 0.05 vs. BzATP (100 µM, 24 h). &
P < 0.05 vs. MCM. The data represent the mean ± SD of three independent
experiments. (D) Bar charts show the viability of the retina microglia cells viability in control, BzATP (100 µM, 1 h), MCM (1 h), pre-added A438079 (100 µM, 2 h) before MCM (1 h), and pre-added Mcc950 (1 µM, 2 h) before MCM (1 h). *P < 0.05 vs. BzATP (100 µM, 1 h). &P < 0.05 vs. MCM (1 h). The data represent the mean ± SD of three independent experiments. (D-F) Bar charts show the changes in Bcl-2, Bax, and Casp-3 mRNA with different treatment. *P < 0.05 vs. BzATP (100 µM, 24 h); &P < 0.05 vs. MCM (24 h). The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments. MCM, retina microglia cell conditioned medium.
Figure. 5 Changes in the production of inflammatory cytokines in rat retina microglia cells. (A–G) Bar charts show the changes in Tnf-α, Cxcl-1, Csf1, Il-6, Il-1β, Il-18, and Il-11 mRNA with different treatments. *P < 0.05 vs. Control. &P < 0.05 vs.
BzATP. The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments. (H-M) Bar charts showing the changes in TNF-α, CXCL-1, IL-6, IL-1β, IL-18, and IL-11 extracellular concentration with different treatment. *P < 0.05 vs Control. &P < 0.05 vs BzATP. The data represent the mean ± SD of three independent experiments.
Figure. 6 Illustration of the proposed pathway where in P2X7R-NLRP3 upregulation induces the processing of cytokines and promotes the RGC death.
Organism Mus musculus Mus musculus Mus musculus Mus musculus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus
Symbol P2x7r Nlrp3 Casp-1 Asc
Table.1 Sequence of primers used for q-PCR NCBI Reference Sequence FORWARD NM_001038839.2 gtg ctc ttc tga ccg gcg ttg NM_001359638.1 gag ctg gac ctc agt gac aat gc NM_009807.2 aca acc act cgt aca cgt ctt gc NM_023258.4 cag gcg agc agc aag ag
REVERSE tag gac acc agg cag aga ctt cac acc aat gcg aga tcc tga caa cac cca gat cct cca gca gca act tc caa gag cgt cca gga tgg cat c
P2x7r
NM_019256.1
cca ctc tgc tgc ctt gtc gtt ac
ctg gta tgc ggt cag atg cga tgg
Nlrp3
NM_001191642.1
gag ctg gac ctc agt gac aat gc
acc aat gcg aga ccc tga caa cac
Casp-1
NM_012762.2
atg gcc gac aag gtc ctg agg
gtg aca tga tcg cac agg tct cg
Asc
NM_172322.1
aga gtc tgg agc tgt ggc tac tg
atg agt gct tgc ctg tgt tgg tc
Tnf-α
NM_012675.3
gca tga tcc gag atg tgg aac tgg
cgc cac gag cag gaa tga gaa g
Cxcl-1
NM_030845.1
acc gaa gtc ata gcc aca ctc aag
gcc agc gtt cac cag aca gac
Csf-1
NM_023981.4
agc aag gaa gcg aac gaa cca g
tca cga gga gac aga cca gca g
Il-6
NM_012589.2
agg agt ggc taa gga cca aga cc
tgc cga gta gac ctc ata gtg acc
Il-1b
NM_031512.2
atc tca cag cag cat ctc gac aag
cac act agc agg ccg tca tca tcc
Il-18
NM_019165.1
tga tat cga ccg aac agc caa cg
ggt cac agc cag tcc tct tac ttc
Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus
Il-11
NM_133519.4
gtg ctg aca agg ctt cga gta gac
gca agg cta ggc gag aca tca ag
Bcl2
NM_016993.1
acg gtg gtg gag gaa ctc ttc ag
ggt gtg cag atg ccg gtt cag
Bax
NM_017059.2
cca gga cgc atc cac caa gaa g
gct gcc aca cgg aag aag acc
Casp-3
NM_012922.2
gta cag agc tgg act gcg gta ttg
agt cgg cct cca ctg gta tct tc
Highlight 1. P2X7R-NLRP3 pathway is activated during chronic ocular hypertension (COH) process 2. P2X7R-NLRP3 proliferation
pathway
3. Retinal microglia cells P2X7R-NLRP3 pathway
activation
mediates
triggers
retinal
cytokine
microglia
production
cells
through
S0014-4835(19)30044-2
DOI:
https://doi.org/10.1016/j.exer.2019.107771
Reference:
YEXER 107771
To appear in:
Experimental Eye Research
Received Date: 17 January 2019 Revised Date:
25 July 2019
Accepted Date: 19 August 2019
Please cite this article as: Zhang, Y., Xu, Y., Sun, Q., Xue, S., Guan, H., Ji, M., Activation of P2X7R- NLRP3 pathway in Retinal microglia contribute to Retinal Ganglion Cells death in chronic ocular hypertension (COH), Experimental Eye Research (2019), doi: https://doi.org/10.1016/ j.exer.2019.107771. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Activation of P2X7R- NLRP3 pathway in Retinal microglia contribute to Retinal Ganglion Cells death in chronic ocular hypertension (COH)
Yujian Zhang1, Yang Xu2, Qing Sun1, Shengding Xue1, Huaijin Guan1**, Min Ji1* 1. Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001; China 2. Institute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, 221002; China.
