Systemic Inflammation Increases the Susceptibility to Levodopa-Induced Dyskinesia in 6-OHDA Lesioned Rats by Targeting the NR2B-Medicated PKC/MEK/ERK Pathway

Background: The long-term administration of levodopa (L-dopa), the gold-standard treatment for Parkinson's disease (PD), is irreparably associated with L-dopa-induced dyskinesia (LID), which dramatically affects the quality of life of patients. However, the underlying molecular mechanisms of how LID exacerbates remain unknown. Neuroinflammation in the striatum plays an active role in LID. These findings prompt an investigation of non-neuronal mechanisms of LID. This study will examine the effects of systemic inflammation in the development and progression of LID. Methods: To evaluate the possible influence of systemic inflammation in the appearance of LID, the PD rats received an intraperitoneal (IP) injection of various concentrations of lipopolysaccharides (LPS, 1, 2, and 5 mg/kg) or saline. One day later, these PD rats started to receive daily treatment with L-dopa (6 mg/kg) along with benserazide (6 mg/kg) or saline for 21 days, and dyskinesia was evaluated at several time points. Moreover, the activation of microglia and astrocytes and the molecular changes in NR2B and mGLUR5 signaling pathways were measured. Results: We found that systemic inflammatory stimulation with LPS exacerbated the intensity of abnormal involuntary movements (AIMs) induced by L-dopa treatment in 6-hydroxydopamine (6-OHDA) lesioned rats. The LPS injection activated the gliocytes and increased the levels of proinflammatory cytokines in the striatum in LID rats. The PD rats that received the LPS injection showed the overexpression of p-NR2B and NR2B, as well as activated PKC/MEK/ERK and NF-κB signal pathways in response to the L-dopa administration. On the contrary, clodronate-encapsulated liposomes (Clo-lipo), which could suppress the inflammatory response induced by peripheral LPS injection, improved behavioral dysfunction, inhibited neuroinflammation, prevented NR2B overexpression, and decreased the phosphorylation of PKC/MEK/ERK and NF-κB signaling pathways. Conclusion: This study suggests that systemic inflammation, by exacerbating preexisting neuroinflammation and facilitating NR2B subunit activity, may play a crucial role in the development of LID. The administration of Clo-lipo restores the effects of LPS and decreases the susceptibility to LID in 6-OHDA lesioned rats.


INTRODUCTION
Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and leads to bradykinesia, rigidity, and tremor (Kalia and Lang, 2015). Levodopa (L-dopa), the gold-standard treatment for PD, alleviates the motor symptoms of PD. However, its long-term administration is irreparably associated with abnormal involuntary movements (AIMs), termed L-dopainduced dyskinesia (LID) (Espay et al., 2018), which dramatically affects the quality of life of patients. Treatment of high doses of L-dopa and the severity of PD mainly contributed to the development and progression of dyskinesia (Cilia et al., 2014).
Recently, non-neuronal mechanisms have been proposed to play important roles in the development of LID ). The immune system has emerged as an important player in LID and was a potential target for pharmacological therapy (Pisanu et al., 2018). Glial cells are significant in the immune reaction of the brain to noxious insults by establishing the inflammatory milieu surrounding activated glial cells (Bortolanza et al., 2015a). Previous researches showed that the L-dopa chronic treatment eliciting AIMs were associated with microgliosis, astrocytosis, and an increased expression of proinflammatory cytokines (Bortolanza et al., 2015a;Del-Bel et al., 2016;Carta et al., 2017). The study of Mulas et al. (2016) showed that a pulsatile treatment with L-dopa resulted in a neuroinflammatory response in the striatum, while continuous delivery of the drug was void of any inflammatory response and dyskinetic outcome. Neuroinflammation is a shared feature of LID in the human brain and in animal models. Conditions that induce neuroinflammatory response and cytokine release could facilitate LID (Mulas et al., 2016). Several reports revealed that peripheral inflammatory response could induce neuroinflammation via the activation of microglia and the production of proinflammatory cytokines, including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the brain (Murta et al., 2015). A well-studied model of systemic inflammation in rodents is the intraperitoneal (IP) injection of Escherichia coli lipopolysaccharide (LPS) from Gram-negative bacteria to induce the innate immune response (Wu et al., 2012). Whether the systematic inflammatory response is involved in LID is unclear.
N-methyl-D-aspartate (NMDA) is a heterotetramer ion channel assembled from a combination of GluN1, GluN2, and GluN3 subunits (Yang et al., 2018). NR2A and NR2B are the most common subtypes of NMDA receptors (NMDARs) found in the central nervous system (CNS) of mammals. Neuronal apoptosis is prevented when NR2A is selectively activated, whereas the activation of NR2B is associated with the inflammatory response . NR2B is best characterized as a regulatory subunit that plays an important role in the inflammatory response (Chen et al., 2008). The proinflammatory cytokines in the brain have been shown to facilitate the NR2B subunit of the NMDA receptor activity (Viviani et al., 2003). The phosphorylation of the NR2B subunit plays a very important role in the regulation of NMDA receptor function and has been connected to alterations of synaptic efficacy of the NMDA receptor. CP-101,606, an NR2B-selective antagonist, has been reported to reduce dyskinesia as measured by clinical scoring (Ba et al., 2019). A possible role for the NR2B receptor in dyskinesia in patients with PD (Herring et al., 2017) or Parkinsonian animals  has been highlighted. However, whether the activation of NR2B in the striatum plays a contributing role in the exacerbation of LID susceptibility after systemic inflammation is currently unknown.
How systematic inflammation aggravated LID remains unclear. Based on the above evidence, we proposed that the systematic inflammatory response, through its actions on NR2B, would influence the development of LID. Furthermore, based on the existence of a correlation between LID and NR2B receptor-dependent signaling pathways, we performed the biochemical analysis in order to elucidate the molecular mechanisms of LPS on LID. To achieve systematic inflammation, we used a model of IP injection of LPS in 6-hydroxydopamine (6-OHDA)-lesioned rats. This study will discuss how systematic inflammation affects LID in 6-OHDA lesioned rats.

