Crosstalk Between Autophagy and Pyroptosis of Microglia contributes to Retinal Ganglion Cells Damage in Chronic Ocular Hypertension

doi:10.21203/rs.3.rs-1516632/v1

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Crosstalk Between Autophagy and Pyroptosis of Microglia contributes to Retinal Ganglion Cells Damage in Chronic Ocular Hypertension

https://doi.org/10.21203/rs.3.rs-1516632/v1

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BACKGROUND. Glaucoma is a group of neurodegenerative diseases characterized by the damage of retinal ganglion cells (RGCs). Chronic ocular hypertension (COH) as one of the critical risk factors of glaucoma, the mechanisms of COH-induced RGCs damage has not been clarified. In recent years, studies have reported altered levels of autophagy and pyroptosis in glaucomatous microglia. However, it is unclear whether autophagy and pyroptosis of microglia are related and whether this relationship mediates COH-induced RGCs damage.

METHODS. A mouse COH experimental glaucomatous model in vivo was established. Besides, we used 2 mmol/L (mM) ATP-treated microglia to simulate COH in vitro. Western blot, enzyme-linked immunosorbent assay, immunofluorescence and electron microscopy were employed to detect the exact relationship between autophagy and pyroptosis of microglia. Moreover, we utilized co-cultivation, live/dead viability assay, neutralizing antibody treatment and intra-vitreous injection to investigate the role of this relationship of microglia in COH-induced RGCs damage.

RESULTS. In COH model in vivo, a transient boost of autophagy and a gradual increase of pyroptosis were observed. And the consistent phenomena were also found in ATP-treated microglia in vitro. We next observed that inhibition of the P2X7R-NLRP3-CASP1-GSDMD pathway and activation of autophagy could attenuate pyroptosis of microglia, thereby counteracting the RGCs damage induced by COH in vivo and ATP in vitro. The intricate molecular mechanism between pyroptosis mediated by the above pathway and autophagy is referred as “crosstalk” in this paper. Furthermore, we revealed the indispensable significance of interleukin-1β (IL-1β) in microglia-mediated RGCs damage.

CONCLUSIONS. Our findings show that the crosstalk between autophagy and pyroptosis of microglia in COH can be achieved through P2X7R-NLRP3 pathway. Moreover, intervening in the crosstalk effectively counteracts COH-induced RGCs damage. Our results provide new insights into developing therapies for glaucoma based on the concept of neuroinflammation.

Crosstalk

autophagy

pyroptosis

microglia

retinal ganglion cells

glaucoma

Glaucoma is the most common cause of irreversible blindness globally, and its common pathological feature is the damage of retinal ganglion cells (RGCs) [1, 2]. Chronic ocular hypertension (COH) is one of the critical risk factors in the occurrence of this disease [3-5]. Nevertheless, the mechanism of COH-induced RGCs damage has not been clarified. The discovery of increased levels of inflammatory markers in glaucomatous retina and the identification of glaucomatous risk genes associated with innate immune functions suggest that neuroinflammation has a prominent role in the pathogenesis of glaucoma [6, 7]. Several studies demonstrate that glaucomatous damage of RGCs is closely relevant to the changes of retinal glial cells, especially microglia [8, 9].

Microglia is the resident macrophage in retina, regulating neuronal homeostasis by clearing dead neurons and trimming non-functional synapses [10, 11]. To our knowledge, activated microglia play as one of the first events in glaucomatous neural damage occurring before RGCs injury [8, 12]. Furthermore, microglial inflammation is reported to play indispensable roles in optic nerve head and retina of glaucomatous models [7, 13].