* Corresponding author. ** Corresponding author. Dr. Min Ji and Professor. Huaijin Guan were the corresponding author of this article. E-mail addresses: Dr. Min Ji, [email protected]; Professor. Huaijin Guan, [email protected]
Abstract
Activation of P2X7R is linked to the occurrence and development of glaucoma. The present study concentrated on the activated P2X7R-NLRP3 pathway underlying the retinal microglia in retinal ganglion cells (RGCs) in chronic ocular hypertension (COH). Mouse COH model was set up to investigate the changes of P2X7R-NLRP3 inflammatory pathway in vivo. Primary microglia cells and primary RGCs were cultured and purified in vitro experiments. The expression of P2X7R, NLRP3, CASP-1, and ASC was detected and analyzed using Western blot, Quantitative polymerase chain reaction (qPCR) and immunofluorescence. Hoechst stains labeled nucleus to count microglia cells after experimental treatment. RGCs survival rate was examined utilizing LIVE/DEAD viability kit. The level of cytokines was measured by qPCR and enzyme-linked immunosorbent assay (ELISA). Consequently, the expression of P2X7R, NLRP3, CASP-1, and ASC was raised in COH mice retina. The number of microglia cells was increased after addition of BzATP, the agonist of P2X7R, to the culture medium of primary rat microglia cells. However, survival rates of RGCs decreased after addition of conditioned media to the RGC cultures. A438079 (100 µM), the inhibitor of P2X7R, and Mcc950 (1 µM), the inhibitor of NLRP3, blocked the effect of P2X7R activation in rat retinal microglia cells. Both inhibitors attenuated RGC death with the treatment of retina microglia cell conditioned medium (MCM). The production of some pro-inflammatory cytokines, such as TNF-α, CXCL-1, CSF-1, IL-6, IL-1β, and IL-18 was increased markedly with the activation of P2X7R in microglia.
However, the effect suffered as a result of A438079 and partially inhibited by Mcc950. These data suggested a role of P2X7R -NLRP3 pathway in activated retinal microglia cell-mediated RGC damages in COH.
Keywords: “microglia”; “glaucoma”; “chronic ocular hypertension”; “P2X7 receptor”; “NLRP3 inflammasome”; “cytokine”; “retinal ganglion cells”.
Introduction
Glaucoma is a disease that damages retinal ganglion cells(RGCs) and the optic nerve, resulting in visual field loss and the leading cause of permanent blindness in the world(Crabb, 2015; Hong et al., 2011). Accumulating evidence suggests that glial cells play a critical role in the development of glaucoma (Feilchenfeld et al., 2008; Seitz et al., 2013; Tezel, 2009). A large number of studies have reported the presence of neuroinflammatory responses by microglia, astrocytes and other immune cells in the optic nerve head (ONH) at different stages of experimental glaucoma (Gramlich et al., 2015; Hernandez, 2000; Rita et al., 2002). P2X7 receptors (P2X7R) are a nonselective cation channel receptor in inflammation and degeneration of the central nervous system(Stokes et al., 2015). Interestingly, P2X7R operates at the intersection of metabolism, inflammation, and cellular response to stress. In previous study, the P2X7R was shown to be activated in Müller cells of rat chronic ocular hypertension (COH) models (Xue et al., 2016; Ying et al., 2016), which might damage the RGCs. However, the role of P2X7R in retinal microglia cells and during the COH process is not yet elucidated. Long-term exposure to P2X7R agonist such as Adenosine 5′-triphosphate (ATP) or 3`-O-(4-benzoylbenzoyl) ATP (BzATP) resulted in formation of larger aqueous pores and finally leads to cell death(Jiang et al., 2013). Constitutive expression of NLRP3 inflammasome has detected in several immune cells (Andersen et al., 2014; Makoto et al., 2012; Segovia et al., 2018). It expressed predominantly in macrophages and plays a central role in response to infections and in general in the
pathogenesis of inflammation(Olaf et al., 2011). Consequently, downstream inflammatory cytokines mature and are released following the activation of the NLRP3 inflammasome. Furthermore, extracellular ATP can activate P2X7R, which is one of the most potent activators of the NLRP3 inflammasome (Franceschini et al., 2015b). This inflammasome is an intracellular protein complex, which mainly includes NLRP3, caspase1 (CASP-1), and apoptosis-associated speck-like protein containing CARD (ASC). The present study aimed to construct and detect the involvement of NLRP3 inflammasome in P2X7R-activated retinal microglia cells and the subsequent effect on RGCs.