Animals
Adult male Sprague-Dawley rats in Specific Pathogen Free (SPF) grade (mean weight 200-250 g) were purchased from the Shanghai Laboratory Animal Center (SLAC; Shanghai, China). The production license number is SCXK 2017-0005. The rats were housed five per cage under a temperature of 22.0 ± 2.0 • C and humid conditions, in a 12 h-light/dark cycle with free access to food and water. All animal works were performed in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals as stipulated by the Institutional Animal Care and Use Committee of the School of Medicine, Shanghai Jiao Tong University, Shanghai, China. All experimental animal procedures followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (https://www.nc3rs.org.uk/arrive-guidelines), and the guidelines of the Regulation for the Administration of Affairs Concerning Experimental Animals of China enacted in 1988.

Drug Treatment and LID
In the first part of our study, the successful PD rats were divided into four groups randomly. Systemic inflammation was induced by one injection of LPS from E. coli O26:B6 (Sigma, St. Louis, MO, USA). These PD rats received a single IP injection of LPS (1, 2, and 5 mg/kg; Sigma-Aldrich Chemical, M O, USA; Cho et al., 2015;Ho et al., 2015;Kosyreva et al., 2018) or 0.9% saline. After 24 h, they were administered with L-dopa (6 mg/kg, IP.) along with benserazide (6 mg/kg, i.p.) once daily for 21 days to induce a model of dyskinesia (Tronci et al., 2017; Figure 1A).
Clodronate-encapsulated liposomes (Clo-lipo) were widely used to deplete peripheral macrophages (Ma et al., 2016;Yan et al., 2018). Clodronate was encapsulated in liposomes with a concentration of 5 mg clodronate/ml, a concentration that ensures the depletion of 80% of macrophages within 72 h after systemic administration (Seiler et al., 1997). Our study found that Clo-lipo can deplete 82% of macrophages in the spleen 72 h after injection (Supplementary Figure 1). In the second part, the successful PD rats received IP injections of phosphate-buffered saline (PBS) liposomes (1 ml/100 g of the order preparation; Nico van Rooijen; Amsterdam, The Netherlands) or Clo-lipo (1 ml/100 g of the order preparation; Nico van Rooijen; Amsterdam, The Netherlands). After 72 h, the two rat groups were administered with LPS (2 mg/kg, IP) and then treated with an administration of L-dopa/benserazide (6/6 mg/kg, IP) once daily for 21 days ( Figure 1B). Two hours after the final L-dopa/benserazide treatment, the rats were sacrificed by CO 2 euthanization, and brain samples were stored at −80 • C until analysis.

