Neuron–Microglia Contacts Govern the PGE2 Tolerance through TLR4-Mediated de Novo Protein Synthesis

Cellular and molecular mechanisms of the peripheral immune system (e.g., macrophage and monocyte) in programming endotoxin tolerance (ET) have been well studied. However, regulatory mechanism in development of brain immune tolerance remains unclear. The inducible COX-2/PGE2 axis in microglia, the primary innate immune cells of the brain, is a pivotal feature in causing inflammation and neuronal injury, both in acute excitotoxic insults and chronic neurodegenerative diseases. This present study investigated the regulatory mechanism of PGE2 tolerance in microglia. Multiple reconstituted primary brain cells cultures, including neuron–glial (NG), mixed glial (MG), neuron-enriched, and microglia-enriched cultures, were performed and consequently applied to a treatment regimen for ET induction. Our results revealed that the levels of COX-2 mRNA and supernatant PGE2 in NG cultures, but not in microglia-enriched and MG cultures, were drastically reduced in response to the ET challenge, suggesting that the presence of neurons, rather than astroglia, is required for PGE2 tolerance in microglia. Furthermore, our data showed that neural contact, instead of its soluble factors, is sufficient for developing microglial PGE2 tolerance. Simultaneously, this finding determined how neurons regulated microglial PGE2 tolerance. Moreover, by inhibiting TLR4 activation and de novo protein synthesis by LPS-binding protein (LBP) manipulation and cycloheximide, our data showed that the TLR4 signal and de novo protein synthesis are necessary for microglia to develop PGE2 tolerance in NG cells under the ET challenge. Altogether, our findings demonstrated that neuron–microglia contacts are indispensable in emerging PGE2 tolerance through the regulation of TLR4-mediated de novo protein synthesis.


Introduction
Microglia, the primary innate immune cells of the brain, maintain the central nervous system (CNS) homeostasis at physiological conditions [1,2]. With their high mobility, The preparation of mesencephalic neuron-glia cultures was performed from the mesencephalon of embryos at gestation day 14 ± 0.5 of the C57/6J mice (n = 18), as previously reported [10,11]. Briefly, after dissection and dissociation of mesencephalic tissues with mild mechanical trituration, cells were seeded to 24-well (5 × 10 5 cells/well) culture plates precoated with poly-D-lysine (20 µg/mL) and maintained in 0.5 mL/well of MEM medium (10% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum (HS), 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids). Cultures were preserved at 37 • C in a humidified atmosphere of 5% CO 2 /95% air. Three days later, 0.5 mL/well of fresh medium was replenished into the cultures. Seven days after seeding, the neuron-glia cultures made up of about 10% microglia, 50% astrocytes, and 40% neurons based on the visual counting of immunostained cells with antibodies against cell-type specific markers: neurons (Neu-N), microglia (OX-42), and astrocytes (GFAP) [28]. The NG cultures were ready for further endotoxin tolerance treatment regimen ( Figure 1A). The neuron-enriched culture contained 99% neurons and less than 1% glia. The dividing glia was depleted from neuron-glia cultures 48 h after seeding with 8-10 µM of cytosine β-d-arabinofuranoside (Ara-C; Sigma-Aldrich, St. Louis, MO, USA) for three days.
Primary mixed glia cultures were prepared from whole brains of postnatal day-1 pups (n = 10) from the C57BL/6J mice [10,11]. After brain tissue disassociation, the cells were seeded onto 6-well (1 × 10 6 cells/well) culture plates and maintained in 1 mL/well of DMEM/F-12 medium (10% FBS, 2 mM of L-glutamine, 1 mM of sodium pyruvate, and 0.1 mM of nonessential amino acids). Before reaching confluence, the medium was changed every 3 days. The mixed glia cultures contained about 80% astrocytes and 20% microglia and were used for endotoxin tolerance treatment regimen.