Materials and Methods Animals All experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines on the Care and Use of Laboratory Animals and the guidelines of Nantong University on the ethical use of the animal. About 60 C57BL/6J (B6) mice for our experiment, including 30 males and 30 females, were used for establishing the COH model. 1-day-old Sprague–Dawley (SD) rats were used for primary retinal cell culture. All the animals in this experiment were ordered from the Nantong Laboratory Animal Company. No. 20160302-007 Table of Animal Experimental Ethical Inspection was approved by the Experimental Animals Center of Nantong University. During this study, all possible efforts were made to minimize the sacrifice to animals and alleviate
their suffering.
COH model The mice COH model was reproduced following a previous research(Huihui et al., 2011), which is described in more detail. All the 8-week-old mice without gender preference were anesthetized by a mixture of ketamine (25 mg/kg) and xylazine (10 mg/kg). After general anesthesia, ocular surface was anesthetized by proxymetacaine hydrochloride eye drops. The intracellular pressure (IOP) was elevated unilaterally in adult mice C57BL/6J (B6) mice after the anesthetic was injected into the anterior chamber using a needle with 5 µm polystyrene microbeads (Invitrogen, Carlsbad, CA) into the right eye at 0 and 14 days. A Hamilton syringe connected to a glass micropipette was used for the injection of small volume (2 µL). Another group was injected with only 2 µL of 0.9% saline to the ipsilateral anterior chamber that served as the control. After COH operation, eyes with cataract, endophthalmia, or other damage after injection were excluded.
IOP measurement of COH mice Mice were anesthetized by 2%– 4% isoflurane inhalation delivered in 100% oxygen, depending on a previous experiment(Yang et al., 2012). After mice lost consciousness, the IOP was measured daily in both eyes with a TonoLab tonometer (Colonial Medical Supply, Franconia, NH) as described previously
(Tadashiro et al., 2008). The IOP was measured in the morning about 3 min after the animal lost consciousness. The IOP was calculated as the mean of six measurements.
Primary rat retinal microglia cell culture 1-day-old SD rats were used to prepare the primary retinal microglia cell cultures. Briefly, fresh retina was aseptically removed from the rats in phosphate-buffered saline (PBS) at low temperature in advance. Then, the tissues were transferred to serum-free Dulbecco’s Modified Eagle’s Medium/Nutrient
Mixture
F-12
Ham
(DMEM/F12)
(Gibco
BRL
Life
Technologies, Rockville, MD, USA), minced to small fragments, and the mixture is filtered through Corning® 40 µm Cell Strainer after digested with 0.25% trypsin(Gibco BRL Life Technologies, Rockville, MD, USA) for about 15 min. Finally, the mixed cells were cultured at 37℃ in 5% CO2/95% air in DMEM/F12 with 10% fetal bovine serum (FBS) (Gibco BRL Life Technologies, Rockville, MD, USA). Mixed RGCs were grown in a humidified atmosphere for 5 days, and the growth medium was refreshed for the first time. Half a month after the implantation, microglia cells was separated from the others using a table concentrator. After 1 h agitation, the cells in the supernatant were collected and replated for the next experiment. Purity of the retina microglia cells was determined by the positive staining of Iba1, a microglia cell marker, in 10 random fields per dish, and the rate of Iba1-positive cells in each field was
calculated using a Leica microscope. A total of 60 fields were selected.
Primary rat retinal mixed neuron culture Primary rat retinal mixed neuron culture was prepared as described previously (Kerrison and Zack, 2006) with minor modification. The cell suspension was plated in poly-D-lysine-coated dishes and cultured in a Neurobasal-A medium (Gibco BRL Life Technologies, Rockville, MD, USA) supplemented with 2 mM glutamine, 25µM glutamic acid, 1% penicillin/streptomycin, B-27 supplement (1:50), insulin (5 µg/mL), 40 ng/mL of each Brain-derived neurotrophic factor (BDNF) (Miltenyi Biotec, Bergisch Gladbach, Germany) and Ciliary neurotrophic factor (CNTF) (Miltenyi Biotec, Bergisch Gladbach, Germany), and 5 mM forskolin (Sango Biotech, Shanghai, China). The RGCs were identified using an antibody against Brn3a, a RGC marker. Subsequently, primary neuron cells were cultured for at least 48 h before further use.