Abnormal Involuntary Movement Ratings
Abnormal involuntary movements were evaluated according to the rat dyskinesia scale described in our previous publications (Gan et al., 2015). On days 1, 2, 4, 6, 9, 13, 17, and 21 of the L-dopa/benserazide treatment, AIMs were individually observed in the 6-OHDA-lesioned PD rats after the L-dopa/benserazide injection by an experimenter who was unaware of the pharmacological intervention. Rats were placed individually in plastic cages and observed for 1 min at 20 min intervals for a duration of 120 min. AIMs were classified into three subtypes according to their distribution as axial movement, limb movement, and orolingual movement. The severity of each AIM subtype was assessed using scores from 0 to 4 (0: absent, 1: present <50% of the time; 2: present >50% of the time; 3: present all the times but interrupted by external stimuli; and 4: present all the times, and not interrupted by external stimuli). For every rat, the ALO (Axical, Limb, Orolingual) AIMs score was calculated by adding each of the three individual scores.

Immunohistochemistry
Immunohistochemical staining of rat brain sections was performed as previously described (Xie et al., 2014). The brains were removed, postfixed for 24 h in 4% paraformaldehyde (PFA), and then cryoprotected in 30% sucrose in PBS (pH 7.4). Subsequently, serial coronal sections (cut thickness: 20 µm) were cut on a freezing microtome. The brain sections were fixed with 4% PFA for 15 min and then incubated in PBS for 10 min. Slides were blocked for 1 h in 10% normal donkey serum and then incubated in a primary antibody at 4 • C overnight. Primary antibodies included rabbit anti-iba1 (1:200, Abcam) and rabbit anti-glial fibrillary acidic protein (GFAP; 1:400, Abcam). After being washed three times with PBS, the sections were incubated with the corresponding donkey anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (1:400 dilution, Life Technologies) for 1 h at room temperature. The brain sections were viewed using a fluorescence microscope (Leica, Solms, Germany). Three fields measuring 150 × 150 µm in the lesioned striatum were imaged in each section. A total of 10 sections (taking every other section spaced 200 µm apart) were evaluated for each rat as previously described (Ma et al., 2016). The mean integrated optical density (IOD) was measured by the ImageJ software (Media Cybernetics, Bethesda, MO). The ratio of the mean IOD between the different groups was used for further analysis.

Statistical Analysis
Behavioral data were non-parametric and analyzed using the Kruskal-Wallis test followed by the Dunn's test for multiple comparisons in the case of comparing data over multiple days, or the Mann-Whitney U-test. Group comparisons were performed using one-way ANOVA with a Tukey's multiple comparisons test. We reported actual values of p from the ANOVA. All values are presented in the study as means ± SEM. Values of p < 0.05 were considered statistically significant. The statistical analysis was performed using the GraphPad software (GraphPad Software, Inc., La Jolla, USA).

Effect of Peripheral LPS Injection on LID
To evaluate whether systematic inflammation can produce an effect on dyskinesia in LID, the successful PD rats received a single IP injection of LPS used at 1 mg/kg, 2 mg/kg, and 5 mg/kg. A lower dose of L-dopa (6 mg/kg, plus 6 mg/kg benserazide, IP) was used in an attempt to better highlight the differences in the AIM score between LPS-and saline-treated PD rats during the L-dopa treatment (Tronci et al., 2017). Our results revealed that 5 mg/kg LPS resulted in the death of PD rats. The sham and PD groups received the saline for 21 days, and these rats did not develop LID. Administration of L-dopa for 21 days induced dyskinesia in both LPS and saline-treated PD rats. In terms of the ALO AIM score, PD+2 mg/kg LPS and PD+1 mg/kg LPS rats were obviously increased compared with the PD+saline rats at the same time point during the course of the L-dopa treatment ( * p < 0.05, * * p < 0.01, * * * p < 0.001 vs. PD+saline+L-dopa. # p < 0.05, ## p < 0.01, vs. PD+saline+L-dopa, Figure 2Aa, n = 12/group). This was the same trend in axial AIM (Figure 2Ab), limb AIM (Figure 2Ac), and orolingual AIM (Figure 2Ad). On the Ldopa treatment days 1, 7, 15, and 21, we also analyzed the ALO AIM score of these five groups at 10, 20, 40, 60, 80, 100, and 120 min after the L-dopa treatment. Interestingly, since day 7 of using L-dopa, an increased ALO AIM score was observed in the LPS-treated rats at 10 min after the Ldopa treatment as compared with the PD+saline+L-dopa group ( * p < 0.05, * * p < 0.01, * * * p < 0.001 vs. PD+saline+L-dopa. # p < 0.05, ## p < 0.01, vs. PD+saline+L-dopa; Figure 2B, n = 12/group), indicating that LPS exacerbates LID. A higher dose of LPS (2 mg/kg) had a greater effect on LID than the lower dose (1 mg/kg). These data suggested that peripheral LPS injection into PD rats showed a higher susceptibility to develop dyskinesia as compared to the saline-treated PD rats during the L-dopa treatment.