Microglia-enriched cultures were prepared from the whole brains of 1-day-old C57/6J pups (n = 45), as previously reported [10,11]. Briefly, after the dissociation of brain tissues, devoid of meninges and blood vessels by mild mechanical trituration, the isolated cells (5 × 10 7 cells) were seeded in 150 cm 2 culture flasks in DMEM/F12 medium (10% FBS, 2 mM of L-glutamine, 1 mM of sodium pyruvate, 0.1 mM of nonessential amino acids, 50 U/mL of penicillin, and 50 µg/mL of streptomycin) and maintained at 37 • C in a humidified atmosphere of 5% CO 2 /95% air. Before reaching confluence, the medium was changed 4 days later. Upon reaching confluence (12-14 days), the enriched microglia (99% pure) were obtained by shaking the flasks for 60 min at 180 rpm.

Cell Treatment
Multiple reconstituted brain cultures, including neuron-glial (NG), mixed glial (MG), microglia-enriched, fixed neurons plus microglia, and neurons plus microglia in Transwell inserts, were pre-incubated with or without LPS (15 ng/mL) for 6 h. After replacing the fresh media and waiting for an additional 6 h, LPS was readded into these cells ( Figure 1A). Thus, endotoxin tolerance (ET) treatment regimen included untreated control, LPS (LPS alone treatment), LPS/LPS (twice LPS treatment), and LPS-untreated control groups. The expressions of COX-2 or PGE 2 were measured at 3, 6, and 24 h in these cells by RT-PCR and ELISA, respectively. Furthermore, serum-free medium (no LPS binding protein (LBP)) and the addition of LBP (1 µg/mL) were used to study TLR4 s role in the development of microglial PGE 2 tolerance in NG cells. Moreover, treated NG cells with cycloheximide, an inhibitor for protein synthesis, was performed to determine involvement of de novo protein synthesis in PGE 2 tolerance of microglia. cytes (GFAP) [28]. The NG cultures were ready for further endotoxin tolerance trea regimen ( Figure 1A). The neuron-enriched culture contained 99% neurons and les 1% glia. The dividing glia was depleted from neuron-glia cultures 48 h after seedin 8-10 μM of cytosine β-d-arabinofuranoside (Ara-C; Sigma-Aldrich, St. Louis, MO for three days.  Neuron-glial (NG) cultures prepared from E14.5 time-pregnant C56/6J mice were pre-treated with vehicle (LPS-treated group) or LPS (15 ng/mL) (LPS/LPS-treated group) for 6 h. These pre-treated NG cultures were replaced with fresh media. Six hours later, LPS (15 ng/mL) was added to these NG cells. The level of COX-2 gene expression and supernatant PGE 2 production was measured at 3, 6, and 24 h after LPS treatment. (B) After 3 h, the mRNA level of the COX-2 gene was measured in these NG cultures with untreated control, LPS-treated group (LPS), LPS/LPS-treated group (LPS/LPS), and LPS-untreated control group by RT-PCR. Three independent experiments were performed in duplicate. Data are expressed as a percentage of the LPS group (mean ± SEM). ** p < 0.01 vs. untreated control; # p < 0.05 vs. LPS. (C) A supernatant level of PGE 2 in these cells with LPS-treated (LPS) and LPS/LPS-treated group (LPS/LPS) was detected at 6 and 24 h after LPS treatment by ELISA. Data are expressed as the mean ± SEM from three independent experiments in duplicate, ** p < 0.01 vs. 6H, ## p < 0.01 vs. LPS.