Western blot Rat retinas were collected in 4℃ PBS and lysed in lysis buffer (containing 1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet P-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After boiling for 15 min, samples were subject to centrifugation at 12,000 rpm for 5 min, and the supernatant was collected. Protein concentration was measured using a NanoDrop 2000
(Thermo Fisher Scientific). An equivalent of 30 µg protein sample was resolved by 8% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were estimated by the Bio-Rad protein assay (Bio-Rad Laboratories, Segrate, Milan, Italy). Also, cell lysates containing an equal amount of protein were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes that were blocked with EZ-Block B in 1× Tris-buffered saline (TBS), followed by probing with primary antibodies against P2X7R (1:1000), NLRP3 (1:800), caspase-1 (1:500), TMS1/ASC (1:200), and GAPDH (1:2000) at 4 ℃ overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5000) at room temperature for 2 h. The immunoreactive bands were detected using a chemifluorescent reagent (ECL) and exposure to X-ray film. The experiments were performed in triplicate, and the protein intensity was quantified by Image J software.
qPCR Total RNA was extracted from mouse retinas or primary rat retinal cells in modified TRIzol reagent (Invitrogen, Carlsbad, CA). Primary rat retina microglia (2 x 104 cells/well) was seeded into 96-well plates and allowed to adhere overnight. On the following day, microglia were primed 1 h before the experiments with serum-free DMEM-F12. Also, inhibitors of P2X7R or NLRP3
were added into the cells simultaneously. The content and purity of our total RNA were detected by Nano Drop 2000. The cDNA synthesis was carried out using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Run corn, Cheshire, UK). Primers involved in this experiment were designed and synthesized by Sangon Biotech. Amplification and qPCR measurements were performed by ABI 7500 real-time PCR systems. The analysis was performed by real-time qPCR using PowerUpTM SYBR® Green Master Mix (Thermo Fisher Scientific, Run corn, Cheshire, UK) according to the standard protocol. The amount of target mRNA in the test samples was normalized to that of Gapdh. 2-∆∆Ct was used to calculate the fold-change in gene expression. The primers are listed in Table 1.
Immunofluorescence (IF) After treatment, mice from different groups were sacrificed, and eyeballs were enucleated and placed in 4% paraformaldehyde for almost 4 h. After overnight dehydration with 30% sucrose, the eyeballs were wetted with SAKURA Tissue-Tek® O.C.T. compound and frozen at -80 ℃ for the next sections. Primary retinal cells were fixed with fresh paraformaldehyde (4%) for about 1 h at room temperature with gentle agitation. Then, samples were blocked with 3% BSA in PBS for at least 1h at room temperature and incubated with Iba1 (1:200, Wako Pure Chemical Industries, Osaka, Japan), NLRP3 (1:200, Abcam, Cambridge, MA, USA), caspase-1 (1:200, Abcam, Cambridge, MA, USA), and
TMS1/ASC (1:200, ProteinTech Group, Chicago, IL, USA) overnight at 4 ℃. Subsequently, the samples were incubated with the corresponding secondary antibodies for 6 h at room temperature in the dark, attended by Hoechst 33342 (1:4000). In addition, equivalent primary rat retina microglia (1 x 104 cells/well) was seeded into 24-well plates and nucleus was marked by Hoechst 33342 (1:5000) after experimental treatment to detect cell proliferation. Fluorescence was detected by a confocal microscope (Leica, Germany) or a fluorescence microscope (Leica).
Co-culture technique We collected supernatant of primary microglia cells treated with 1 h 100 µM BzATP (Sigma-Aldrich, St. Louis, MO, USA). We centrifuge the supernatant for 10 min at 4℃ until 4000 g. Sediment would be deserted and supernatant will be kept for the follow-up experiment. We co-cultured RGCs with the different conditioned medium for 2 h.
LIVE/DEAD viability assay The survival rate of RGCs was determined by the LIVE/DEAD viability assay kit (Invitrogen, Carlsbad, CA), which can simultaneously distinguish between live and dead cells as described previously (Harvey and Chintala, 2007). Briefly, cells plated on sterile glass coverslips were incubated (without fixation) for 30 min with a mixture of a solution containing 1 µM green-fluorescent
calcein-AM and 0.5 µM red-fluorescent ethidium homodimer-1 prepared in 1 mL Dulbecco’s PBS (D-PBS). After incubation, the mixture was removed, a drop of a D-PBS was added, and a coverslip was placed on the slides to be captured by a fluorescence microscope. The live and dead cells were individually counted from at least 100 cells (from each condition) and expressed as mean ± SD.
Enzyme-linked immunosorbent assay（ （ELISA） ） ELISA was conducted on microglia cell culture media collected after experimental treatment. The samples were subjected to centrifugation for 5 min at 4000 ×g; the conditioned supernatant was collected and stored at -80 °C until further use. The secretion of inflamma tory cytokines such as rat TNF-α, IL-1β, IL-18, CXCL1/CINC-1/KC (IL-8), IL-6, and IL-11 using ELISA kits (NeoBioscience Technology Co. Shenzhen, China) according to manufacturer’s instructions.