Emerging evidence indicate that abnormal autophagy and pyroptosis in microglia broadly contribute to neurodegenerative and neuroinflammatory diseases [14-16]. Autophagy, as a protective factor for inflammatory diseases generally, is essential for the clearance of pathogens [17, 18]. LC3B and P62 as common autophagic markers, both of which are involved in the formation of autophagosomes. Glaucoma exacerbates the effects of oxidative stress in the retina and subsequently causes impaired autophagic function [19-21]. Moreover, pyroptosis, as a kind of inflammation-related programmed cell death, has attracted much attention of ophthalmologic researchers recently. Importantly, gasdermin (GSDM) family members arbitrate the process of pyroptosis, of which GSDMD is a relatively definite pyroptotic executor [22]. Cleavage of caspase-1 (CASP1) transmits inflammatory signals to the resting-state GSDMD, causing its activation and releasing of the GSDMD-N terminal (GSDMD-NT). Additionally, IL-1β has been reported to be a key cytokine in the inflammatory cascade reaction of pyroptosis [23, 24]. Pyroptosis of microglia was detected to rise significantly in the retina of a glaucomatous model, whereas inhibiting the decisive factors of pyroptosis alleviate the glaucomatous RGCs damage [25, 26].In recent studies, it is worth noting that there is an inverse relationship between autophagy and the inflammasomes that induce pyroptosis under some backgrounds of inflammatory diseases [27, 28]. However, this inverse relationship is poorly studied in glaucomatous pathological changes.

A sustained elevation of extracellular adenosine triphosphate (ATP) is observed to occur in the posterior eye of rat, mouse and primate models of COH [29]. Besides, several studies showed that injection of ATP analogues into the vitreous cavity could induce glaucoma-like damage of RGCs by activation of retinal microglia [30]. Additionally, the elimination of ATP effects alleviates the COH-induced RGCs damage [31]. Therefore, we have good reason to utilize ATP-treated microglia to mimic the activated microglia in COH. ATP-based intercellular communication is mediated by P2 purinergic receptors, especially P2X purinergic receptor 7 (P2X7R) [32]. Besides, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a cytosolic immune signaling complex that can induce inflammation and pyroptosis. Inflammasome complex responds to a variety of pathogens, as well as danger or homeostasis-altering signals, exerting crucial roles in the development of autoinflammatory conditions [33, 34]. In our previous studies, we found that P2X7R-NLRP3 pathway was activated in the retina of mouse COH model, and in vitro experiments proved that activation of this pathway in microglia could induce the release of inflammation cytokines and contribute to the death of RGCs [35]. We also found some remarkable phenomena, for example: retinal Müller cells' gliosis, mGluR5 activation, Kir channels suppression, Microglia-derived TNF-α mediated Müller cell activation and so on, which greatly inspired our current research [36-39]. However, the precise molecular mechanism among P2X7R-NLRP3 pathway, pyroptosis and autophagy remains elusive.

In present study, we use the COH model in vivo and ATP-treated microglia in vitro to investigate the relationship among pyroptosis, autophagy and P2X7R-NLRP3 pathway, and speculate what role this relationship plays in the COH-induced RGCs damage.

2.1 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 animals. All mice in our experiments were ordered from Nantong Laboratory Animal Company. Animal Experimental Ethical Inspection (No. 20160302-007) was approved by the Experimental Animals Center of Nantong University. About 200 C57BL/6J (B6) mice were used to establish COH model and extract mouse primary cells.

2.2 Antibodies and reagents

Polystyrene microbeads (cat. no. F8829, Invitrogen, Carlsbad, CA, USA); Compound tropicamide eye drops (CR double-crane Pharmaceutical co, Shenyang, China); Ofloxacin eye ointment (Santen Pharmaceutical Co, Japan); RIPA lysis buffer (Solarbio, Beijing, China); ATP (HY-B2176, Med Chem Express, USA); A438079 (Sigma-Aldrich, USA); Mcc950 (HY-12815A, Med Chem Express, USA); VX765 (S2228, Selleck, USA); Disulfiram (DSF, S1680, Selleck, USA); 3-Methyladenine (3MA, S2767, Selleck, USA); Rapamycin (RAPA, S1039, Selleck, USA); GSDMD small interfere RNA (GSDMD siRNA, siG170424110525, Ribo Life Science, Guangzhou, China); 4% Fluorogold (FG, Biotium, Hayward, CA, USA); goat serum (Solarbio, Beijing, China); DAPI (Beyotime, Shanghai, China)

