RESEARCH Open Access A novel role of NLRP3-generated IL ......RESEARCH Open Access A novel role of...

RESEARCH Open Access

A novel role of NLRP3-generated IL-1β inthe acute-chronic transition of peripherallipopolysaccharide-elicitedneuroinflammation: implications for sepsis-associated neurodegenerationZhan Zhao1,2,3†, Yubao Wang2,3*†, Ran Zhou1,2, Yi Li2, Yun Gao2, Dezhen Tu2, Belinda Wilson2, Sheng Song2,Jing Feng1,2*, Jau-Shyong Hong2 and Jerrel L. Yakel2

Abstract

Background: Sepsis-associated acute brain inflammation, if unresolved, may cause chronic neuroinflammation andresultant neurodegenerative diseases. However, little is known how the transition from acute to chronicneuroinflammation, which is critical for the following progressive neurodegeneration, occurs in sepsis. The goal ofthis study was to investigate potential immune factors regulating the transition process using a widely usedendotoxemia LPS mouse model. This model shows distinct acute and chronic phases of neuroinflammation andrecapitulates many cardinal features of Parkinson’s disease, thus, providing a unique opportunity for studying phasetransition of neuroinflammation.

Methods: C57BL/6 J, NLRP3−/−, and IL-1R1−/− mice were employed. Mild and severe endotoxemia were producedby LPS ip injection at 1 or 5 mg/kg. Neuroinflammation in vitro and in vivo was assessed with proinflammatorycytokine expression by qPCR or ELISA and microglial activation by immunohistochemical analysis.Neurodegeneration was measured by manual and stereological counts of nigral dopaminergic neurons andimmunohistochemical analysis of protein nitrosylation and α-synuclein phosphorylation.Results: LPS-elicited initial increases in mouse brain mRNA levels of TNFα, IL-6, IL-1β, and MCP-1, and nigral microglialactivation were not dose-related. By contrast, the delayed increase in brain mature IL-1β levels was dependent on LPSdoses and protracted nigral microglial activation was only observed in high dose of LPS-treated mice. LPS-elicited increasein brain mature IL-1β but not IL-1α level was NLRP3-dependent. After high dose LPS treatment, deficiency of NLRP3 orIL-1R1 did not prevent the initiation of acute neuroinflammation but abolished chronic neuroinflammation. Genetic orpharmacological inhibition of the NLRP3-IL-1β axis repressed LPS-stimulated upregulation of chronic neuroinflammatorymediators including MHC-II, NOX2, and Mac1, and protected dopaminergic neurons. Ten months after LPS-elicited severeendotoxemia, nigral persisted microglial activation, elevated nitrosylated proteins and phosphorylated α-synuclein, andsignificant neuronal degeneration developed in wild-type mice but not in NLRP3−/− or IL-1R1−/− mice.

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© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: [email protected]; [email protected]†Zhan Zhao and Yubao Wang contributed equally to this work.2Neurobiology Laboratory, National Institute of Environmental HealthSciences, National Institutes of Health, Research Triangle Park, NC 27709, USA1Respiratory Department, Tianjin Medical University General Hospital, Tianjin300052, ChinaFull list of author information is available at the end of the article

Zhao et al. Journal of Neuroinflammation (2020) 17:64 https://doi.org/10.1186/s12974-020-1728-5

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Conclusions: This study uncovers a novel role of the NLRP3-IL-1β signaling pathway in gauging the severity of sepsis-associated inflammation and determining whether acute neuroinflammation will resolve or transition to low grade chronicneuroinflammation. These findings also provide novel targets for developing therapy for severe systemic infection-relatedneurodegeneration.

Keywords: Sepsis, Neuroinflammation, Acute-chronic transition, Neurodegeneration, NLRP3 inflammasome, IL-1β, IL-1R1,Parkinson’s disease

BackgroundEpidemiological investigations revealed that infections,such as sepsis (severe systemic infections) or viralinfections, increase the risk of long-term brain dysfunc-tion [1] and chronic neurodegeneration, includingParkinson’s disease (PD), Alzheimer’s disease (AD), andmultiple sclerosis [2–7]. Symptoms of neurodegenera-tion in patients usually occur months or years after theincidence of infection. Strong evidence indicates anessential role of neuroinflammation in the pathogenesisof progressive neurodegeneration [8–16].To model the sepsis-associated neuropathology, we

have previously created a mouse model by a single intra-peritoneal (ip) injection of sublethal dose of lipopolysac-charide (LPS, 5 mg/kg) [17–19]. Unlike most of acutePD animal models, which often kill neurons too fast[20], this model shows distinct acute and chronic phasesof neuroinflammation and recapitulates many cardinalfeatures of Parkinson’s disease, thus providing a uniqueopportunity for studying phase transition of neuroin-flammation [21–23].Numerous reports indicate that brain microglia are the