Statistical analysis All data are presented as means ± SD. The differences among groups involved in this study were analyzed by ANOVA or Student’s t-test. The statistical analyses were carried out using SPSS 22.0 software packages and GraphPad Prism version 7.0. P < 0.05 indicated statistical significance.
Results 1. COH activated P2X7R-NLRP3 pathway in the mice retina The mouse COH model was successfully established, and the changes in the IOP of the operated eyes were similar to that reported previously (Qiang et al., 2012) (Figure 1A and 1B). The expression of Iba1 was used to evaluate the microglia cell activation in the full-thickness retina. The sustained expression of microglia could be observed during 3 days in the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL) during COH process (Fig. 1C). The expression level of P2X7R, NLRP3, cleaved caspase-1, and ASC proteins was elevated during the COH process (Fig. 1D). Levels of P2x7r and Nlrp3 mRNA rose gradually and peaked at 14 days during COH (Fig. 1E and 1F). Cleaved caspase-1, ASC, and the other two parts of NLRP3 inflammasome showed varying degrees of elevation at different stages of the COH process. Cleaved caspase-1 and ASC were enhanced in the early stages (Fig. 1G and 1H). Consistent results could be acquired in qPCR (Fig. 1 I–L) as assessed by Western blot.
2. P2X7R activation enhanced expressions of NLRP3 inflammasome in microglia cells The purity of retinal microglia cells was deduced by immunofluorescent staining with the antibodies against Iba1, the representative cell marker for retinal microglia cells. The percentage of purified microglia cells was 89.2 ± 4.9%
(Fig. 2A). Consequently, the cell number was altered during BzATP treatment (Fig. 2B). After treatment with 100 µM BzATP for 1 h, microglia cells increased obviously detected by immunofluorescence (Fig. 2C). The expression of P2x7r, Nlrp3, Casp-1, and Asc mRNA in rat retinal microglia cells was measured by qPCR. The average mRNA level of P2x7r, Nlrp3, Casp-1, and Asc mRNA elevated after treatment with 100 µM BzATP for 1 h as compared to the control (Fig. 2D–2G).
3. Inhibiting P2X7R-NLRP3 pathway reduced NLRP3 inflammasome expression in microglia cells Compared to the BzATP groups (100 µM, 1 h), microglia cell number decreased significantly after addition of A438079 (100 µM, 2 h,Sigma-Aldrich, St. Louis, MO, USA) or Mcc950 (1 µM, 2 h, Sigma-Aldrich, St. Louis, MO, USA)) (Fig. 3A-3C). Each group of cells was subjected to serum starvation for 2 h before the use of inhibitors against P2X7R or NLRP3. 100 µM A438079 and 1 µM Mcc950 were selected as the appropriate dose for this experiment. After pre-addition of A438079 or Mcc950, the fluorescence intensity of NLRP3, CASP-1, and ASC was decreased as compared to BzATP groups (100 µM, 1 h) as assessed by immunofluorescence staining (Fig. 3D). A similar result was obtained by qPCR (Fig. 3E–3G).
4. Inhibiting P2X7R-NLRP3 pathway in microglia cells increased the
survival rate of RGCs in MCM The ratio of RGCs was identified by immunofluorescence staining with the antibodies against Brn3a, the representative cell marker for RGCs. The percentage of Brn3a-positive cells was about 67.9 ± 8.9% of the total cells (Fig. 4A). Compared to BzATP group, MCM group significantly reduced the survival rate of RGCs (Fig. 4B). Correspondingly, a sharp decrease was noted in the Bcl2 mRNA of the RGCs with MCM as compared to the BzATP groups (Fig. 4D). However, the Bax and Casp-3 mRNAs of the RGCs showed opposite results (Fig. 4E and 4F). These phenomena could be inhibited after pre-addition of A438079 or Mcc950 (Fig. 4B–4F).
5. P2X7R-NLRP3 pathway was involved in the production of cytokines of microglia cells The production of some cytokines, associated with microglia cells, was examined using qPCR and ELISA. Strikingly, A438079 significantly inhibited the transcription of these cytokines after treatment with 100 µM BzATP (Fig. 5A–5G). Nevertheless, Mcc950 could not effectively decrease the mRNA level of Il-6, Il-1β, Il-18, and Il-11 (Fig. 5D–5G). The extracellular cytokines in the supernatant post-treatment showed a trend consistent to that of the intracellular mRNA (Fig. 5H–5M).
Discussion
In recent years, an increasing number of studies have been dedicated to understanding the role of neuroinflammation in glaucoma. Some studies even pointed out that therapies modulating the immune and cell response might represent a promising approach for reducing the loss of retinal ganglion cells associated with glaucoma (Adornetto et al., 2019).