Quantitative Real Time-PCR
According to the manufacturer's instruction, the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) and the MuLV reverse transcriptase (Applied Biosystems, Foster City, CA, USA) were used to isolate the total cellular RNA of cells and synthesize the first-strand cDNA. After reverse transcription reaction, the SYBR-Green Master Mix (Applied Biosystems, Foster City, CA, USA) was used to perform real-time quantitative PCR analysis with the following PCR conditions: hold at 95 • C for 10 min and start 40 cycles at 95 • C for 15 s and 60 • C for 1 min. Data were normalized to a GAPDH expression. Vector NTI Advance 11.5 software (Invitrogen, Waltham, MA, USA) was used to design the primers. The sequences of the primers were the following: mouse COX-2 forward primer 5 -TGA-TAT-GTC-TTC-CAG-CCC-ATT G-3 ; mouse COX-2 reverse primer 5 -AAC-GGA-ACT-AAG-AGG-AGC-AGC-3 ; mouse GAPDH forward primer 5 -TTC-AAC-GGC-ACA-GTC-AAG-GC-3 ; mouse GAPDH reverse primer 5 -GAC-TCC-ACG-ACA-TAC-TCA-GCA-CC-3 .

Measurement of PGE 2
PGE 2 in the culture medium was measured with the commercial ELISA kits from R&D Systems (Minneapolis, MN, USA).

Statistical Analysis
All data are expressed as the mean ± standard error of mean (SEM) and were compared between groups using the Student's t test, as well as one-way or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparisons test (Prism 7; GraphPad Software, San Diego, CA, USA). A p value of <0.05 was considered statistically significant. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results
To determine whether endotoxin tolerance (ET) of a microglial COX-2-PGE 2 axis occurred, the ET treatment regimen (as described in Section 2.4; Figure 1A) was performed in primary neuron-glial (NG) cultures, containing 40% neurons, 50% astroglia, and 10% microglia. The expressions of COX-2 mRNA and supernatant PGE 2 were measured at 3, 6, and 24 h in the NG cells by RT-PCR and ELISA, respectively. RT-PCR data showed that the LPS treatment induced mRNA levels of the COX-2 gene in the NG cells (1 vs. 9.41 ± 1.25, p < 0.01, one-way ANOVA, Figure 1B). Conversely, the NG cells received with 6 h LPS pre-incubation had decreased the expression of the subsequent endotoxin-induced COX-2 mRNA by 50% (9.41 ± 1.24 vs. 4.55 ± 0.81, p < 0.05, one-way ANOVA, Figure 1B). Our data indicated that the refectory to up-regulation of COX-2 mRNA occurred in the ET-treated NG cells ( Figure 1B). The NG cells with a treatment regimen of saline (untreated control) or the LPS plus untreated control had no effect on the COX-2 induction ( Figure 1B). Furthermore, ELISA data revealed that the production of PGE2 was induced in the supernatant of the LPS-treated NG cells at 24 h (225 ± 16.86 ng/mL vs. 883.67 ± 58.03 ng/mL, p < 0.01, two-way ANOVA, Figure 1C). Similar to the expression profile of the COX-2 mRNA, the NG cells with LPS pre-incubation had lower PGE 2 production following subsequent LPS treatment (LPS/LPS) at 24 h in comparison with the NG cells with LPS alone treatment (LPS) (883.67 ± 58.03 ng/mL vs. 294 ± 19.15 ng/mL, p < 0.01, two-way ANOVA, Figure 1C). Accordingly, our data indicated that microglia were capable of programing COX-2-PGE 2 axis tolerance in NG cells.
