Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor

Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation. Here, we aimed to determine the role of autophagy in α7nAChR-mediated inhibition of neuroinflammation and its underlying mechanism. Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively. The severity of EAE was evaluated with neurological scoring. Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot. Autophagosomes were observed using transmission electron microscopy and tandem fluorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux. The mRNA levels of interleukin-6 (IL-6), IL-1β, IL-18, and tumor necrosis factor-α (TNF-α) were detected by real-time PCR. We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively. Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice. PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45- or CD68-positive cells with LC3) both in spinal cord and spleen from EAE mice. The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor. In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux. Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1β, IL-18, and TNF-α mRNA. Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE.

Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation. Here, we aimed to determine the role of autophagy in α7nAChRmediated inhibition of neuroinflammation and its underlying mechanism. Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively. The severity of EAE was evaluated with neurological scoring. Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot. Autophagosomes were observed using transmission electron microscopy and tandem fluorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux. The mRNA levels of interleukin-6 (IL-6), IL-1β, IL-18, and tumor necrosis factor-α (TNF-α) were detected by real-time PCR. We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively. Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice. PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45-or CD68-positive cells with LC3) both in spinal cord and spleen from EAE mice. The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor. In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux. Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1β, IL-18, and TNF-α mRNA. Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE.
Keywords: alpha7 nicotinic acetylcholine receptor, autophagy, neuroinflammation, experimental autoimmune encephalomyelitis, microglia inTrODUcTiOn It is commonly believed that microglia contribute to the triggering of inflammatory responses in central nervous system (CNS), mainly through the induction of inflammatory cytokine production as well as the immune responses (1,2). However, the overactivation of microglia may lead to the pathogenesis and aggravation of CNS damage in inflammatory diseases including multiple sclerosis (MS) (3)(4)(5). MS is recognized as a chronic inflammatory autoimmune disorder featuring CNS demyelination and neurodegeneration (6,7). Accumulation and overactivation of microglia greatly aggravates the severity of symptoms and demyelination in experimental autoimmune encephalomyelitis (EAE) mice (8,9). Thus, suppressing microglia-mediated inflammation serves as a potential therapeutic strategy for MS or EAE.
Alpha7 nicotinic acetylcholine receptor (α7nAChR) is a subtype of nAChRs, which is a member of superfamily of cysloop cationic ligand-gated channels (10,11). Studies from our lab previously showed that α7nAChR was associated with various cardiovascular diseases (12,13). Recently, there is evidence that activation of α7nAChR contributes to the alleviation of neuroinflammation in EAE model (14)(15)(16), but the underlying mechanism has not been fully clarified.
Autophagy is a self-protecting cellular catabolic pathway relying on lysosomes. Some long-lived proteins, as well as damaged organelles and misfolded proteins are degraded and recycled through autophagy process (17)(18)(19). It is widely acknowledged that autophagy is closely associated with CNS disorders, including cerebral ischemia, Parkinson's disease, and MS (20)(21)(22)(23). It has been reported that inducing autophagy could ameliorate several neurodegenerative diseases (24,25). Studies demonstrated that inducing autophagy could inhibit inflammation, especially in inflammatory or immune cells such as macrophages and dendritic cells (26,27). Recently, it has been reported that enhancement of autophagy could ameliorate the pathogenesis of MS or EAE disease through the limit of inflammation (21,28,29). However, whether autophagy plays a role in α7nAChR-mediated alleviation of neuroinflammation remains unclear.
In this study, we raised the hypothesis that activation of α7nAChR could promote monocyte/microglia autophagy, which inhibited the production of inflammatory cytokine and thus contributed to the attenuation of EAE severity. Our study may provide a novel therapeutic strategy for the treatment of MS. In another set of experiments, BV2 microglia were preincubated with compound C (10 µM, Sigma-Aldrich, St. Louis, MO, USA), an AMPK inhibitor, for 10 min before LPS and PNU282987 treatment. For the blockade of autophagy in vitro, bafilomycin A1 (5 nM, Selleckchem, Houston, TX, USA) or autophagy-related gene 5 small-interfering RNA (Atg5 siRNA) were applied for study.