proximal factor for sepsis-associated acute neuroinflam-mation. Once activated by sepsis via humoral pathway,neural pathway, and blood–brain barrier (BBB) alterations,microglia release a variety of cytokines, chemokines, andfree radicals such as nitric oxide (NO) and reactive oxygenspecies (ROS) [24]. We have demonstrated that tumornecrosis factor alpha (TNFα) is necessary for initiatingacute neuroinflammation after endotoxemia [17]. Al-though much is known about how endotoxemia affectsbrain immune functions, a crucial question as to how thetransition of acute to chronic neuroinflammation occursremains unanswered. The purpose of this study was toidentify possible immune factors and investigate theirmechanisms underlying this immune transition, whichcould offer new insights and potential therapies for sepsis-associated neurodegeneration.Interleukin-1β (IL-1β), one of the potent proinflamma-

tory cytokines, plays crucial roles in amplifying innateand adaptive immunity through its functional receptorIL-1 receptor 1 (IL-1R1) [25]; however, this cytokinecould exacerbate tissue injuries in pathologicalconditions [26–32]. IL-1β precursor protein requires

proteolytic cleavage by activated caspase-1 through theinflammasome, a large intracellular multiproteincomplex, to generate mature IL-1β, the releasable andbioactive form [28, 30]. Nod-like receptor protein 3(NLRP3) inflammasome can be activated by diversestimuli, such as ATP, crystals, and endoplasmicreticulum (ER) stress, serving as a gate to produce ma-ture IL-1β and involving multiple immune disorders[32–43]. The important role of NLRP3 inflammasome inmediating neurodegeneration produced by severaltoxins, including intracranial injection of LPS [44, 45],has been described. However, how precisely NLRP3-IL-1β impacts endotoxemia-elicited acut-chronic neuroim-mune phase transition, which dominates the followingprogressive neurodegeneration, is still unknown. Thus,this study will provide the first evidence unveiling anovel role NLRP3-IL-1β axis in mediating sepsis-associated brain immune dynamic changes.In this study, we investigated roles of the NLRP3-IL-

1β signaling pathway in the transition of acute tochronic neuroinflammation using the peripheral LPS-induced mouse model [17, 21, 23]. We uncoveredseveral unique changes of microglial NLRP3-generatedIL-1β in response to LPS treatment in vitro and in vivo,including a delayed increase in the production and re-lease of mature IL-1β compared with most otherproinflammatory cytokines, and differential regulatedprecursor processing pattern in response to differentdoses of LPS. Moreover, genetic or pharmacologicalinhibition of either NLRP3 or IL-1R1 did not preventthe initiation of acute neuroinflammation but abolishedlong-term chronic neuroinflammation and subsequentneurodegeneration. To the best of our knowledge, thisstudy provides the first evidence showing a novel role ofNLRP3-IL-1β in mediating the transition from acute tochronic neuroinflammation incited by sepsis.

MethodsAnimalsMale C57BL/6 J, NLRP3−/−, and IL-1R1−/− mice at theage of 10- to 12-week-old were obtained from TheJackson Laboratory (Bar Harbor, Maine). All housingand breeding procedures and experimental protocols

Zhao et al. Journal of Neuroinflammation (2020) 17:64 Page 2 of 19

were approved by IACUC (Institutional Animal Careand Use Committee) of NIH (National Institutes ofHealth).

ReagentsLPS (Escherichia coil O111:B4) used for cell culturestudies was purchased from Calbiochem (San Diego,CA; cat# 437627) and for animal studies was purchasedfrom Sigma-Aldrich (St. Louis, Mo; cat# L3012). Anti-tyrosine hydroxylase (TH) and anti-Iba-1 were pur-chased form EMD Millipore (Burlington, MA) andWako (Richmond, VA), respectively. The secondary anti-bodies were purchased from Vector Laboratories (Bur-lingame, CA). The rat anti-mouse CD-11b antibody waspurchased from abD Serotec (Raleigh, NC, cat#MCA711G). Anti-pro-IL-1β, anti-alpha-synuclein (α-sy-nuclein), and anti-3-Nitrotyrosine (3-NT) antibodieswere purchased from Abcam (Cambridge, MA). Mouseinterleukin-1 receptor antagonist (IL-1Ra), NLRP3 in-hibitor MCC950, caspase-1 inhibitor Z-YVAD, IRE1α(inositol-requiring enzyme 1α) inhibitor 4μ8C, TNF-α,and IL-1β ELISA kits were purchased from R&D Sys-tems (Minneapolis, MN). Tauroursodeoxycholic acid(TUDC) was from Selleckchem (Houston, TX). MouseIL-1β pro-form ELISA kit was from eBioScience (SanDiego, California). Recombinant mouse IL-1β was fromBioLegend (San Diego, CA). Cell culture ingredientswere obtained from Invitrogen (San Diego, CA). Allother reagents came from Sigma Chemical Co. (St.Louis, MO).