Table 1

Antibodies

Target	Product information	Diluted concentration
P2X7R	Proteintech; #28207-1-AP	WB: 1:1000; IF: 1:200
NLRP3	Abcam; #ab263899	WB: 1:1000
LC3B	Abcam; #ab48394	WB: 1:1500
CASP1	ABclonal; #A0964	WB: 1:1000
GSDMD	ABclonal; #A20197 Abcam; #219800	WB: 1:800
IL-1β	ABclonal; #A1112	WB: 1:1000
RBPMS	Proteintech; #15187-1-AP	IF: 1:200
Iba1	Wako; #019-19741	IF: 1:500
IL-1β neutralizing antibody	eBioscience; #14-7012-85	Neutralization: 1:1000
P62	Abcam; #ab56416	IF: 1:200
GAPDH	ABclonal; #AC001	WB: 1:6000
Anti-Rabbit HRP	Jacksonimmuno Research; #111-005-003	WB: 1:10000
Anti-mouse HRP	Jacksonimmuno Research; #115-035-003	WB: 1:10000
Alexa Fluor 568 Goat anti-rabbit IgG	Invitrogen; #2155282	IF: 1:200
Alexa Fluor 488 Goat anti-mouse IgG	Invitrogen; #2140660	IF: 1:200

IF: immunofluorescence; WB: Western blot

2.3 COH model and intraocular pressure (IOP) measurement

The construction of mouse COH model was based on previous studies [40, 41]. COH group and sham operation group mice were anesthetized by sodium pentobarbital by abdominal injection at a dose of 60mg/kg. After general anesthesia, ocular surfaces were anesthetized by proparacaine hydrochloride eye drops. Subsequently, a Hamilton syringe was used to connect 30gauge (G) needles, and 2 μL of polystyrene microbeads were injected into the anterior chamber of COH group. Sham operation group only injected 2μL of 0.9% saline into the anterior chamber as a control. After the operation, surgical eyes were smeared with ofloxacin eye ointment to prevent infection. Finally, we placed mice in a warm and humid environment, waiting for them to wake up naturally.

For measure IOP, the mice were anesthetized by inhaling 2-4% isoflurane. A TonoLab tonometer (Colonial Medical Supply, Franconia, NH) was used to measure the IOP of both eyes at 3, 7, 14, 21, 28 days (d) after COH surgery. After the measurement was completed, apply ofloxacin eye ointment to prevent infection. IOP was calculated as the average of 6 measurements.

2.4 Cell culture and treatment

The mouse BV2 microglial cell line was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Rockville, MD, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin in 37 °C incubator at 5% CO₂. Cells were sub-cultured for further passages when they reached 80% confluence. Cells were pretreated with A438079 (P2X7R inhibitor, 50 μM), Mcc950 (NLRP3 inhibitor, 1 μM), 3MA (autophagic inhibitor, 100 μM), RAPA (autophagic agonist, 100 nM), VX765 (CASP1 inhibitor, 10 μM) and disulfiram (DSF, pyroptotic pore formation inhibitor, 10 μM) for 2 h and treated with or without 2 mM ATP for indicated times.

2.5 Primary cell Cultures

The primary microglial culture was prepared basing on previous research [42]. Briefly, retinal tissues were stripped from postnatal day 3 B6 mice (provided by Laboratory Animal Center of Nantong University, Nantong, China). Subsequently, the retinal tissues were digested with trypsin (0.25%, Gibco) for 8 minutes (min) at 37 °C. The cells were centrifugated followed by incubation with 10% FBS in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F12, Gibco) under humidified 5% CO₂ at 37°C. The medium was changed every 3 d, and the flasks were shaken in a shaker at 250 rpm/min for 2 hours (h) after 10 d incubation. Through centrifugation, the primary microglia cells were collected from the supernatant for continued experiments. The purity of retinal microglia was determined by randomly measuring the microglia-specific antibody Iba1 in 10 fields in each culture dish, and the ratio of Iba1 positive cells in each field was calculated using a Thunder Leica microscope [43].