The upregulation of P2X7R has been demonstrated to contribute to the neuroinflammatory and neurodegenerative disease progression(Savio et al., 2018). Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X7R expression in the neuroinflammatory foci also containing activated microglia(Monif et al., 2009). Also, P2X7R expresses ubiquitously in RGCs, thereby increase the mortality of the cells (Xue et al., 2016). Accumulating evidence demonstrated that antagonists of the P2X7R prevented the ATP-induced neuronal apoptosis in the retina (Jacobson and Civan, 2016). Reportedly, P2X7R interacts directly with the NLRP3 inflammasome scaffold protein
(Franceschini
et
al.,
2015a).
Supposedly,
the
COH-activated
P2X7R-NLRP3 pathway led to a series of neuroinflammatory responses, which finally contributed to RGC death.
In another study, 300 µM BzATP was directly injected into the eyeballs of the rat to establish the animal COH model. The level of Iba-1, the characteristic marker of microglia, was increased significantly after P2X7R activation in retina
implied that the P2X7R stimulation could drive the microglia cells into a reactive form. We speculated that COH activated the P2X7R on the microglia cells and increased their viability. Also, several studies demonstrated that microglia activation could reduce number of RGCs(Bosco et al., 2012; Ke et al., 2015; Wu et al., 2014).
Microglia
are
the
resident
macrophages
that
maintain
retinal
homeostasis(Lloyd et al., 2017). Microglia are distributed in the outer plexiform layer (OPL), outer nuclear layer (ONL), IPL, GCL, and nerve fiber layer (NFL) of the primate retina (Ling et al., 2002). The microglia cells were extended and retracted in all directions randomly and repeatedly and monitored the whole retina homeostasis continuously. Herein, we found that the microglia could be activated in the INL, IPL, and GCL under a COH condition (Fig. 1C). In addition, the P2X7R-NLRP3 pathway was activated in the whole retina due to the COH. The role of microglia-mediated neuroinflammation in COH needs further investigation. Previous studies have shown that selective A2A receptor antagonist prevents microglia-mediated neuroinflammation and protects RGCs (Liu et al., 2016; Yang and Zhong, 2015). The current study showed that the P2X7R-NLPR3
pathway
was
involved
in
the
microglia-mediated
neuroinflammation (Fig 1. D–L).
Whether inhibiting the microglia activation could exert a protective effect on
RGCs by inhibiting the P2X7R-NLPR3 pathway is yet to be elucidated. Next, the primary rat retina microglia were cultured in a nutritive medium containing 10% FBS like in the in vitro model. We observed a sharp increase in cell number after treated with 100µM BzATP for 1 h (Fig. 2B). The Herve´Le Corronc group found proliferation of microglia in mammalian spinal cord depended almost entirely on P2X7R(Rigato et al., 2012). In addition, some other research also proved P2X7R played an important role in microglia cell proliferation(Monif et al., 2010; Reichenbach and Bringmann, 2016), which is consistent with our study.
The P2X7Rs effectuate the RGCs to lead to cytokine release, including IL-3. The RGCs also have IL-3Rs, and hence, the P2X7R agonism leads to auto-stimulation and changes in the Ca2+ levels (Lu et al., 2007). The mechanosensitive release of ATP stimulates the P2X7Rs on RGCs and ONH astrocytes. Moreover, the P2X7R stimulation on astrocytes or RGCs leads to IL-6 release; the stretch also leads to IL-6 release, the latter of which can be blocked by the P2X7R antagonists(Lu et al., 2017). Albalawi Farraj and et al (Albalawi et al., 2017) established acute ocular hypertension to evaluate the NLRP3 inflammasome and P2X7R interaction with respect to glaucoma. The increased expression of in vitro NLRP3 inflammasome and IL-1β was measured from ONH astrocytes following stretch or swelling. Although the glaucoma animal model is different, the P2X7R antagonist could effectively
decrease the expression of IL-1β in the study by Albalawi et al., which was consistent with the current results. In the central nervous system (CNS), the microglia responded to the damage by activating the ATP receptor and then extend to the injury area(Stefka and Traynelis, 2013). In the retina, the microglia cells were driven by extracellular ATP that was released from astrocytes and Müller cells (Wang and Wong, 2014). The cell interactions appear to be a mode of bi-directional communications that shape the overall injury response in the retina(Wang and Wong, 2014). Consequently, the ocular hypertension can drive the astrocytes or Müller cells to release the ATP to activate the microglia cells in glaucoma. However, additional experiments are essential to designate the cells found predominantly in vivo ocular hypertension.