eae induction and assessment
Mice were carefully grouped and selected randomly for studies. During animal experiments, a design of single-blind study was applied. EAE was induced in mice (8-10 weeks old, male) as previously reported (32,33). In brief, mice were subcutaneously immunized with 200 µg MOG35-55 in Complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) contained with heat-killed Mycobacterium tuberculosis (H37RA strain, 5 mg/ml) (BD Diagnostics, Franklin Lakes, NJ, USA). Pertussis toxin (Calbiochem, Billerica, MA, USA) in a dose of 200 ng for each mouse was injected on days 0 and 2 via i.p. Experimental mice were examined and evaluated every day for clinical signs and were scored according to the following criteria: "0, " no clinical signs; "1, " paralyzed tail; "2, " paresis; "3, " paraplegia; "4, " paraplegia with forelimb weakness or paralysis; "5, " moribund or death. For the treatment of drugs, PNU282987 (0.1 mg/kg, i.p.) or 3-methyladenine (3-MA) (10 mg/kg, i.p.) (Sigma-Aldrich, St. Louis, MO, USA) was injected once a day from day 3 till the end of the study. Mice were treated with saline as vehicle control (100 µl for each mouse).

histopathological analysis
Experimental mice were anesthetized with phenobarbital sodium (35 mg/kg, i.p.) and sacrificed by cervical dislocation. Animals were then perfused with PBS (pH 7.4, 20 ml) followed by 4% (w/v) paraformaldehyde (20 ml). Spinal cords were subsequently excised and further fixed in 4% (w/v) paraformaldehyde overnight. Five-μm-thick sections of spinal cords embedded with paraffin were stained by hematoxylin and eosin as well as luxol fast blue to measure spinal inflammatory infiltration or demyelination, respectively.

immunoblot analysis
BV2 microglia or spinal cord and spleen from EAE mice were washed with PBS for one time and lysed in lysis buffer on ice for 30 s. Protein concentration was detected by the bicinchoninic acid method (Thermo Scientific, Pittsburgh, PA, USA). Samples were loaded in 10% or 12% Tris/Gly gels, subjected to SDS-PAGE, and transferred on NC membranes (Millipore, Billerica, MA, USA). Immunoblot was conducted using the rabbit anti- After that, the membranes were incubated with a Donkey anti-Rabbit or Donkey anti-mouse secondary antibody (1:5,000, LI-COR Biosciences, Lincoln, NE, USA) accordingly. Images were obtained and analyzed using the Odyssey infrared imaging system (LI-COR Bioscience, Lincolin, NE, USA).

reverse Transcription and real-time Pcr
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for the extraction of total RNA from BV2 microglia. Reverse transcription was conducted for the extracted RNA to obtain the cDNA with PrimeScript™ RT Master Mix (Takara, Otsu, Shiga, Japan).

Transmission electron Microscopy
Murine BV2 microglia were cultured at 37°C on glass coverslips overnight, followed by the treatments mentioned above. BV2 microglia were harvested and fixed overnight at 4°C in 2.5% glutaraldehyde in 0.1 M PBS, and then post-fixed in 1% buffered osmium tetroxide for 2 h. Specimens were processed in routine procedure and examined under a transmission electron microscope (H-700; Hitachi, Tokyo, Japan).