Animal treatmentMice, housed in a 12 h light/dark cycle for 1 week,received a single intraperitoneal injection of LPS[1 mg/kg (3 × 106 EU/kg) or 5 mg/kg (15 × 106 EU/kg)] or vehicle (PBS solutions). At different timepoints after LPS injection, mice were euthanized byFatal-Plus overdose followed by cardiac perfusion withPBS (for mRNA and protein analysis) or formalde-hyde (for IHC) and brains were collected. Brains forIHC were further post-fixed with 4% paraformalde-hyde at 4 °C for 48 h, and subsequently immersed in30% sucrose until the brains sank to the bottom ofthe container. Coronal sections (35 μm) encompassingSN pars compacta (SNpc) and hippocampus (Hip)were cut at 35 μm and stored in PBS.

Primary mouse mesencephalic neuron-glial culturesNeuron-glial cultures were prepared from the ventralmesencephalic tissues as previously described [46].Ventral mesencephalic tissues were dissected from em-bryonic day 14 ± 0.5 and then dissociated with a mildmechanical trituration in ice-cold MEM. Cells wereseeded to poly-D-lysine-coated 24-well (6.5 × 105/well)

plates with 0.5 ml/well of maintenance medium andplace it in a humidified 37 °C, 5% CO2 incubator. Threedays later, neuron-glia cultures were replenished with0.5 ml/well fresh medium and were used for treatmentat 7 days after their initial seeding. The composition ofmajor cell types at the time of treatment was estimatedby visual counting of immunostained cells withantibodies against cell-type specific markers: 11%microglia, 48% astrocytes, and 41% neurons, where ap-proximately 1% of neurons were tyrosine hydroxylase-immunoreactive (TH-ir).

Primary mouse mixed glial culturesPrimary mixed glial cultures were prepared by a previ-ously described method [47]. Whole brains of postnatalday 1 neonates of C57BL/6 J mice, after stripping bloodvessels and meninges, were dissociated by trituration inDMEM/F12 media. Cells were seeded to poly-D-lysine-coated 24-well (5.5 × 105/well) plates with 0.5 ml/well ofDMEM/F12 mixed glial culture media and maintainedin a humidified 37 °C, 5% CO2 incubator. The mediumwas changed every 3 days with 1 ml/well of DMEM/F12mixed glial culture media. Cultures were ready fortreatment at 14 days after initial seeding. Based on theestimation by immunostained cells with specific micro-glia marker (Iba-1) and astrocyte marker (GFAP), mixedglia cultures contain about 20% microglia and 80%astrocytes.

Immunohistochemistry and double-labelingimmunofluorescence of brain slicesFree-floating 35 μm coronal brain slices encompassingSNpc and Hip regions were subjected to immunostain-ing as described previously [12]. After washing (twotimes) with PBS, the brain slices were treated with 1%hydrogen peroxide for 10 min. The slices were againwashed (three times) with PBS and then incubated for20 min with blocking solution (PBS containing 1%bovine serum albumin, 0.4% Triton X-100, and 4%appropriate serum to block the non-specific binding).The slices were incubated overnight at 4 °C with rabbitpolyclonal antibody against tyrosine hydroxylase (TH)diluted (1:5000, Dopaminergic neuron marker), Iba-1 (1:5000, microglia marker), Phospho alpha-synuclein(S129) (1:5000), or rat monoclonal antibody againstmouse CD11b (1:15000, microglia marker), in antibodydiluents (DAKO), and then the slices were washed (threetimes) for 10 min each time in PBS. The slices were nextincubated for 1 h with PBS containing 0.3% Triton X-100 and the appropriate biotinylated secondary antibody(goat anti-rabbit antibody, 1:227; horse anti-rat, 1:227).After washing (three times) with PBS, the slices wereincubated for 1 h with the Vectastain ABC reagents(Vector Laboratory, Burlingame, CA) diluted in PBS


containing 0.3% Triton X-100. To visualize the signal,the slices were incubated with 3,3′-diaminobenzidineand urea-hydrogen peroxide tablets dissolved in water.For morphological analysis, digital images were acquiredunder microscope (Nikon) connected to a Leica AperioAT2 scanner.For immunofluorescence, brain slices were incubated

for 20 min in blocking solution to block non-specificbinding. Slices were immunostained overnight at 4 °Cwith rabbit polyclonal antibodies against TH (1:5000)and co-labeled with a mouse monoclonal antibodyagainst 3-nitrotyrosine (3-NT) (1: 1000). Antibodieswere detected and visualized using Alexa Fluor 594 goatanti-rabbit IgG (1:750), or Alexa Fluor 488 goat anti-mouse IgG (1:750) secondary antibodies. The imageswere acquired using an inverted confocal microscope(Zeiss LSM 780). The intensities of Iba-1, CD-11b, Ser-129 phosphorylated α-synuclein, and immunofluores-cence were analyzed using ImageJ software.

Immunocytochemistry of cell culturesCultures were fixed with 3.7% formaldehyde in PBS for20 min. After washing (two times) with PBS, the cultureswere treated with 1% hydrogen peroxide for 10 min. Thecultures were again washed (three times) with PBS andthen incubated for 20 min with blocking solution. Thefixed cultures were immunostained overnight at 4 °Cwith rabbit polyclonal antibodies against either TH (1:5000) or rat monoclonal antibody against mouse CD-11b (1:15000) in antibody diluents. Secondary antibody(1:227; Vector Laboratory) was added, amplified withVectastain ABC reagents (Vector Laboratory), and visu-alized with 3,3′-diaminobenzidine (DAB). For morpho-logical analysis and cell counting, images were acquiredunder microscope (Nikon) connected to a Leica AperioAT2 scanner. The intensity of CD-11b was analyzedusing ImageJ software.