For primary RGCs culture, the protocol was followed as described previously [44, 45]. After retina was digested into cell suspension by 0.25% trypsin, the cell suspension was plated in poly-D-lysine-coated dishes and cultured in a Neurobasal-A medium (Gibco) containing 2 mM glutamine, 25 μM glutamic acid, 1% penicillin/streptomycin, 1% B-27 supplement, 5μg/mL insulin, 40 ng/mL Brain-derived neurotrophic factor (BDNF, Miltenyi Biotec, Bergisch Gladbach, Germany), 40 ng/mL Ciliary neurotrophic factor (CNTF, Miltenyi Biotec) and 5 mM forskolin (Sango Biotech, Shanghai, China). Cells were maintained at 37°C in an incubator at 5% CO₂ for 3 d, and the medium was refreshed every day. RGCs were identified using an antibody against RBPMS, which is a specific marker for RGCs [46]. The identification diagram is shown in the supplemental figure 2c.

2.6 Microglia culture and collection of conditioned medium

Microglia were pretreated with GSDMD siRNA, RAPA and IL-1β neutralizing antibody respectively for 2 h before ATP treatment. Culture medium was collected as microglia condition medium (MCM), centrifuged at 4000 g for 10 min. After filter-sterilized (PES 0.22 μm), 250 μL supernatants were used for RGCs culture in 24-well plates. The same normal medium (NM) composition and conditions were used for the production of control media without cultured microglia.

2.7 LIVE/DEAD viability assay

The survival rate of RGCs was determined by the LIVE/DEAD viability assay kit (Invitrogen, Carlsbad, CA). Briefly, the cells plated on sterile dishes were incubated for 30 min with a mixture of a solution containing 1 mM green-fluorescent calcein-AM and 0.5 μM red-fluorescent ethidium homodimer-1 prepared in PBS. Live and dead cells images were captured by fluorescent microscope (Leica, Germany) and counted from at least 100 cells (from each condition).

2.8 Western blot (WB)

The retina tissues and cells were lysed with RIPA lysis buffer (Solarbio, Beijing, China) supplemented with protease inhibitors (Promega, Madison, WI, USA). Bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China) was utilized to measure protein concentration. Then, equal quality of protein was separated via 8%–12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride (PVDF) membranes (Roche Diagnostics GmbH, Manheim, Germany). The PVDF membranes were blocked and incubated with indicated primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. Finally, ChemiDocTM Imaging System (Bio-rad, California, USA) was used to detect the strips and Image J software (National Institutes of Health, Bethesda, MD, USA) was used for analysis.

All antibody details are listed in Table 1.

2.9 Immunofluorescence (IF)

Thawed tissue cryosections or grown cells were fixed by 4% paraformaldehyde (PFA), After permeabilized and blocked with 1x PBS containing 5% goat serum and 0.5% TritonX-100 for 30 min, tissue sections and cells were incubated with indicated primary antibodies (Table 1) overnight at 4°C. Subsequently, appropriate secondary antibodies were incubated for 1 h. Finally, samples were incubated with 2 µg/mL DAPI in 1x PBS for 2 min, washed and detected by a fluorescent microscope Microphotographs were made by using the THUNDER Imaging Systems (Lecia Microsystems).

2.10 Retrograde labeling and counting of RGCs

The COH group and sham group mice were anesthetized by sodium pentobarbital at a dose of 60mg/kg. After anesthetized, mice were placed in the stereotactic apparatus and the brain surface was exposed by perforating the parietal bone. Next, to label RGCs, 4% fluorogold was injected into both sides of the superior colliculi. After 7 d, the mice were sacrificed and their eyes were fixed with 4% PFA on ice overnight before retinal flat mounting. The whole retinas were then carefully separated, flattened and mounted with the vitreous side up on slides. Photographs were captured using a fluorescent microscope and FG-labeled RGCs were counted by the same investigator using automated particle counting software in Image Pro Version 6.0 (Media Cybernetics, Bethesda, USA). The number of labeled cells in 12 photographs of each retina (three photographs per retinal quadrant) at 1/6, 3/6, and 5/6 of the retinal radius were summed together and expressed as mean RGC densities/mm² for each group.