autophagy Flux assessment
Murine BV2 microglia were seeded on the cultural slides and transfected with tandem fluorescent mRFP-GFP-LC3 plasmid (HanBio, Shanghai, China) when the confluence reached to 50-70% (34,35). In brief, after the culture in DMEM supplemented with 10% (vol/vol) FBS for 24 h, cells were incubated with plasmids for 6 h and then changed back to fresh DMEM supplemented with 10% (vol/vol) FBS for the cultivation of another 36 h to ensure the expression of the genes. After transfection, cells were challenged with 100 ng/ml LPS for 12 h in the presence or absence of PNU282987 (10 µM) for 10 min. Cellular autophagosomes (G + R + ) and autolysosomes (G − R + ) were detected by confocal microscopy (Leica TCS SP8, Leica, Biberach, Germany). Total number of puncta (>1 μm) per cell was counted.
cell Viability assay Cell viability was evaluated using a non-radioactive cell counting kit-8 (CCK-8; Dojindo, Kamimashiki-gun Kumamoto, Japan) as described previously (36). In brief, murine BV2 microglia at the density of 1 × 10 4 were seeded in a 96-well plate in DMEM supplemented with 10% (vol/vol) FBS. After cultivation for 6 h, cells were treated as described above. After 12 h treatment, CCK-8 culture medium was added for 1 h additional cultivation. Absorbance was assayed with a microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at the wavelength of 450 nm for the analysis of cell viability.
statistics Data were presented as mean ± SEM. A two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test for  resUlTs activating α7nachr ameliorates eae severity and spinal inflammatory infiltration in eae To assess the role of α7nAChR on CNS disorders, we treated EAE mice with α7nAChR agonist PNU282987 in the doses of 0.03 and 0.1 mg/kg body weight, respectively, from days 3 postimmunization (PI) till the end of study, and found that PNU282987 at the dose of 0.1 mg/kg body weight could effectively reduce the peak severity and cumulative clinical score of EAE while only slight decreases in the peak severity and cumulative clinical score were detected at the dose of 0.03 mg/kg body weight ( Figure 1A). Histological study of spinal cord was performed at day 17 PI. Compared with vehicle, PNU282987 (0.1 mg/kg) caused a significant reduction of leukocyte infiltration and alleviative demyelination in spinal cord shown by H&E and luxol fast blue staining (Figures 1B,C).
activating α7nachr increases autophagy in spinal cord and spleen from eae Mice It has been previously demonstrated that autophagy plays a protective and ameliorative role in EAE through the regulation of inflammatory cytokine production (21). We thus ask whether the protective effect of activating α7nAChR on EAE is associated with the augmented autophagy. First, we tested the  effect of activating α7nAChR on the levels of autophagy-related proteins in spinal cord and spleen from EAE mice and found that activating α7nAChR with PNU282987 significantly increased the LC3-II/I ratio and Beclin 1 abundance and decreased the p62/ SQSTM1 abundance in spinal cord and spleen from EAE mice (Figures 2A,B). Second, to determine the change of monocyte autophagy including macrophages in periphery and microglia in central (hereafter referred to as "monocyte/microglia autophagy") upon activation of α7nAChR, we tested the colocalization of CD45-or CD68-positive cells with LC3, respectively, in spinal cord and spleen from the control and EAE mice with or without the treatment of PNU282987. We found that compared with the control group, EAE mice had more colocalization of CD45or CD68-positive cells with LC3 in spinal cord and spleen. PNU282987 treatment further increased the colocalization of CD45 (Figures 2C,D) or CD68 (Figures 2E,F) with LC3, suggesting that activating α7nAChR contributed to the enhancement of monocyte/microglia autophagy in spinal cord and spleen from EAE mice.