Strikingly, the RGCs suffered deep damage after co-culturing with MCM, implying that the damage was not caused by P2X7R activation of RGCs. In addition, the inhibition of P2X7R-NLPR3 pathway protected the RGCs (Fig. 4). Also, it has been proved that P2X7R antagonist exerts a protective effect on the RGCs by inhibiting the microglia activation in a rat COH model(Dong et al., 2018), which was consistent with the current results. Herein, MCM aggravated the RGC damage, which indicated that the amount of pro-inflammatory cytokines might be greater than that of the anti-inflammatory cytokines. Moreover, the pro-inflammatory effect played a dominant pattern. Hence, we evaluated some pro-inflammatory cytokines, including TNF-α, CXCL-1, CSF1,
IL-6, IL-1β, IL-18, and IL-11, related to P2X7R and NLRP3 in the microglia cells. Among these cytokines, TNF-α, CXCL-1, and CSF1 were correlated to the P2X7R-NLRP3 pathway. The inhibition of both P2X7R and NLRP3 can decrease the production of these three cytokines. Moreover, the production and process of IL-6, IL-1β, IL-18, and IL-11 after P2X7R activation occurs in an NLRP3-independent manner.
In summary, COH contributes to the activation of P2X7R-NLRP3 inflammatory pathway in vivo. The inhibition of P2X7R-NLRP3 pathway is an effective method to protect and support RGCs. However, the underlying mechanism needs to be addressed in future investigations. This study might provide an in-depth understanding of retinal immunity and provide a new perspective for targeted therapy of glaucoma.
Acknowledgements This study was supported by Natural Science Foundation of China (NSFC, No.81670852); China Postdoctoral Science Foundation (No. 2017M610343); Postdoctoral Science Foundation of Jiangsu Province, China (No. 1701009A). This is my first SCI paper. I would like to thank my mother and my dad for their selfless love during my life. I would like to thank Dr. Min Ji for her patient guidance and encouragement to me during my graduate student period.
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Figure Legends Figure. 1 Changes of P2X7R and NLRP3 inflammasome in mice COH retina. (A) IOP measurements in mice COH eyes as compared to the 0 d group. *P < 0.05 vs. 0 d. The data represented the mean ± SD of three independent experiments. (B) Anterior segment photography of the mouse after COH surgery. (C) Immunofluorescence labeling showed changes in Iba1 protein expression in COH retina extracts at different time points. The microglia identified in the Iba1 panel are marked by white arrows. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (D) Representative immunoblots show the changes in P2X7R, NLRP3, cleaved-caspase-1, and ASC protein in mice COH retina extracts at different time points. A representative of three experiments. (E–H) Bar charts summarized the average densitometric quantification of
immunoreactive bands of P2X7R, NLRP3, cleaved-caspase-1, and ASC expression at different time points as shown in (D). *P < 0.05 vs. 0 d. The data represent the mean ± SD of three independent experiments. (I–L) Bar charts show the changes in P2x7r, Nlrp3, Casp-1, and Asc mRNA in COH retina for different time points. *P < 0.05 vs. 0 d. The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments. IOP, intraocular pressure; COH, chronic ocular hypertension.
Figure. 2 Primary rat microglia cell identification and cell response after treatment with BzATP. (A) Microphotographs of primary cultured rat retinal microglia cells (a3), stained with the antibody against Iba1 (red, a1). a2 is Hoechst image. (B) Microphotographs of retinal microglia cell nucleus marked by Hoechst before and after 100 µM BzATP treatment. (C) Bar chart summarizes the average cell numbers under different conditions as shown in (B). *P < 0.05 vs. Control. (D– G) Bar charts showing the changes in P2x7r, Nlrp3, Casp-1, and Asc mRNA with 100 µM BzATP with different time points. *P < 0.05 vs. Control. The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments.
Figure. 3 Effect of the P2X7R-NLRP3 pathway inhibition on rat retinal
microglia cells. (A) Microphotographs of retinal microglia cell nucleus marked by Hoechst in control, BzATP (100 µM, 1 h), and pre-added A438079 (10, 50, 100 µM, 2 h) before BzATP (100 µM, 1 h) groups. (B-C) Bar chart summarizes the average cell numbers under different conditions as shown in (A). *P < 0.05 vs. Control. &
P < 0.05 vs. BzATP (100 µM, 1 h). The data represent the mean ± SD of three
independent experiments. (D) Immunofluorescence labeling shows the changes in NLRP3, CASP-1, and ASC protein expression in the retina microglia cells with different treatments. (E–G) Bar charts show the changes in Nlrp3, Casp-1, and Asc mRNA with different treatments. *P < 0.05 vs. Control; &
P < 0.05 vs. BzATP (100 µM, 1 h). The mRNA expression of genes was
normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments.