2.11 Secretion of mature IL-1β

The culture mediums of each group were collected and centrifugated. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentrations of mouse IL-1β in cellular supernatants following the manufacturer instructions (pc-biotech, Shanghai, China). The 96-well plate was examined using a microplate reader (Biotech, New Brunswick, USA) at the absorbance of 450 nm. The results were normalized to cell number of each group.

2.12 Treatment regimen

Intraocular injections were performed according to previous research [47]. After anterior chamber injection, the vitreous cavities of experimental eyes were injected with negative control (NC), GSDMD siRNA and RAPA. Mice's surgical eyes were smeared with ofloxacin eye ointment to prevent infection. Finally, put mice in a warm and humid environment, waiting for them to wake up naturally. The mice were sacrificed 7 d after intravitreal injection.

2.13 Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

For TEM, BV2 microglial cells were fixed with 4% glutaraldehyde in phosphate buffer at 4℃ for 12 h. After the glutaraldehyde was removed, the cells were embedded in 1% agarose. Before samples were embedded in epoxy resin, they were sequentially dehydrated in 50%, 70%, 90% and 100% acetone. Sections (70 nm thick) were cut and stained with uranylacetate for 15 min.

For SEM, BV2 microglial cells were inoculated on a crawler and were fixed the same as TEM. After the cells were dehydrated through critical point drying, they were coated with gold-palladium. Finally, images of TEM and SEM were acquired with a transmission electron microscope (HITACHI, Tokyo, Japan).

2.14 Statistical analysis

All data were presented as mean ± SD and were analyzed using GraphPad Prism 8 Software. Statistical evaluation was performed via two-way analysis of variance (ANOVA). p< 0.05 was considered statistically significant. All experiments were repeated at least three times.

3.1 COH induce altered autophagy and pyroptosis in retinal microglia in vivo

COH model was successfully established, and the IOP was significantly higher than that of the sham-operated model (Figure 1A). We first extracted the whole retina tissue proteins at 3, 7 and 14 d after COH surgery to explore the changes of P2X7R-NLRP3, autophagy and pyroptosis in the COH retina. We observed that the protein levels of P2X7R and IL-1β increased continuously; NLRP3, CASP1 and GSDMD-NT increased gradually. Notably, the autophagic marker LC3B increased at 3 d after COH surgery but then decreased (Figure 2B-H). We also labeled the retinal sections that existed the highest autophagic (3 d) and pyroptotic (7 d) levels by histochemical immunofluorescence. We found that P62 and GSDMD were co-located with Iba1, a specific microglial marker (Supplemental figure 1A-B). These data indicate that COH can induce a gradual increase of pyroptosis and a short-term boost of autophagic flux in retinal microglia.

3.2. ATP-treated induce altered autophagy and pyroptosis in cultured microglia in vitro

Next, we established an in vitro model using mouse microglia subjected to 2 mM ATP, which activates P2X7R, to mimic COH. The concentration of ATP refers to previous literatures[31, 41, 48, 49]. However, due to the limited number of primary mouse retinal microglia, which was not enough to provide cells required for the entire experiments, we only used primary microglia to verify that P2X7R activation could induce autophagy and pyroptosis by western blot, as shown in supplemental figure 1C. We subsequently used mouse BV2 microglial cell line to further study the possible functions of ATP-treated microglia. The identification of mouse BV2 microglial cell line and primary microglia are shown (supplemental figure 2A-B). Western blot bands showed that ATP treated for 0.25, 2, 16 h kept NLRP3, CASP1, IL-1β and GSDMD-NT expression increased gradually, while LC3B boosted at 0.25 h, and then decreased (Figure 2A-E). The changes are similar to those in COH. In addition, representative SEM images (Figure 2F) showed the cells swelling gradually over time. After 16 h of ATP treatment, microglia showed a typical pyroptotic morphology, that is, membrane pores formation, cell swelling and rupturing. Representative TEM images (Figure 2G) showed that normal microglia are intact generally, with abundant mitochondria and few autophagosomes. Numerous of autophagosomes could be observed in the cytoplasm at 0.25 h of ATP treatment. Subsequently, 2 h ATP caused a large number of mitochondria swelling and autophagosomes reducing. Finally, lysosomes wrapped in various cellular debris and severely disrupted cellular structures could be observed after 16 h. In short, we described the dynamic changes in autophagy and pyroptosis of microglia from both protein expression and morphology by SEM and TEM. We found the sustaining increase of pyroptosis in retina and a short-term boost of autophagic flux were similar to the changes of COH retinas.