Blockade of autophagy attenuates the Protective effects of α7nachr activation on eae Mice
To determine the influence of autophagy on the α7nAChRmediated protective effects on the MS course in vivo, we examined neurobehavioral deficits in 3-MA, PNU282987, 3-MA + PNU282987, and vehicle-treated mice following MOG35-55-induction of EAE. As shown in Figure 1, the disease course in the EAE mice model was a chronic progressiverelapsing phenotype. PNU282987 (0.1 mg/kg, i.p.) treatment significantly reduced the severity of neurobehavioral deficits, cumulative scores, and maximum neurological disability in EAE mice compared with these vehicle-treated mice. This protective effect of PNU282987 was abolished by 3-MA (10 mg/kg), an autophagy inhibitor (Figures 3A-C), suggesting that autophagy at least partly played a role in the protective effects of activating α7nAChR on EAE.
As shown above, in EAE mice, the protective effects of PNU282987 treatment was associated with reduced inflammatory infiltration and demyelination. We then tested if 3-MA abolished the protective effects of PNU282987 by upregulating inflammatory infiltration and demyelination. We found that blockade of autophagy with 3-MA greatly attenuated the alleviative effects of PNU282987 on inflammatory infiltration and demyelination (Figures 4A,B). We further investigated the association between autophagy and α7nAChR-mediated anti-inflammatory effects in vivo by examining the production of IL-6, IL-1β, IL-18, and TNF-α in mRNA level in spinal cord and spleen obtained from mice. In EAE mouse model, the treatment of PNU282987 significantly decreased the mRNA levels of IL-6, IL-1β, IL-18, and TNF-α in spinal cord and spleen, while blockade of autophagy with 3-MA greatly abolished this effect (Figures 4C,D). Taken together, these data indicated that blockade of autophagy with 3-MA greatly attenuated the protective effect of α7nAChR activation on the alleviation of EAE symptoms and inhibition of inflammation in EAE mice.
activating α7nachr increases autophagy in BV2 Microglia stimulated with lPs Upon LPS stimulation, the levels of LC3-II/I ratio and Beclin 1 abundance were significantly increased in BV2 microglia. Whereas p62/SQSTM1, a cargo receptor targeting the substrates into forming autophagosomes, was significantly decreased (Figures 5A,B). Preincubation with PNU282987 (0.1, 1, and 10 µM) dose-dependently enhanced these LPS-induced effects in BV2 microglia (Figures 5A,B). Since autophagosomes are basic functional units for autophagy, here we further detected the number of autophagosomes in vitro. Under the stimulation of LPS, the number of autophagosomes was greatly increased which was detected by transmission electron microscopy. In addition, PNU282987 (10 µM) treatment further enhanced this effect (Figures 5C,D). activating α7nachr Promotes the level of autophagy Flux in BV2 Microglia stimulated with lPs Autophagy is considered as a recycling process that includes the maturation of autophagosomes and subsequently the fusion of autophagosomes and lysosomes for the formation of the degradative autolysosomes. Autophagy flux depicts this entire dynamic process. In vitro, the autophagy flux was previously reported to be detected by the transfection of adenovirus harboring mRFP-GFP-LC3 (35). After transfection, autophagosomes were shown as yellow punta (the combination of red and green fluorescence), and autolysosomes were shown as red punta (the extinction of GFP in the acid environment of lysosomes). As shown in Figures 6A-C, LPS challenge increased both the number of yellow autophagosomes and red autolysosomes (the extinction of GFP in the acid environment of lysosomes). Preincubation with PNU282987 further enhanced this effect induced by LPS, suggesting that activating α7nAChR increased the conversion from autophagosomes to autolysosomes and induced a high level of autophagy flux.  Blockade of autophagy by Atg5 sirna or Bafilomycin a1 greatly attenuates the anti-inflammatory effect of PnU282987 in BV2 Microglia stimulated with lPs Upon LPS stimulation, mRNA levels of IL-6, IL-1β, IL-18, and TNF-α in BV2 microglia were significantly increased. PNU282987 treatment significantly decreased the mRNA levels of inflammatory cytokines (IL-6, IL-1β, IL-18, and TNF-α). Atg5, an E3 ubiquitin ligase, is necessary for autophagy due to its role in autophagosome elongation. Knockdown of Atg5 using Atg5 siRNA (siRNA1 according to Figure 7A) significantly attenuated the inhibitory effect of PNU282987 on the levels of IL-6, IL-1β, IL-18, and TNF-α in mRNA (Figures 7B-E). Similar changes were found in the mRNA levels of IL-6, IL-1β, IL-18, and TNF-α with the application of bafilomycin A1 ( Figure S1A in Supplementary Material). In addition, bafilomycin A1 also significantly inhibited the effect of PNU282987 on LC3-II/I ratio in LPS-stimulated BV2 microglia (Figures S1B,C in Supplementary Material). The administration of LPS, PNU282987, or bafilomycin A1 did not produce significant effect on cell viability ( Figure S1D in Supplementary Material). Collectively, those results suggested that autophagy at least partly mediated the anti-inflammatory effect of PNU282987 in LPS-stimulated BV2 microglia.