Figure. 4 Effect of retina microglia cell conditioned medium (MCM) on RGCs. (A) Microphotographs of primary mixed cultured rat retinal cells (a3) stained with the antibody against Brn3a, an RGC marker (red, a1). a2 is a Hoechst image. (B) Microphotographs show representative confocal images of Live signals (green) and Dead signals (red) in the retina microglia cells with different conditions in the control group. Control group: RGCs was cultured with normal microglia cell medium; BzATP group: RGCs was cultured with
microglia cell medium containing 100 µM BzATP; MCM group: RGCs was cultured with collected supernatant; MCM+A438079: After treated with 2 h 10 µM A438079, RGCs was cultured with collected supernatant; MCM+Mcc950: After treated with 2 h 1 nM Mcc950, RGCs was cultured with collected supernatant. (C) Bar chart summarizes the average ratio of Live/Dead cells under different conditions as shown in (B). *P < 0.05 vs. BzATP (100 µM, 24 h). &
P < 0.05 vs. MCM. The data represent the mean ± SD of three independent
experiments. (D) Bar charts show the viability of the retina microglia cells viability in control, BzATP (100 µM, 1 h), MCM (1 h), pre-added A438079 (100 µM, 2 h) before MCM (1 h), and pre-added Mcc950 (1 µM, 2 h) before MCM (1 h). *P < 0.05 vs. BzATP (100 µM, 1 h). &P < 0.05 vs. MCM (1 h). The data represent the mean ± SD of three independent experiments. (D-F) Bar charts show the changes in Bcl-2, Bax, and Casp-3 mRNA with different treatment. *P < 0.05 vs. BzATP (100 µM, 24 h); &P < 0.05 vs. MCM (24 h). The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments. MCM, retina microglia cell conditioned medium.
Figure. 5 Changes in the production of inflammatory cytokines in rat retina microglia cells. (A–G) Bar charts show the changes in Tnf-α, Cxcl-1, Csf1, Il-6, Il-1β, Il-18, and Il-11 mRNA with different treatments. *P < 0.05 vs. Control. &P < 0.05 vs.
BzATP. The mRNA expression of the genes was normalized against that of Gapdh. The data represent the mean ± SD of three independent experiments. (H-M) Bar charts showing the changes in TNF-α, CXCL-1, IL-6, IL-1β, IL-18, and IL-11 extracellular concentration with different treatment. *P < 0.05 vs Control. &P < 0.05 vs BzATP. The data represent the mean ± SD of three independent experiments.
Figure. 6 Illustration of the proposed pathway where in P2X7R-NLRP3 upregulation induces the processing of cytokines and promotes the RGC death.
Organism Mus musculus Mus musculus Mus musculus Mus musculus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus
Symbol P2x7r Nlrp3 Casp-1 Asc
Table.1 Sequence of primers used for q-PCR NCBI Reference Sequence FORWARD NM_001038839.2 gtg ctc ttc tga ccg gcg ttg NM_001359638.1 gag ctg gac ctc agt gac aat gc NM_009807.2 aca acc act cgt aca cgt ctt gc NM_023258.4 cag gcg agc agc aag ag
REVERSE tag gac acc agg cag aga ctt cac acc aat gcg aga tcc tga caa cac cca gat cct cca gca gca act tc caa gag cgt cca gga tgg cat c
P2x7r
NM_019256.1
cca ctc tgc tgc ctt gtc gtt ac
ctg gta tgc ggt cag atg cga tgg
Nlrp3
NM_001191642.1
gag ctg gac ctc agt gac aat gc
acc aat gcg aga ccc tga caa cac
Casp-1
NM_012762.2
atg gcc gac aag gtc ctg agg
gtg aca tga tcg cac agg tct cg
Asc
NM_172322.1
aga gtc tgg agc tgt ggc tac tg
atg agt gct tgc ctg tgt tgg tc
Tnf-α
NM_012675.3
gca tga tcc gag atg tgg aac tgg
cgc cac gag cag gaa tga gaa g
Cxcl-1
NM_030845.1
acc gaa gtc ata gcc aca ctc aag
gcc agc gtt cac cag aca gac
Csf-1
NM_023981.4
agc aag gaa gcg aac gaa cca g
tca cga gga gac aga cca gca g
Il-6
NM_012589.2
agg agt ggc taa gga cca aga cc
tgc cga gta gac ctc ata gtg acc
Il-1b
NM_031512.2
atc tca cag cag cat ctc gac aag
cac act agc agg ccg tca tca tcc
Il-18
NM_019165.1
tga tat cga ccg aac agc caa cg
ggt cac agc cag tcc tct tac ttc
Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus
Il-11
NM_133519.4
gtg ctg aca agg ctt cga gta gac
gca agg cta ggc gag aca tca ag
Bcl2
NM_016993.1
acg gtg gtg gag gaa ctc ttc ag
ggt gtg cag atg ccg gtt cag
Bax
NM_017059.2
cca gga cgc atc cac caa gaa g
gct gcc aca cgg aag aag acc
Casp-3
NM_012922.2
gta cag agc tgg act gcg gta ttg
agt cgg cct cca ctg gta tct tc
Highlight 1. P2X7R-NLRP3 pathway is activated during chronic ocular hypertension (COH) process 2. P2X7R-NLRP3 proliferation
pathway
3. Retinal microglia cells P2X7R-NLRP3 pathway
activation
mediates
triggers
retinal
cytokine
microglia
production
cells
through