Real-time RT-PCR analysisTotal RNA was extracted from the mouse brain or cellcultures by using a RNeasy Mini Kit from QIAGEN(Valencia, CA) to detect the level of TNF-α, IL-1β,interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), major histocompatibility complex II (MHC-II), and NOX2 (gp91) according to the previousdescription [47]. Total RNA was reversely transcribedwith MuLV reverse transcriptase and oligo-dT primers.Then SYBR green PCR master mix was used for real-time PCR analysis. The primer sequences were as follow:GAPDH F (5′ TTC AAC GGC ACA GTC AAG GC 3′),GAPDH R (5′ GAC TCC ACG ACA TAC TCA GCACC 3′), TNF-α F (5′ GAC CCT CAC ACT CAG ATCATC TTC T 3′), TNF-α R (5′ CCT CCA CTT GGTGGT TTG CT 3′), IL-1β F (5′ CTG GTG TGT GAC

GTT CCC ATT A 3′), IL-1β R (5′ CCG ACA GCACGA GGC TTT 3′), MHC-II F (5′ CCG TCA CAGGAG TCA GAA AGG 3′), MHC-II R (5′ CGG AGCAGA GAC ATT CAG GTC 3′), NOX2 F (5′ GAT TCAAGA TGG AGG TGG GAC 3′), NOX2 R (5′ GGTCAG TGT GAA TGG GTG CC 3′), MCP-1 F (5′ TTTGAA TGT GAA GTT GAC CCG 3′), and MCP-1 R (5′GAA GTG CTT GAG GTG GTT GTG 3′). Real-timePCR amplification was performed using SYBR GreenPCR Master Mix and QuantStudio 6 Flex Real-TimePCR System (Applied Biosystems, Foster City, CA, USA)according to manufacturer’s protocols. Amplificationswere done at 95 °C for 10 s, 55 °C for 30 s, and 72 °C for30 s for 40 cycles. All samples were tested in duplicateand normalized with GAPDH using the 2-ΔΔCt method.Fold changes for each treatment were normalized to thecontrol.

Pro-IL-1β Western blot analysisTissues were homogenized in lysis buffer containingprotease inhibitor cocktail. Equal protein from eachhalf brain was separated by Bis-Tris-polyacrylamideelectrophoresis gel, and then transferred to polyvinyli-dene difluoride (PVDF) membranes. Membranes wereblocked with 5% non-fat milk and probed overnightat 4 °C with anti-pro-IL-1β antibody. Membraneswere followed by incubating with secondary anti-rabbit IgG for 1 h. ECL Plus reagents were used as adetection system. Relative band intensities were quan-tified by ImageJ.

Stereological and manual counting assessments ofneurodegenerationDopaminergic neurons of the SNpc were identifiedthrough TH immunohistochemistry. The number ofTH-immunoreacted (TH-ir) neurons in SNpc betweensaline and LPS-injected groups was counted with bothoptical fractionator and manual counting ways. For ste-reological counts, a total of eight sections containing theSNpc were sampled at 105-μm intervals. We used anoptical fractionator method on an Olympus BX50 ste-reological microscope to estimate TH-ir neurons withinuser-defined boundaries [21–23]. Systematic randomsampling of sites with an unbiased counting frame(100 μm× 100 μm) was conducted within definedboundaries of the SN. The guard zone was set at2 μm and the dissector height was 11 μm. The countswere performed with an Olympus BX50 microscopewith a 60 × 1.4NA oil immersion objective. The coeffi-cient of error values was less than 0.1. For manualcount, three individuals performed counting in adouble-blind manner [46].


Statistical analysisData was presented as the mean ± SEM. Comparison ofmore than two groups was performed using one-wayANOVA followed by Bonferroni post hoc multiple com-parison test. Comparisons of more than two parameterswere performed by two-way ANOVA analysis followedby Bonferroni post hoc multiple comparison test. Datawere analyzed using Prism (v7.00, GraphPad, San Diego,CA). P values less than or equal to 0.05 were consideredstatistically significant.