Participation of the aMPK-mTOr-p70s6K in the Protective effects of PnU282987 in BV2 Microglia stimulated with lPs
It has been previously demonstrated that AMPK activation inhibits mTOR and reduces p70S6K phosphorylation, thus promoting autophagy (37,38). Here, we investigated whether the autophagy-inducing effect of activating α7nAChR was through the AMPK-mTOR-p70S6K signaling pathway in LPS-stimulated BV2 microglia. We found that LPS stimulation increased the phosphorylation of AMPK while decreased the phosphorylation of mTOR and p70S6K. PNU282987 treatment further increased the changes of phosphorylation of AMPK, mTOR, and p70S6K (Figures 8A-D). Furthermore, compound C (an AMPK inhibitor) significantly inhibited the PNU29287-mediated attenuation of   IL-6, IL-1β, IL-18, and TNF-α production in LPS-stimulated BV2 microglia (Figures 8E-H). Taken together, these data indicated that the anti-inflammatory effects mediated by α7nAChR via inducing autophagy were at least partly through the AMPK-mTOR-p70S6K signaling pathway.

DiscUssiOn
Our previous works have demonstrated that activating α7nAChR plays a protective role in several kinds of cardiovascular diseases including ischemic stroke, hypertension, and myocardial ischemia via the "cholinergic anti-inflammatory pathway. " It has been demonstrated previously that activating α7nAChR alleviates EAE through the inhibition of inflammatory reaction (14)(15)(16). For example, Hao et al. (16) demonstrated that α7nAChR was a key mediator in the process of nicotine-mediated reduction of the CNS inflammatory response and protection against EAE through the inhibition of auto-reactive T-cell proliferation and cytokine production from helper T cell. Consistent with those reports, in our present study, we demonstrated that activating α7nAChR significantly reduce the severity in clinical score and histological examination. The production of several inflammatory cytokines including IL-6, IL-1β, IL-18, and TNF-α was attenuated with the activation of α7nAChR in EAE mice. In combination, those data indicate that activating α7nAChR contributes greatly to the alleviation of EAE. Autophagy has been recognized as a self-protective mechanism through degrading and recycling long-lived proteins, damaged organelles and misfolded proteins (39). Previous studies demonstrated that autophagy played an important role in neuroprotection through the modulation of inflammatory or immune reaction in CNS in MS or EAE (6,(40)(41)(42)(43). For instance, it has been demonstrated (42) that the mammalian target of rapamycin (mTOR), a negative regulator of autophagy, was involved in microglial pro-inflammatory activation. Rapamycin, an mTOR inhibitor, ameliorated the clinical course of the relapsing-remitting as well as the chronic EAE through the induction of microglia autophagy. Autophagy level has also been demonstrated to be enhanced in EAE mice (34,42). In our previous work, we demonstrated that activation of cannabinoid receptor 2 could induce autophagy, which subsequently led to the inhibition of NLRP3 inflammasome in CNS in EAE as well as colitis mice models (6,44). Here, we reported that blocking autophagy with 3-MA could attenuate the anti-inflammatory and alleviative effects mediated by α7nAChR activation in EAE mice. Similar results were found in LPS-stimulated BV2 microglia when transfected with Atg5 siRNA or the application of bafilomycin A1. Taken together, those data indicate that autophagy plays an important role in the amelioration of EAE as well as the anti-inflammatory effects in LPS-challenged BV2 microglia mediated by the activation of α7nAChR.
Recently, activating α7nAChR has been reported to play a protective role in CNS inflammation. The association between activating α7nAChR and induction of autophagy in neurons has been demonstrated. Jeong and Park reported that melatonin, a secretory hormone produced from various organs, enhanced the activation of autophagy process in neurons through the upregulation of α7nAChR signaling pathway, thus playing an important role in neuroprotection in prion-mediated neurodegenerative diseases (45). In their other article, the authors showed that the activation of α7nAChR stimulated by cellular prion protein expression contributed to the induction of