Under the LPS challenge, microglia are the main resource of the brain in producing PGE 2 . Then, we determined if the development of PGE 2 reduction also occurred in microglia during the ET challenge. Microglia-enriched cultures were prepared and subjected to the same ET treatment regimen, shown in Figure 1A. Our data showed that the production of PGE 2 in LPS pre-treated microglia (LPS/LPS group) was significantly increased in comparison with the microglia without LPS pre-treatment (LPS group) at 6 h of endotoxin treatment (116.6 ± 46.98 ng/mL vs. 2674.6 ± 680.35 ng/mL, p < 0.01, two-way ANOVA, Figure 2A). Meanwhile, similar to the microglia with once LPS treatment, the microglia with LPS pre-treatment produced a certain amount of PGE 2 production at 24 h of endotoxin treatment (2701.2 ± 364.94 ng/mL vs. 2540.2 ± 386.34 ng/mL, Figure 2A). These results suggested that microglia alone failed to develop PGE 2 tolerance during the ET challenge. Furthermore, to determine whether astroglia played a role in PGE 2 reduction in tolerant microglia, the mixed glial cultures containing microglia and astroglia were prepared and applied to the same ET experimental procedure ( Figure 1A). Our data revealed that compared to the cells with LPS treatment (LPS group), the pre-treatment of mixed glial (MG) cells with LPS (LPS/LPS group) increased the production of PGE 2 at 6 h (374 ± 78.9 ng/mL vs. 3365.6 ± 495.66 ng/mL, p < 0.05, two-way ANOVA, Figure 2B) and failed to show PGE 2 reduction at 24 h after endotoxin treatment (2910.4 ± 624.88 ng/mL vs. 2942.2 ± 1008.49 ng/mL, Figure 2B). The expression profile of PGE 2 in microglia-enriched cultures and MG cells during endotoxin tolerance were similar ( Figure 2). In other words, the presence of astroglia was unable to program PGE 2 reduction in tolerant microglia. ng/mL, Figure 2B). The expression profile of PGE2 in microglia-enriched cultures an cells during endotoxin tolerance were similar ( Figure 2). In other words, the prese astroglia was unable to program PGE2 reduction in tolerant microglia. According to Figures 1 and 2, while PGE2 tolerance occurred in NG cultures not occur in microglia-enriched and MG cultures, implying that the presence of ne may participate in PGE2 reduction in tolerant microglia. We further determined w soluble factors were secreted by neuron-regulated, tolerant microglia for PGE2 redu Thus, the condition media from neuron-glial cells (NGCM) were collected and adde the mixed glial cultures ( Figure 3A). After 24 h of incubation, these MG cells were a to the same ET treatment regimen ( Figure 1A). Our data revealed that the incuba MG cells with NGCM failed to restore the tolerant capacity of microglia in PGE2 red (1822 ± 388.5 ng/mL vs. 1984 ± 268 ng/mL, p = 0.74, Student's t-test, Figure 3A). A tively, by using the Transwell culture system, the microglia in the upper inserts h direct cell-cell contacts with neurons grown in the lower compartment of the cultur ( Figure 3B, upper panel). However, soluble factors were permeable between the upper and lower compartments ( Figure 3B, upper panel). Further, our data showe the production of PGE2 in these microglia with either once LPS (LPS group) or twi treatment (LPS/LPS group) were comparable (1010 ± 75.35 ng/mL vs. 1006 ± 112 ng = 0.97, Student's t-test, Figure 3B, bottom panel), suggesting that neural soluble were not sufficient for PGE2 reduction in tolerant microglia. Subsequently, we exa whether physical contact with neurons was involved in PGE2 reduction in tolerant glia. Neuron-enriched cultures were fixed with 4% formaldehyde solution and w out with PBS three times. Although the fixed, dead neurons were unable to produ soluble factors, they still presented antigen on their cell surface. Microglia were into the fixed neurons for 24 h of incubation ( Figure 3C, upper panel) and applied same ET treatment regimen ( Figure 1A). Our data showed that PGE2 reduction oc in microglia with fixed neurons in