Peripheral LPS Injection Produces Long-Lasting Effects on Neuroinflammation in the Striatum of LID Rats
To investigate if only a single IP injection of LPS is involved in neuroinflammation in normal rats, the inflammatory response in the striatum was tested. Our study found that LPS could significantly increase the expression of glia markers Iba-1 (Supplementary Figure 2Aa,b) and GFAP (Supplementary Figure 2Aa,c) and the mRNA levels of IL-1β, IL-6, and TNF-α (Supplementary Figure 2B) in the striatum of 24 h after injecting the normal rats. Furthermore, we investigated the effects of one IP injection of LPS on neuroinflammation in the striatum of LID rats. As shown in Figure 3, in the PD+saline+L-dopa group, the microglia and astrocytes in the lesioned hemispheres were activated as compared to the PD group and the sham group ( # p < 0.05, ## p < 0.01 vs. sham, n = 4/group). To characterize the effect of peripheral LPS injection on neuroinflammation in the striatum, the activation of microglia and astrocytes, IL-1β, IL-6, and TNF-α mRNA levels were quantified. The PD rats pretreated with a challenge with LPS showed an increase of Iba1+ and GFAP+ cells in the lesioned striatum as compared to the PD+saline+L-dopa group after the last L-dopa treatment, ( * p < 0.05, * * p < 0.01, * * * p < 0.001 vs. PD+saline+L-dopa, Figures 3A,B, n = 4/group). Moreover, the Q-PCR analysis of mRNA for the proinflammatory cytokines was performed on day 21 of the L-dopa chronic administration. Results showed the release of peripheral LPSinfusion induced inflammatory cytokines IL-1β, IL-6, and TNF-α in the PD+LPS+L-dopa group ( * p < 0.05, * * p < 0.01 vs. PD+saline+L-dopa, Figure 3C). These results showed that IP infusion of LPS induced neuroinflammation in the striatum, characterized by significant activation of microglia and astrocytes, and increased in the expression of IL-1β, IL-6, and TNF-α.

LPS Treatments Increased the Expressions of p-NR2B and NR2B in the Striatum of LID Rats
Our study found that LPS-treated rats showed significantly higher levels of phosphorylation of NR2B and total NR2B in the striatum 24 h after the injection (Supplementary Figures 3A,B).
No differences were found in the mGluR5 levels in the striatum injected with LPS compared to the saline-injected group (Supplementary Figure 3C). To examine whether a single IP injection of LPS to the PD rats influenced the protein expression of NR2B and mGluR5 in the striatum in L-dopa induced animals, we performed Western blotting on the striatum tissue collected at 2 h after the last L-dopa injection. The LPS-treated PD rats showed significantly higher levels of phosphorylation of NR2B and total NR2B, in response to L-dopa, compared with the PD+saline+L-dopa group ( * p < 0.05 vs. PD + saline+ Ldopa, Figures 4A,B, n = 4/group). No difference was found in the expression of mGLUR5 levels in the lesioned striatum injected with LPS compared with the PD+saline+L-dopa group ( Figure 4C; n = 4/group). We thought that the overexpression of the NR2B subunit induced by a single IP injection of LPS might play an important role in the induction of dyskinesia during the course of the L-dopa injection.