ResultsBrain-delayed mature IL-1β levels, but not initial acuteproinflammatory response, are peripheral LPS dose-dependentMice received a single intraperitoneal injection of either1 or 5 mg/kg of LPS to produce either mild or severeendotoxemia. No difference in the increases in brainmRNA levels of TNFα, IL-6, MCP-1, and IL-1β were ob-served between LPS 1 and 5 mg/kg groups at 1 h afterinjection, indicating that brain initial proinflammatoryresponses are not related to LPS doses (Fig. 1a–d).Different from most proinflammatory cytokines, theproduction of mature IL-1β is inflammasome-dependentvia limited proteolysis of its precursor protein. There-fore, we compared time-course expression of not onlyIL-1β mRNA and precursor but also IL-1β mature form,to determine whether endotoxemia severity affects brainmature IL-1β levels. We found that 1 and 5 mg/kg ofLPS-elicited increases in IL-1β mRNA and precursorlevels are similar (Fig. 1d, e and Additional file 1: FigureS1). By contrast, high dose of LPS produced muchhigher brain mature IL-1β than that of low dose of LPSduring 7–11 h after LPS injection (Fig. 1f). After 24 h,brain mature IL-1β concentrations were too low to de-tect by the ELISA kit.For more detailed mechanistic studies on the process-

ing of IL-1β protein, we employed primary mouse mix-glial cultures, which mainly contain 20% microglia and80% astroglia. There are two reasons for using mix-glialcultures, instead of enriched microglial cultures. First,reports indicate that microglia are the main source ofIL-1β production in the brain after peripheral LPS injec-tion [48–50] and in cell cultures upon LPS stimulation[39]. Second, the presence of astroglia helps the survivaland stabilization of microglia in cultures [51]. Two con-centrations of LPS (10 and 1000 ng/ml), which producedifferent degrees of inflammatory responses, were addedto the cultures. And then IL-1β mRNA, precursor pro-tein, and mature IL-1β were measured at different timesafterwards. Similar to brain tissues, mix-glial culturesproduced comparable increases in the expression of IL-1β mRNA and precursor levels between high and lowconcentrations of LPS treatment (Fig. 1g, h). By contrast,

high concentrations of LPS caused a larger delayed in-crease in the supernatant IL-1β level than that of lowconcentrations of LPS (Fig. 1i).

High dose, but not low dose, of peripheral LPS causeslong-lasting nigral microglial activationIn vitro and in vivo findings prompted us to furtherdetermine whether the dose-related increase in matureIL-1β could be related to the transition of acute tochronic inflammation. To obtain evidence supportingthis hypothesis, we investigated the persistence of micro-glia activation after 1 and 5 mg/kg LPS treatment. Formonitoring microglial activation, immunostaining ofIba-1 (ionized calcium binding adaptor molecule 1) wasperformed at acute stage (6 h after LPS) and CD-11b (α-chain of Mac1 receptor) was used as a marker for thechronic stage (1 week after LPS). The reason for usingtwo different markers at different stages is based on ourprevious reports indicating that microglial Iba-1 but notCD-11b expression was acutely enhanced after LPS in-jection. During the acute inflammation stage, the level ofCD-11b at 6 h after LPS was too low for immunostain-ing. By contrast, the increase of CD-11b expression wasmuch higher than that of Iba-1 at 1 week and later timepoints [21, 52]. In this study, Iba-1 staining quantifica-tion revealed that acute microglial activation in the sub-stantia nigra was apparent at 6 h after LPS injection, butthere were no differences between LPS 1 and 5 mg/kggroups (Fig. 1j). One week later, nigral microglialmorphology in 1 mg/kg LPS group was similar to that ofsaline group, suggesting that acute neuroinflammationhad resolved (Fig. 1k). In contrast, protracted neuroin-flammation occurred in 5 mg/kg LPS group, whichshowed clear sustained activation of microglia in the SNregion (Fig. 1k). Thus, brain-delayed mature IL-1β levelsand prolonged neuroinflammation are both affected byLPS in a dose-dependent manner, which is consistentwith the idea that IL-1β may serve as a critical factor inthe transition from acute to chronic neuroinflammation.

Deficiency of NLRP3 reduces LPS-elicited increase in IL-1β,but not IL-1α, levelsAmong inflammasome family members, NLRP3 inflam-masome is abundantly expressed and plays a key role inIL-1β processing in the brain [53–55]. However, whetherelevated brain IL-1β levels incited by endotoxemia areNLRP3-dependent remain unclear. Since brain matureIL-1β levels peaked around 7–11 h after LPS injections(Fig. 1f), we compared 9 h brain mature IL-1β betweenNLRP3-deficient (NLRP3−/−) and WT mice and foundthat NLRP3−/− mice generated significantly less brainmature IL-1β than that of WT mice (Fig. 2a). In mix-glial cultures, either genetic knockout of NLRP3 orpharmacological inhibition of NLRP3 by MCC950 (an