response to the ET treatment (4450.66 ± 297.37 vs. 2125.33 ± 375.36 ng/mL, p < 0.01, two-way ANOVA, Figure 3C, bottom panel). neurons had no effect on PGE2 production ( Figure 3C, bottom panel). In other wor loss of PGE2 tolerance in microglia alone was recovered when it contacted with ne According to Figures 1 and 2, while PGE 2 tolerance occurred in NG cultures, it did not occur in microglia-enriched and MG cultures, implying that the presence of neurons may participate in PGE 2 reduction in tolerant microglia. We further determined whether soluble factors were secreted by neuron-regulated, tolerant microglia for PGE 2 reduction. Thus, the condition media from neuron-glial cells (NGCM) were collected and added into the mixed glial cultures ( Figure 3A). After 24 h of incubation, these MG cells were applied to the same ET treatment regimen ( Figure 1A). Our data revealed that the incubation of MG cells with NGCM failed to restore the tolerant capacity of microglia in PGE 2 reduction (1822 ± 388.5 ng/mL vs. 1984 ± 268 ng/mL, p = 0.74, Student's t-test, Figure 3A). Alternatively, by using the Transwell culture system, the microglia in the upper inserts had no direct cell-cell contacts with neurons grown in the lower compartment of the culture plate ( Figure 3B, upper panel). However, soluble factors were permeable between the plate's upper and lower compartments ( Figure 3B, upper panel). Further, our data showed that the production of PGE 2 in these microglia with either once LPS (LPS group) or twice LPS treatment (LPS/LPS group) were comparable (1010 ± 75.35 ng/mL vs. 1006 ± 112 ng/mL, p = 0.97, Student's t-test, Figure 3B, bottom panel), suggesting that neural soluble factors were not sufficient for PGE 2 reduction in tolerant microglia. Subsequently, we examined whether physical contact with neurons was involved in PGE 2 reduction in tolerant microglia. Neuron-enriched cultures were fixed with 4% formaldehyde solution and washed out with PBS three times. Although the fixed, dead neurons were unable to produce any soluble factors, they still presented antigen on their cell surface. Microglia were added into the fixed neurons for 24 h of incubation ( Figure 3C, upper panel) and applied to the same ET treatment regimen ( Figure 1A). Our data showed that PGE 2 reduction occurred in microglia with fixed neurons in response to the ET treatment (4450.66 ± 297.37 ng/mL vs. 2125.33 ± 375.36 ng/mL, p < 0.01, two-way ANOVA, Figure 3C, bottom panel). Fixed neurons had no effect on PGE 2 production ( Figure 3C, bottom panel). In other words, the loss of PGE 2 tolerance in microglia alone was recovered when it contacted with neurons (Figures 2A and 3C). Moreover, our data indicated that the neuron-microglia contacts were critically involved in the development of the microglial ET capacity on PGE 2 reduction.  Previous studies demonstrate that the activation of toll-like receptor 4 (TLR4) by LPS is critical for downstream inflammatory [29,30], anti-inflammatory [10], and tolerance responses [31]. Thus, we determined whether the TLR4-derived signal participated in the modulation of microglial PGE2 tolerance by neurons. Due to LPS-contained hydrophobic multi-acyl chains forming aggregates or micelles in aqueous solutions, the accessory LPSbinding proteins (LBPs) are required to mediate the sensitive recognition of LPS as well as their efficient transfer to the TLR4 [32,33]. After binding to LPS, the TLR4 signaling cascades are activated in the host immune response [30]. Therefore, by using serum-free medium (no LBP) with or without addition of recombinant LBP protein to incubate NG cells, the role of TLR4 signal in PGE tolerance was studied ( Figure 4A, left panel). Our data revealed that during the ET treatment, PGE2 reduction occurred in NG cells at 24 h in the presence of serum medium-contained LBP (100 ± 3.11 vs. 23.79 ± 1.35, p < 0.01, two- Previous studies demonstrate that the activation of toll-like receptor 4 (TLR4) by