Lipopolysaccharide Treatments Further Activated the Striatal PKC/MEK/ERK and NF-κB Signal Pathways in the Striatum After Pulsatile L-Dopa Treatment
Consistent with the previous report, we found the phosphorylation of PKC/MEK/ERK was significantly increased in the lesioned striatum of the PD+saline+L-dopa group as compared to the PD group ( # p < 0.05, ## p < 0.01 vs. sham, Figure 5, n = 4/group). Our data showed that peripheral LPS injection could increase the expression of phosphorylation of NR2B and total NR2B in the striatum of the L-dopa-treated group. Thus, the impact of LPS injection on the striatal NR2B receptor was evaluated by measuring PKC/MEK/ERK and NF-κB signal pathways. Our results demonstrated that the administration of peripheral LPS to the PD rats could significantly increase the phosphorylation of PKC/MEK/ERK and NF-κB compared with the saline-treated PD rats during the course of the L-dopa injection ( * p < 0.05, * * * p < 0.001 vs. PD+saline+L-dopa, Figure 5, n = 4/group). These results showed that a single acute intraperitoneal LPS administration to the PD rats could further activate PKC/MEK/ERK and NF-κB signaling pathways in the striatum during the L-dopa treatment.
induced by the L-dopa treatment in the 6-OHDA-lesioned rats.