Fig. 1 Peripheral LPS dose-dependently increases brain mature IL-1β production and causes sustained nigral microglial activation. In vivo cytokinemeasurements (a–f): At 1 h after injection of LPS (1 or 5 mg/kg, ip) or saline vehicle in C57BL/6 J mice, mRNA levels of TNFα (a), IL-6 (b), andMCP-1 (c) were detected by qPCR in brain tissues (n = 4/group). ***p < 0.001 and ****p < 0.001 compared with saline vehicle group. No significantdifference (NS) between LPS 1 and 5 mg/kg groups. One-way ANOVA followed by Bonferroni post hoc multiple comparison test. Levels of IL-1βmRNA (d), and its precursor (d) and mature IL-1β (F) were measured in brain tissues at indicated time points by qPCR, Western blotquantification, and ELISA, respectively (n = 3–5/group for each timepoint). ***p < 0.001, #p < 0.0001, or NS compared with LPS 1 mg/kg group.Two-way ANOVA followed by Bonferroni post hoc multiple comparison test. In vitro IL-1β measurements (g–i): In C57BL/6 J mix-glia cultures afterLPS (10 ng/ml or 103 ng/ml), or vehicle medium treatment, mRNA (g), supernatant precursor (h), and mature form (i) of IL-1β were determined atindicated time points by qPCR or ELISA, respectively. Results were from three independent experiments. ****p < 0.001 compared with LPS 10 ng/ml group. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test. In vivo microglial activation (j, k): At 6 h and 1 week afterinjection of LPS (1 or 5 mg/kg, ip) or saline vehicle in C57BL/6 J mice (n = 3/group for each timepoint), nigral microglial Iba-1 (j) or CD-11b (k)immunostaining was performed, respectively. Representative images were shown. Scale bar = 300 μm. The histograms showed the density of Iba-1 (j) or CD-11b (k) quantified by with ImageJ. **p < 0.01 and ***p < 0.001 compared with saline vehicle group, and NS between LPS 1 and 5 mg/kg groups. One-way ANOVA followed by Bonferroni post hoc multiple comparison test


NLRP3 inhibitor) [56] significantly reduced LPS-stimulated mature IL-1β release (Fig. 2c, d). We alsomeasured LPS-elicited increase in brain and culturesupernatant levels of IL-1α, another IL-1 family member,which is the ligand of IL-1R1 too [29], to determinewhether processing of this cytokine is NLRP3-dependent. LPS-elicited IL-1β levels were decreased bothin brain tissues and the supernatant of mix-glial culturesin the absence of NLRP3. By contrast, brain IL-1αproductions were independent of NLRP3 (Fig. 2b, c).Similarly, post-treatment of MCC950 reduced the pro-duction of IL-1β but not IL-1α (Fig. 2d).

ER stress mediates LPS alone-induced NLRP3-dependentIL-1β productionWe first determined whether LPS enhances the expres-sion of NLRP3 and found that mRNA levels of NLRP3were greatly increased and peaked at 3 h after LPStreatment in mixed glial cultures (Additional file 2:Figure S2a). Additional studies showed that YVAD (acaspase-1 inhibitor) significantly repressed LPS-inducedincrease in the release of mature IL-1β (Additional file 2:Figure S2b). These results indicate the involvement ofthe NLRP3-caspase-1 pathway in LPS alone-inducedmicroglial mature IL-1β release. In fact, we have

Fig. 2 Lacking NLRP3 lessens endotoxemia-elicited increase in IL-1β, but not IL-1α, production. At 9 h after LPS 5 mg/kg or saline ip injection,levels of mature IL-1β (a) and IL-1α (b) were measured in 0.2 mg total protein from brain homogenization of WT and NLRP3−/− mice by ELISA(n = 5–10/group). ***p < 0.001 for (a); NS for (b) between LPS-treated WT and NLRP3−/− groups. Two-way ANOVA followed by Bonferroni post hocmultiple comparison test. c Levels of IL-1β and IL-1α in the supernatant of NLRP3−/− mix-glial cultures at 24 h after vehicle or LPS treatment. Datawere from three independent experiments. *p < 0.05, ***p < 0.001, and NS compared to respective vehicle medium group. One-way ANOVAfollowed by Bonferroni post hoc multiple comparison test. d Mix-glial cultures were treated with 10 ng/ml or 103 ng/ml of LPS or vehiclemedium. MCC950 (10 μM) were added to cultures 6 h later. Culture supernatant levels of IL-1β and IL-1α were assessed at 24 h after LPStreatment. Data were from three independent experiments. *p < 0.1, ***p < 0.001, and NS compared with the same dose of LPS treatment groupwithout MCC950. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test


performed Western blot analysis to measure levels ofNLRP3, caspase-1 (in pro and mature form), but notASC (antibody used did not work), in both microglia-enriched cultures and in intact brains and dose-dependent increases in both NLRP3 and mature form ofcaspase-1. LPS-elicited increase in activated caspase-1and mature IL-1β levels were significantly reduced inthe presence of the NLRP3 inhibitor MCC950 inprimary microglia-enriched cultures. These data were in-cluded in a separate paper submitted to another journalfor publication (Gao et al., submitted for publication).Most investigations have demonstrated that NLRP3

inflammasome-dependent processing of IL-1β precursorto mature form requires secondary stimulating agents,such as ATP, or pore-forming toxins, to activate theNLRP3-caspase-1 pathway [39]. Interestingly, LPS alonein our study can trigger microglial mature IL-1β releasein the absence of known NLRP3 activators. We werewondering the mechanism underlying LPS alone-induced microglial IL-1β maturation. Furthermore, arecent report indicated that ER stress can activateNLRP3 inflammasome in macrophage stimulated bypathogen alone in the absence of NLRP3 inflammasomeactivators [43]. Additionally, increased levels of ER stressmakers were observed in the brain after LPS ip injectionand relief of ER stress decreased brain cytokine produc-tion including IL-1β [57]. Therefore, we investigated apotential role of ER stress in mediating LPS alone-induced NLRP3-dependent mature IL-1β production inmicroglia. Our data showed that LPS-induced matureIL-1β levels declined significantly by post-treatment withER stress inhibitors either tauroursodeoxycholic acid(TUDC, a chemical chaperon mitigating ER stress) or4μ8C (a specific IRE1α inhibitor), suggesting a role ofER stress in LPS alone-induced microglial activation ofNLRP3 inflammasome (Additional file 2: Figure S2c).