LPS is critical for downstream inflammatory [29,30], anti-inflammatory [10], and tolerance responses [31]. Thus, we determined whether the TLR4-derived signal participated in the modulation of microglial PGE 2 tolerance by neurons. Due to LPS-contained hydrophobic multi-acyl chains forming aggregates or micelles in aqueous solutions, the accessory LPSbinding proteins (LBPs) are required to mediate the sensitive recognition of LPS as well as their efficient transfer to the TLR4 [32,33]. After binding to LPS, the TLR4 signaling cascades are activated in the host immune response [30]. Therefore, by using serum-free medium (no LBP) with or without addition of recombinant LBP protein to incubate NG cells, the role of TLR4 signal in PGE tolerance was studied ( Figure 4A, left panel). Our data revealed that during the ET treatment, PGE 2 reduction occurred in NG cells at 24 h in the presence of serum medium-contained LBP (100 ± 3.11 vs. 23.79 ± 1.35, p < 0.01, two-way ANOVA, Figure 4A, right panel). Conversely, in serum-free media (no LBP), PGE 2 tolerance disappeared (even higher PGE 2 production) in NG cells at 24 h during the ET challenge (100 ± 17.43 ng/mL vs. 300.53 ± 8.15 ng/mL, p < 0.01, two-way ANOVA, Figure 4A, right panel). Furthermore, a recombinant LBP protein was added into serum-free media of NG cultures to confirm whether the TLR4 signal activation was crucial for the development of PGE 2 tolerance. Our results revealed that adding recombinant LBP protein at 1 µg/mL concentration entirely reversed the failure of PGE 2 tolerance in NG cultures at a serum-free condition (100 ± 2.4 ng/mL vs. 44.02 ± 2.75 ng/mL, p < 0.01, two-way ANOVA, Figure 4A, right panel). In addition, our data indicated that the TLR4-derived signal is necessary for PGE 2 tolerance in microglia. Moreover, to determine whether inducing de novo protein synthesis by TLR4 activation was required for programming PGE 2 tolerance, NG cells were treated with a protein synthesis inhibitor cycloheximide at 1 µg/mL concentration and subsequently applied to the same ET treatment regimen ( Figure 4B, left panel). Our data showed that the inhibition of protein synthesis by cycloheximide disrupted the PGE 2 tolerance in NG cells during ET (1882.66 ± 67.62 ng/mL vs. 2394.33 ± 252.02 ng/mL, Figure 4B, right panel). Moreover, our results suggested that the TLR4-dependent de novo protein synthesis participated in neuron-mediated PGE 2 tolerance in microglia.  Figure 4A, right panel). In addition, our data indicated that the TLR4-derived signal is necessary for PGE2 tolerance in microglia. Moreover, to determine whether in ducing de novo protein synthesis by TLR4 activation was required for programming PGE tolerance, NG cells were treated with a protein synthesis inhibitor cycloheximide at 1 μg/mL concentration and subsequently applied to the same ET treatment regimen ( Figure  4B, left panel). Our data showed that the inhibition of protein synthesis by cycloheximide disrupted the PGE2 tolerance in NG cells during ET (1882.66 ± 67.62 ng/mL vs. 2394.33 ± 252.02 ng/mL, Figure 4B, right panel). Moreover, our results suggested that the TLR4-de pendent de novo protein synthesis participated in neuron-mediated PGE2 tolerance in mi croglia.

Discussion
As the first responder to the immune challenge, microglia secrete a wide spectrum and various inflammatory factors at inflammatory conditions, including IL-1β, TNF-α Experimental procedure for studying the role of de novo protein synthesis in PGE 2 reduction in response to endotoxin tolerance. Right panel: After treatment with cycloheximide (1 µg/mL) for 1 h, the NG cultures were applied to the procedure of the endotoxin tolerance challenge (LPS-treated versus LPS/LPS-treated group). PGE 2 level in the supernatant was detected 24 h after treatment by ELISA. Data are expressed as the mean ± SEM from three independent experiments in duplicate, LPS group versus the LPS/LPS group. ** p < 0.01, NS, not significant.