DISCUSSION
A growing body of studies demonstrated that the systemic inflammatory response could strongly influence the CNS (Holmes, 2013;Anthony and Couch, 2014). In our study, we used a single peripheral LPS injection to mimic the systemic inflammatory response to research the relationship between the periphery and the brain. Our study showed that systemic inflammation could increase the susceptibility to LID in 6-OHDA lesioned rats. Several findings are of interest in our study. Firstly, the peripheral LPS administration could induce neuroinflammation in the striatum of LID rats as demonstrated by the activation of glia cells and the increase in proinflammatory cytokines. Secondly, the exacerbation of LID in the LPSinduced systemic inflammation was also accompanied by the upregulation of both phosphorylated and total forms of NR2B in the striatum. Thirdly, systemic inflammation activated the PKC/MEK/ERK and NF-κB signaling pathways in LID rats. Clolipos ameliorated the effect of peripheral LPS injection, improved behavioral dysfunction, prevented NR2B overexpression, and decreased the phosphorylation of PKC/MEK/ERK and NF-κB signaling pathways.
There is increasing evidence that neuroinflammation may play a critical role in the etiogenesis of LID (Pisanu et al., 2018). Researches showed that inflammatory cytokines in the peripheral immune system might amplify neuroinflammation and contribute to the pathogenesis of a neurodegenerative disease (Perry et al., 2007;Cunningham et al., 2009;Ferrari and Tarelli, 2011). However, the role of systemic inflammation is unknown in LID. Thus, we wanted to study whether systemic inflammation would worsen the L-dopa-induced neuroinflammatory response in the striatum and exacerbate LID. To achieve this, systemic inflammation was induced by a single IP injection of LPS, an easy and well-characterized model of systemic inflammation. LPS is a major Gram-negative bacterial endotoxin that can trigger various cellular activities that contribute to the pathogenesis of inflammatory responses and induce the production of proinflammatory cytokines, such as TNF-α and IL-1β (Fu et al., 2014). Our study found that administration of LPS to the PD rats resulted in an increased susceptibility to the development of AIMs during the L-dopa treatment. In addition, several previous studies demonstrated that neuronal proinflammatory factors have been found to correlate with the LID expression in the 6-OHDA rat models of PD, where the development of AIM was accompanied by an increased immunoreactivity for the microglial marker OX-42, together with GFAP and with increases of nNOS, cyclooxygenase-2 (COX2), and NF-κB levels in the striatum (Bortolanza et al., 2015a,b;Dos-Santos-Pereira et al., 2016). Therefore, we analyzed the activation of striatal glial cells and the expression of pre-inflammatory cytokines in LPSpretreated LID rats and controls. Our results revealed that LPS administration increased the number of activated microglia and astrocytes and promoted the pre-inflammatory cytokines TNF-a, IL-1β, and IL-6 in the striatum of LID rats.
Another striking finding of this study was the role of NR2B, a subunit of the NMDA receptor, in the LPS-treated LID rats. NMDARs are one of the major excitatory receptors within the CNS and have been the focus of potential therapeutics in a variety of neurological disorders (Yang et al., 2018). In the adult mammalian brain, NMDARs are comprised of different combinations of subunits of two classes, NR2A and NR2B, that are differentially distributed throughout the brain. Glycine confers neuroprotection through non-ionotropic activation of NR2A and subsequent enhancement of Akt activation . On the other hand, the activation of NR2B plays an important role in the development and maintenance of chronic inflammatory response (Chen et al., 2008). NR2B was also found involved in the occurrence of LID (Blandini and Armentero, 2012). Many early studies performed in animals with dyskinetic conditions found an increase in the expression of NR2B in the striatum (Ahmed et al., 2011). NR2B antagonist CP-101,606 inhibits NR2B phosphorylation and provides benefit for the therapy of the LID rat model (Kong et al., 2015). A previous study showed that a single acute IP injection of LPS to postnatal male rats caused long-lasting (over 2 months) and possibly permanent changes in both the hippocampal and the cortex at the mRNA level of NR2B (subunit of NMDA; Harre et al., 2008). Our study found that the expression of phosphorylated and total forms of NR2B was markedly increased in the PD+LPS+Ldopa group compared with the PD+saline-L-dopa group. Thus, our findings confirmed that LPS-induced systemic inflammation could activate the NR2B submit. In addition, there is other evidence showing that peripheral inflammation can induce the activation of NR2B. For example, IL-1β can directly alter NR2B function via tyrosine kinase-mediated phosphorylation of NR2B (Viviani et al., 2003). NR2B subunits are overexpressed in an inflammatory model of Alzheimer's disease (AD; Maher et al., 2014). Therefore, we can speculate that a single IP injection of LPS would alter the NR2B subunit expression in the striatum of PD rats and lead to an increase in the susceptibility to LID in 6-OHDA lesioned rats. The downstream molecules resulting from NR2B activation may be responsible for behavioral deficits in the LPS-treated LID rats. Previous researches demonstrated that NMDA receptors could activate the PKC/ERK signaling pathway (Jia et al., 2007;Rodriguez-Duran and Escobar, 2014;Jiang et al., 2018), which may be due to positive coupling of the NR2B subunit to ERK (Sava et al., 2012). Furthermore, it was reported that hyperactivation of PKC/ERK and NF-κB by prolonged Ldopa treatment contributed to the development of LID (Bishnoi et al., 2008;Lin et al., 2017). Our results demonstrated that the administration of peripheral LPS could further increase the phosphorylation of PKC/MEK/ERK and NF-κB in LID rats.
Clodronate-liposome administration is a well-described method of depleting macrophages and reducing the experimental acute inflammatory response in an animal model. In the following experiment, we used Clo-lipos to ameliorate systemic inflammation induced by an LPS injection. Our results indicated that Clo-lipo administration significantly attenuated the AIM score of the LPS-treated LID rats. Furthermore, Clo-lipos decreased the LPS-induced activation of glial cells and the production of proinflammatory factors in the striatum. In addition, Clo-lipo administration reduced the overexpression of both phosphorylated and total forms of NR2B, which inhibited the phosphorylation of PKC/MEK/ERK and NF-κB signal pathways in the LPS-treated LID rats. These data suggested that Clo-lipos, which ameliorated the effect of LPS, improved behavioral dysfunction, prevented NR2B activation, and decreased the phosphorylation of PKC/MEK/ERK and NF-κB signaling pathways.
Our data indicated that systemic inflammation, by exacerbating neuroinflammation and facilitating the NR2B subunit activity, increased the susceptibility to LID in 6-OHDA lesioned rats, suggesting a possible pathophysiological role of the systemic inflammatory response in the appearance of dyskinesia, at least in the 6-OHDA-lesioned rat model of PD. On the other hand, the administration of Clo-lipo restored the effects of LPS, which further attenuated dyskinesia in the LPS-treated PD rats during the L-dopa treatment.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

AUTHOR CONTRIBUTIONS
ZL and AY designed experiments. AY, LS, YZ, and XW performed the experiments. AY analyzed the results. AY wrote the manuscript with contributions from ZL. All authors read and approved the final manuscript. Values are presented as the mean ± SEM. n = 4 per group. * p < 0.05, * * p < 0.01, and * * * p < 0.001 vs. PBS lipo+LPS. && p < 0.01, &&& p < 0.001 vs. PBS lipo+saline.