3.3. the crosstalk between autophagy and pyroptosis of microglia can be achieved through P2X7R-NLRP3 pathway

To further study the relationship between autophagy and pyroptosis caused by ATP treatment, we pretreated microglia with A438079, Mcc950, 3MA, RAPA, VX765 and DSF for 2 h before adding 2 mM ATP. As shown in Figure 5, NLRP3 inflammasomes were stimulated by ATP and could be suppressed by A438079, RAPA, Mcc950 and disulfiram. Meanwhile, ATP treatment significantly suppressed the expression of LC3B, while its expression was restored after the addition of A438079, Mcc950 and RAPA. Moreover, the expressions of CASP1 and GSDMD were intensified activated by 3MA, but the other reagents alleviated their activation. Furthermore, SEM images showed that ATP-induced microglial pyroptotic morphology could be alleviated by A438079, Mcc950, RAPA, VX765 and DSF, but not 3MA (Figure 6). The results indicated that the most likely pathway for ATP induced pyroptosis was ATP-P2X7R-NLRP3-CASP1-GSDMD, while autophagy might play an anti-pyroptotic effect by eliminating the accumulation of NLRP3 inflammasome early on the ATP stimulation. However, over time passed, autophagy is destroyed by the gradually accumulated NLRP3 inflammasomes.

3.4. Intervention in autophagy, pyroptosis and IL-1β of microglia alleviated the RGCs damage in vitro

We next verify the mechanism of the above crosstalk on the RGCs damage. In our previous experiments, the survival of RGCs co-cultured with microglia was protected by antagonism of P2X7R-NLRP3 pathway. Therefore, we can easily speculate based on the crosstalk that upregulating autophagy and downregulating pyroptosis can protect RGCs from injury. Thus, we performed ELISA, co-cultivation, antibody neutralization and live/dead cell imaging methods to verify our speculation. Moreover, we also pondered what are the mediators of microglia pyroptosis leading to RGCs injury. It has been reported that IL-1β is one of the major facilitators to the pyroptotic cascade response[50]. Thus, we examined the IL-1β expressions from microglial supernatants after ATP addition in combination with other treatments by ELISA (Figure 4A-B), and found that the changes of IL-1β were consistent with CASP1 and GSDMD. This observation suggests that IL-1β releasing is positively correlated with pyroptosis. Moreover, compared to the NM group, MCM group accelerated the RGCs damage indicating the adverse effects of microglia on RGCs. The observations that GSDMD siRNA (supplemental figure 1D) and RAPA attenuated RGCs damage support our speculation (Figure 4C-D). Furthermore, using IL-1β neutralizing antibody could also alleviate microglia-induced RGCs damage (Figure 4C-D) demonstrating its indispensable role in microglial pyropstosis contributed RGCs injury.

3.5. Vitreous injection of RAPA and knockout of GSDMD genes alleviate COH-induced RGCs damage

It has been shown that glaucoma-induced RGCs damage was ameliorated in P2X7 and NLRP3 knockout mice[30, 51]. Similarly, we suspect that injecting RAPA and GSDMD siRNA into vitreous body in vivo could prevent RGCs damage caused by COH. As expected, results showed an increased survival rate of RGCs in mice injected with 10 μM RAPA and GSDMD siRNA compared to the sham operation mice (Figure 5). To sum up, the results indicated that inhibiting the pyroptotic actuator GSDMD, or enhancing autophagic flux might have protective effects on RGCs under COH. Moreover, the crosstalk between autophagy and pyroptosis activated by P2X7R contributes to RGCs death in COH as shown in Figure 6.