Discussion
As the first responder to the immune challenge, microglia secrete a wide spectrum and various inflammatory factors at inflammatory conditions, including IL-1β, TNF-α, PGE 2 , and BDNF, to prevent invading pathogens [34]. However, the uncontrolled and unresolved inflammation induced by microglia can damage the neurons [34]. It is relatively difficult to distinguish the functional role of microglia as either "protective" or "injurious" to the neurons during the neuroinflammatory process. Having a better understanding of heterogenous microglial activation during the inflammatory process, such as the occurrence of microglial endotoxin tolerance, has become a critical issue in developing microgliabased therapy for inflammation-related brain diseases [35,36]. Through using multiple reconstituted brain cell cultures, including neuron-glial, mixed glial, neuron-enriched, and microglia-enriched cultures, the main strength of the current study is to uncover the regulatory mechanisms of microglial PGE 2 tolerance by interacting with other brain cells, such as neurons and astroglia. However, this NG culture system does not contain oligodendrocytes, which are the myelinating cells of the CNS. Interestingly, endotoxin tolerance of PGE 2 occurs in NG cells (Figure 1), implying that oligodendrocytes do not participate in the regulation of PGE 2 tolerance in microglia. Together, this study explored the immune-suppressive mechanism of PGE 2 production mediated by neuron-microglia interactions via TLR4 signal-derived de novo protein synthesis in response to repeated LPS exposure ( Figure 5).
omedicines 2022, 10, x FOR PEER REVIEW 9 understanding of heterogenous microglial activation during the inflammatory pro such as the occurrence of microglial endotoxin tolerance, has become a critical issu developing microglia-based therapy for inflammation-related brain diseases [35 Through using multiple reconstituted brain cell cultures, including neuron-glial, m glial, neuron-enriched, and microglia-enriched cultures, the main strength of the cur study is to uncover the regulatory mechanisms of microglial PGE2 tolerance by interac with other brain cells, such as neurons and astroglia. However, this NG culture sy does not contain oligodendrocytes, which are the myelinating cells of the CNS. Inte ingly, endotoxin tolerance of PGE2 occurs in NG cells (Figure 1), implying that oligo drocytes do not participate in the regulation of PGE2 tolerance in microglia. Together study explored the immune-suppressive mechanism of PGE2 production mediate neuron-microglia interactions via TLR4 signal-derived de novo protein synthesis i sponse to repeated LPS exposure ( Figure 5). In addition to electrical signal transmission, neurons are important immune reg tors in restraining immune activation of homeostatic microglia at normal conditions ferred to as immune checkpoint [37,38]. The communications among neurons and m glia are bidirectional and reciprocal through various soluble factors and in receptor and interactions [38,39]. With a volume transmission manner, neurons release the sol factors out of the synaptic cleft to trigger receptor-mediated signals in microglia [40 The neural soluble factors, such as ATP, glutamate, GABA, CSF-1, and TGF-β, are cap of regulating phagocytosis, motility, and viability of microglia [40,42,43]. On the o hand, many receptor ligands (i.e., CD47, CD200, CD22, and HSP60) on the surface of rons directly bind with their corresponding surface receptors on microglia (i.e., CD1 In addition to electrical signal transmission, neurons are important immune regulators in restraining immune activation of homeostatic microglia at normal conditions, referred to as immune checkpoint [37,38]. The communications among neurons and microglia are bidirectional and reciprocal through various soluble factors and in receptor-ligand interactions [38,39]. With a volume transmission manner, neurons release the soluble factors out of the synaptic cleft to trigger receptor-mediated signals in microglia [40,41]. The neural soluble factors, such as ATP, glutamate, GABA, CSF-1, and TGF-β, are capable of regulating phagocytosis, motility, and viability of microglia [40,42,43]. On the other hand, many receptor ligands (i.e., CD47, CD200, CD22, and HSP60) on the surface of neurons directly bind with their corresponding surface receptors on microglia (i.e., CD172a, CD200R, CD45, and TREM2) that represent the classical contact-dependent communications [44][45][46][47][48]. Overall, these humoral or contacts signals from neurons not only lead microglia to prune neural synapses and neurites, and remove the apoptotic neurons during early brain development [45,47], but they also modulate motility, surveillance, and immunity of microglia at inflammatory conditions [44,46,48]. Our data showed that, in response to the ET challenge, microglial PGE 2 tolerance occurred in the presence of neurons (Figure 1), while microglia alone or microglia co-cultured with astroglia failed to develop PGE 2 tolerance (Figure 2). Furthermore, neuron-microglia contacts participate in neuron-mediated PGE 2 tolerance in microglia (Figure 3). Receptor-ligand interactions among neurons and microglia may exert their functions to control microglial PGE 2 tolerance. However, molecular details in neural contacts for microglia ET development remain an open question that will be further investigated.