The NLRP3- IL-1R1 pathway dictates peripheral LPS-elicited transition from acute to chronicneuroinflammationIL-1β acts on two receptors: IL-1R1, a receptor respon-sible for IL-1β signal transduction via MyD88-NF-κBand MAPK pathways, and IL-1R2, a decoy receptor thatblocks the function of IL-1β [25]. To further elucidatethe role of IL-1β in LPS-induced acute-chronic transi-tion of neuroinflammation, we injected wild-type (WT),NLRP3−/−, and IL-1R1−/− mice with LPS (5 mg/kg, ip).This dose of LPS produced prolonged neuroinflamma-tion in WT mice (Fig. 1k). Increased levels of brainmRNA including TNFα, IL-6, and MCP-1 measured at1 h after LPS injection were similar among these threestrains of mice (Additional file 3: Figure S3a-c), exceptthe IL-1β mRNA levels, which were slightly higher inNLRP3−/− mice than those in WT mice, perhaps due to

a compensatory effect (Additional file 3: Figure S3d).Furthermore, during the acute phase of LPS-elicitedneuroinflammation (6 h after LPS), no difference inmicroglial activation as shown by enhanced Iba-1 immu-noreactivity in the SN region was observed betweenthese three strains of mice (Fig. 3a). Thus, sepsis stilllaunches acute proinflammatory response in the brainwithout the participation of NLRP3 or IL-1R1. By con-trast, NLRP3 and IL-1R1 are required for long-termmaintenance of microglial activation after sepsis. Duringthe chronic neuroinflammatory phase (1 week, 4 and10 months after LPS), higher levels of CD-11b immuno-reactivity and activated morphology of nigral microgliawere still evident in WT mice, but not in both NLRP3-or IL-1R1-deficent mice (Fig. 3b–d). Taken together, ourresults from different doses of LPS (Fig. 1), plus datafrom mutant mice (Figs. 2 and 3), strongly indicate acritical role of the NLRP3-IL1β-IL-1R1 pathway in medi-ating the transition from acute to chronic neuroinflam-mation incited by severe endotoxemia.

Genetic/pharmacological inhibition of NLRP3 or IL-1R1represses LPS-elicited production of chronic inflammatorymediators in neuron-glial culturesTo determine potential inflammatory factors mediatingNLRP3-IL-1β-elicited chronic neuroinflammation, weperformed in vitro studies using mouse primarymidbrain neuron-glial cultures containing approximately40% neurons, 10% microglia, and 50% astroglia. To ob-serve clearer neuronal damage and reactive microgliosis,we treated neuron-glial cultures with a higher con-centration of LPS (20 ng/ml). In this series of studies,we assessed the consequence of lacking either NLRP3or IL-1R1 on the induction of acute factors such asTNFα, and delayed factors including MHC-II, NOX2(gp91), and Mac1. These delayed factors are necessaryfor sustained reactive microgliosis and long-lastingneuroinflammation [11, 21, 58–67].We first demonstrated that the lack of NLRP3 or IL-

1R1 did not impede the initiation of LPS-elicited acuteimmune response. LPS stimulated a large increase ofTNFα level measured at its peak release level (4 h afterLPS) in cultures prepared from WT, NLRP3−/−, and IL-1R1−/− mice; however, no difference in the amount of in-crease was observed among these three groups (Fig. 4a).There was a delayed increase in the release of IL-1β inWT and IL-1R1−/−, but not in NLRR3−/− cultures(Fig. 4b). Furthermore, addition of a NLRP3 inhibitorMCC950 to the cultures diminished LPS-elicited IL-1βproduction (Fig. 4c).Several late-expressed genes, including MHC-II,

NOX2, and Mac1, which are involved in mediatingchronic neuroinflammation were determined usingNLRP3 or IL-1R1 inhibitors. To avoid interfering with


LPS-elicited acute neuroinflammatory response,MCC950 was added 6 h and an IL-1 receptor antag-onist (IL-1Ra) was added 9 h after LPS treatment.These two posttreatment time points reflect the peak

of IL-1β processing and the peak of IL-1β release, re-spectively. The blockade of the NLRP3-IL-1β pathwayby either preventing the production or inhibiting theaction of IL-1β greatly reduced LPS-induced