COH is sometimes painless and easily overlooked, resulting in an army of patients heading for the ophthalmologic clinic until their vision is affected at the terminal of the disease. Studies have shown that progression of glaucoma is usually suspended when IOP is reduced by 30%-50% from baseline [48, 49]. The prevailing clinical treatments for lowering IOP include various eye drops and anti-glaucoma surgeries, yet some patients still have uncontrolled IOP, which subsequently leads to more severe and irreversible visual impairments. Thus, a clear understanding of the mechanisms underlying COH induced RGCs damage is required in order to develop novel therapeutic alternatives. As two major studied mechanisms of cell death, autophagy and pyroptosis of microglia were found to be involved in the glaucomatous retina [43]. Simultaneously, their crosstalk has gradually become a hot spot [52, 53]. However, the effect of autophagy in COH remains controversial, and little is known about the crosstalk between autophagy and pyroptosis. In current study, we established a mouse model of COH to explore the crosstalk between autophagy and pyroptosis in microglia.

Microglia is known as a primary guardian of the immune defenses in central nervous system, broadly contributing to neurodevelopment, degeneration and inflammation[12, 54]. Indeed, the sensing and housekeeping functions of microglia are required for neuronal homeostasis [55]. Current therapeutic strategies for seizure targeted to microglia, proposing the neuro-macrophages interactions during epileptic phenotypes [56, 57]. P2X7R, mainly expressed in macrophages, can be turned on by binding to extracellular ATP, subsequently mediating inflammasome activation, cytokine releasing and cell death [58, 59]. Studies have shown that ATP activates microglia through P2X7R, while targeted inhibition of P2X7R could effectively reverse microglial activation [60, 61]. Additionally, BzATP is reported to be agonists of P2X1, P2X2, P2X3 and P2X7 receptors. Some experiments used BzATP instead of ATP to activate P2X7R because its activity is substantially greater than ATP[59, 62]. When the central nervous system is damaged, the damaged neurons could release ATP to act on microglia, causing microglia to migrate to the damaged areas. Thus, ATP was used to stimulate microglia in this study, and a stable increase of P2X7R expression was found within 16 h, achieving the same effect as 100μM BzATP of our previous study [35].

Researchers showed that there is microglial activation and redistribution in the glaucomatous retina, as well as microglia proliferate near RGCs [63, 64]. Recruitment and activation of microglia were observed before RGCs damage, indicating that microglial activation is associated with glaucomatous RGCs damage [64]. Autophagy is a high sensitivity dynamic process that is triggered in response to many stresses for maintaining homeostasis [65, 66]. Inhibition of microglial activation by RAPA significantly reduces RGCs loss in rat COH model [67]. Here we show that autophagic marker LC3B boosted on 3 d after COH surgery, but dampened subsequently. Additionally, we also observed that microglia co-located with another autophagic marker, P62. Moreover, our findings showed that activation of autophagy significantly reduced RGCs death in vitro and in vivo.

Exposing to elevated IOP represents a stressful environment, which activating microglia, increasing IL-1β expression and contributing to RGCs injury [68, 69]. Pyroptosis is a kind of inflammatory-related cell death, showing disadvantages broadly in body health. As reported, NLRP3, CASP1 and IL-1β expression elevated and RGCs damage were found in a high IOP mouse model [70]. Moreover, inhibition of NLRP3 reversed the upregulation of CASP1, microglial activation and RGCs damage in an acute glaucomatous rat model [71]. Therefore, pyroptotic pathway in which NLRP3, CASP1 and inflammatory responses are crucial in microglia-mediated RGCs injury. P2X7R-NLRP3-CASP1 pathway is indispensable in aseptic inflammation, inhibiting this pathway above could suppress microglial pyroptosis [72]. Our findings showed that expression of NLRP3, CASP1 and GSDMD were gradually increased in both COH model and ATP-induced microglia. Intervention in GSDMD expression significantly improved the RGCs livability. Furthermore, we also found a significant reduction in microglia-induced RGCs damage when neutrlizing IL-1β in extracellular fluid.