Toll-like receptors (TLRs) function as the prime cellular sensors in innate immune cells for microbial components. Thus, its activation must be properly controlled by various mechanisms to maintain homeostasis. For instance, the induction of endotoxin tolerance by TLR4-ligand lipopolysaccharide (LPS) is one mechanism to prevent overstimulation from continuous exposure to the same and related danger signals [49]. The activation of the LPS receptor complex induces TLR4 dimerization/oligomerization with rapid activation of the MyD88-dependent signaling and TRIF-dependent signaling pathway, and further triggers various transcription factors, leading to strong production of pro-inflammatory cytokines [50]. Additionally, the activity of the microRNA miR-146a-known to target key elements of the myeloid differentiation factor 88 (MyD88) signaling pathway-including IL-1 receptor-associated kinase (IRAK1), IRAK2, and tumor necrosis factor (TNF) receptorassociated factor 6 (TRAF6), has been reported to establish and sustain tolerance [51]. Our data revealed that TLR4 activation and de novo protein synthesis are required for developing neuron govern PGE 2 tolerance in microglia during ET (Figure 4). It is important to further study the mechanisms underlying neurons that modulate microglial TLR4 activation, its downstream signaling pathways, and de novo protein synthesis to preserve PGE 2 tolerance.
Although the mechanism of ET formation in the brain and cultured brain slices or microglial cells have been reported [52][53][54][55][56], microglial PGE 2 tolerance has not been fully investigated. Dr. Ajmone-Cat and his colleagues have been the first to report that the production of TNF-α, nitric oxide (NO), PGE 2 , and 15-deoxy-∆12,14-PGJ2 (15d-PGJ2) was measured in primary rat microglial cultures received to one, two, or three consecutive LPS stimulations [53]. The results indicated that the ability of microglial cells to produce NO, TNF-α, and 15d-PGJ2 upon the first LPS challenge rapidly declined after the second and third stimulations, whereas cyclooxygenase-2 and PGE 2 synthesis remained constantly elevated [53]. Further mechanistic studies in the transcription factors nuclear factor kappa B and CREB and the p38 MAPK revealed that the single or multiple LPS stimulations evoke profoundly different signaling pathways [53]. Even if the ET treatment regimens and species are distinct, similar results in this study also showed the failure of PGE 2 tolerance (even having higher PGE 2 production) in mouse microglia-enriched cultures with repeated LPS exposure ( Figure 2). Accordingly, these data suggested that the circumstance of the CNS microenvironment, such as the presence of healthy neurons, plays an important regulating role in developing microglial ET [11]. Alternatively, they can determine if neurodegeneration-associated molecular patterns (NAMPs) participate in the disruption of microglial ET and whether its mechanism may provide attracted immune therapeutic targets for neurodegenerative disease.
In this study, we identified a distinct and essential role of non-immune brain cells, i.e., neurons in the development of PGE 2 tolerance in microglia. In the absence of neurons, microglia-enriched and mixed glial cultures failed to form PGE 2 tolerance. Notably, neural contacts program microglial PGE 2 tolerance-not its soluble factors. To the best of our knowledge, our study provides the first evidence that non-immune cells, i.e., neurons, can influence the capacity of microglial PGE 2 . Moreover, this study revealed a novel regulatory role of neuron-microglia contacts in the development of microglial PGE 2 .