autophagy flux in neurons, which played a pivotal role in neuroprotection (46). Their studies suggest an important role of α7nAChR -induce autophagy, which play a protective role in neuroprotection. Here, in our work, we demonstrated that activating α7nAChR contributed to the enhancement of autophagy both in spinal cord and spleen from EAE mice and BV2 microglia stimulated with LPS, and blockade of autophagy greatly attenuated its anti-inflammatory effect. Collectively, we believe that activating α7nAChR may suppress the inflammatory reaction through the induction of monocyte/microglia autophagy, thus playing an important role in neuroprotection as well as alleviation of EAE. Together with the recently proved neuroprotective effects of α7nAChR-mediated autophagy in neurons, we believe that enhancing α7nAChR-mediated autophagy may probably serve as a potential and promising therapeutic strategy in the treatment of CNS diseases.
In the in vivo studies, we used 3-MA to inhibit autophagy process for the detection of autophagy participation in the pathogenesis and progression of EAE, since 3-MA was widely used as an autophagy blocker (20,47). Our results showed that 3-MA significantly attenuated the anti-inflammatory effects of activating α7nAChR in EAE model, thus demonstrating the involvement of α7nAChR-mediated autophagy in EAE disease. However, as discussed previously, the application of 3-MA as an autophagy blocker was not without its problems. It was reported that 3-MA could inhibit all clarifications of PtdIns3K and the related downstream signaling cascades, indicating that its specificity as an autophagy suppressor should be carefully considered (6,20). It was further noted that the long-term application of 3-MA might slightly enhance autophagy level since 3-MA was not a satisfactory autophagy-specific inhibitor (48). In consideration of those limitations of 3-MA, Atg5 siRNA was applied in the present study, since Atg5 was regarded as a critical and necessary autophagyrelated gene, participating the formation of autophagosome (49). We found that Atg5 siRNA or the application of bafilomycin A1 significantly attenuated the inhibitory effect of PNU282987 on the mRNA levels of IL-6, IL-1β, IL-18, and TNF-α in BV2 microglia, indicating that autophagy at least partly mediated the anti-inflammatory effects of PNU282987 in LPS-stimulated BV2 microglia. According to those discussions, to further clarify the exact role of monoctye/microglia autophagy in EAE, other different kinds of autophagy inhibitors are demanded and more detailed works are warranted.
cOnclUsiOn Taken together, we demonstrated the α7nAChR-mediated protective roles against neuroinflammation via AMPK-mTOR-p70S6K related autophagy in monocyte/microglia ( Figure S2 in Supplementary Material). We first reported that activating α7nAChR contributed to the alleviation of EAE in severity and inflammatory infiltration. Furthermore, activating α7nAChR significantly led to the induction of autophagy both in spinal cord and spleen from EAE mice and LPS-challenged BV2 microglia. In addition, blockade of autophagy greatly attenuated the α7nAChRmediated anti-inflammatory effects both in EAE mice and BV2 microglia stimulated with LPS. Finally, we reported that this process involved the AMPK-mTOR-p70S6K signaling pathway. These results demonstrated a novel mechanism for α7nAChR in MS or EAE, which might provide a potential therapeutic target in the treatment of MS and even other inflammation-or autoimmune-related disorders.

FigUre s2
| schematic illustration of the protective mechanism of α7nachr activation through the induction of autophagy in monocyte/ microglia. (a) Under the occurrence of experimental autoimmune encephalomyelitis (EAE) or the stimulation of lipopolysaccharide (LPS), inflammatory response is greatly triggered in monocyte/microglia which significantly overwhelms the induced protective autophagy, thus leading to the deterioration of EAE severity. (B) However, activating α7nAChR by PNU282987, a specific α7nAChR agonist, largely increased the level of autophagy in monocyte/microglia, which contributes to the inhibition of inflammation and consequently the alleviation of EAE, restoring the balance between the protective autophagy and inflammation. PNU, PNU282987.