As reported, the knockout of autophagic genes 16 (Atg16) leads to overactivation of pyroptotic pathway and inflammatory cytokines [73]. Besides, Atg7 knockout mice showed increased activity of inflammasomes in macrophages, accompanied by elevated levels of IL-1β in blood. Moreover, reactive oxygen species (ROS) increased autophagic flux through NLRP3-PYCARD pathway to establish a negative regulation of pyroptosis. Therefore, the crosstalk between pyoptosis and autophagy is crucial to the pathology of inflammatory diseases [52]. Herein, our results showed that autophagy was activated in the early stage of COH, which in turn suppressing NLRP3. While in the later stage, NLRP3 inflammasomes accumulation destructed autophagic function resulting in severe inflammatory cell death. Our observations demonstrated that the crosstalk between autophagy and pyroptosis could be linked by P2X7R-NLRP3, indicating that microglia are sensitive and manageable to glaucomatous inflammation.

Our observations show that the crosstalk between autophagy and pyroptosis of microglia in COH is linked by P2X7R-NLRP3 pathway. Furthermore, intervention in the crosstalk above effectively counteracts the COH-induced RGCs damage. Our results provide new insights into developing therapies for glaucoma based on neuroinflammation.

ATP: Adenosine Triphosphate;

CASP1: Casepase-1;

COH: chronic ocular hypertension;

DSF: disulfiram;

GSDMD: Gasdermin D;

IF: immunofluorescence;

IL-1β: interleukin-1β;

IOP: Intraocular pressure;

MCM: microglial condition medium;

NM: normal medium;

NLRP3: NOD-like receptor family pyrin domain containing 3;

P2X7R: P2X7 receptor;

RAPA: Rapamycin;

RGCs: Retinal ganglion cells;

SEM: scanning electron microscopy;

TEM: Transmission electron microscopy;

WB: Western blot;

3MA: 3-Methyladenine

Acknowledgments

We thank Prof. Zhongfeng Wang of Department of Neurology, Institutes of Brain Science, Zhongshan Hospital, Fudan University, Shanghai, China for the critical comments. And we would thank Dr. Jianfeng Yu and Doc. Bai Qin of Eye Institute, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China for their theoretical and technical guidance.

Authors contributions

Conception and design: MJ and ZYH; performed the research and data collection: ZYH, HYZ, MW and JJX; data analysis and interpretation: ZYH, GWZ, LMC and MJ; drafted the manuscript: ZYH, GWZ, HJG and MJ. All authors read and approved the final manuscript.

Funding

This work was supported by Natural Science Foundation of China (No.81670852) and

Availability of data and materials

The data supporting the findings of this study are available within this article and its additional information files. All other relevant data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Declarations

Ethics approval and consent to participate

All experimental procedures described in this article were approved by the animal care committee of Nantong University.

Consent for publication

All authors have approved the contents of this manuscript and approved consent for publication.

Competing interests

The authors declare no competing interests.

Author details

¹ Eye Institute, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, Jiangsu, 226000, China

² Neurology department, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, Jiangsu, 226000, China

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Crosstalk Between Autophagy and Pyroptosis of Microglia contributes to Retinal Ganglion Cells Damage in Chronic Ocular Hypertension

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Abstract

Figures

1. Background

2. Materials And Methods

2.1 Animals

2.2 Antibodies and reagents

2.3 COH model and intraocular pressure (IOP) measurement

2.4 Cell culture and treatment

2.5 Primary cell Cultures

2.6 Microglia culture and collection of conditioned medium

2.7 LIVE/DEAD viability assay

2.8 Western blot (WB)

2.9 Immunofluorescence (IF)

2.10 Retrograde labeling and counting of RGCs

2.11 Secretion of mature IL-1β

2.12 Treatment regimen

2.13 Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

2.14 Statistical analysis

3. Results

3.1 COH induce altered autophagy and pyroptosis in retinal microglia in vivo

3.2. ATP-treated induce altered autophagy and pyroptosis in cultured microglia in vitro

3.3. the crosstalk between autophagy and pyroptosis of microglia can be achieved through P2X7R-NLRP3 pathway

3.4. Intervention in autophagy, pyroptosis and IL-1β of microglia alleviated the RGCs damage in vitro

3.5. Vitreous injection of RAPA and knockout of GSDMD genes alleviate COH-induced RGCs damage

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

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Supplementary Files

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