Fig. 3 High dose LPS injection produces long-lasting microglial activation in WT mice, but not in NLRP3−/− or IL-1R1−/− mice. Following a singleinjection of LPS (5 mg/kg; ip) or saline vehicle, mice were perfused 6 h, 1 week, 4 and 10 months thereafter for nigral microglial Iba-1 or CD-11bimmunostaining (n = 3/group for each time point). Representative images at each time point were shown (a–d). Scale bar = 300 μm. Results areexpressed as folds of time-matched vehicle control. Histograms represent degree of microglial activation quantified by measuring the density ofIba-1 or CD-11b staining with ImageJ. ***p < 0.001, ****p < 0.0001, and NS compared to respective saline vehicle group. Two-way ANOVAfollowed by Bonferroni post hoc multiple comparison test


upregulated expression of MHC-II and NOX2 mRNAlevels and immunoreactivity of CD-11b during thechronic phase of neuroinflammation (Fig. 4d–g). To-gether, results from in vitro studies are consistentwith our in vivo data and further support the conceptthat the NLRP3-IL-1β signaling pathway is essentialin mediating the transition of acute to chronicneuroinflammation.

Genetic mutation or pharmacological inhibition of NLRP3or IL-1R1 prevents LPS-elicited dopaminergic neuronsdeathStrong emerging evidence supports the notion that sus-tained neuroinflammation drives chronic neurodegener-ation as mentioned in the introduction. Since thedeficiency of NLRP3-IL-1β pathway prevented chronic,but not acute, neuroinflammation after sepsis (Figs. 3, 4,

Fig. 4 Genetic or pharmacological inhibition of NLRP3 or IL-1R1 represses LPS-elicited production of chronic inflammatory mediators in neuron-glial cultures. Neuron-glial cultures prepared from WT, NLRP3−/−, and IL-1R1−/− mice were treated with LPS (20 ng/ml) or vehicle medium. Datawere from three independent experiments. After LPS treatment, supernatant levels of 4 h TNFα (a), 24 h mature IL-1β (b), and 24 h mature IL-1βwith post 6 h addition of MCC950 at 10 μM (c) were detected by ELISA. ***p < 0.001 compared with respective vehicle medium group and NSamong LPS groups. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test for (a, b). One-way ANOVA followed byBonferroni post hoc multiple comparison test for (c). At 4 days after LPS, mRNA levels of MHC-II (d) and NOX2 (e) were measured by qPCR.MCC950 (10 μM) or IL-1Ra (0.5 μg/ml) was post-added to WT neuron-glial cultures at 6 h and 9 h. ND not detectable. At 7 days after LPS andpost-treatment of MCC950 or IL-1Ra, representative images of microglial CD-11b (the α-chain of Mac1, indicating Mac1 expression here)immunostaining (f) and the intensity of CD-11b (g) measured by ImageJ were shown. Bar = 50 μm. d–g NS, #p < 0.001 compared with WT vehiclegroup, and ***p < 0.001 compared with WT LPS group. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test


and Additional file 2: Figure S2), we next studiedwhether blockage of generation or action of IL-1β wouldavoid sepsis-associated neurodegeneration once acuteneuroinflammation began.We first performed an in vitro study by using

mouse primary midbrain neuron-glia cultures pre-pared from WT, NLRP3−/−, or IL-1R1−/− mice as de-scribed above. A 40–50% tyrosine hydroxylase (TH)-immunoreactive (ir) neurons (dopaminergic neurons)loss was found at 7 days after LPS treatment in WTcultures. By contrast, LPS failed to produce significantloss of dopaminergic neurons in either NLRP3−/− orIL-1R1−/− cultures (Fig. 5a, b). Next, following LPStreatment, we post-treated cultures with MCC950 at6 h or IL-1Ra at 9 h after LPS (Fig. 4d, e). The re-sults showed that 7 days later, LPS-elicited loss ofTH-ir neurons was prevented by these two inhibitors(Fig. 5c, d). Therefore, either genetic or pharmaco-logical inhibition of the NLRP3-IL-1β pathwayhampered LPS-induced neurotoxicity. We furtherconducted a rescue study by adding small amounts ofrecombinant IL-1β to NLRP3−/− neuron-glial culturesat 12 h after LPS. Lacking NLRP3 prevented LPS-elicited loss of dopaminergic neurons, whereas addingIL-1β at 40 pg/ml resulted in about 50% dopamin-ergic neuron loss at 7 days in LPS-treated NLRP3−/−

neuron-glial cultures (Fig. 5e, f). Although the amountof mature IL-1β released to the supernatant aremerely ~ 40 pg/ml (about 2.2 × 10−13 M) (Fig. 4b), thislittle amount of IL-1β is sufficient for causing long-lasing neuroinflammation and neuronal loss. The es-sential role of IL-1β in chronic neuroinflammation-mediated neuronal damage was further confirmed inanimal studies. The gradual loss of nigral dopamin-ergic neurons is one of the cardinal features of PD.Ten months after a single injection of LPS, a 30% lossof nigral dopaminergic neuron was found in WT;however, there was no significant difference betweensaline and LPS treatment in NLRP3−/− or IL-1R1−/−

mice (Fig. 6a–c).To provide evidence indicating that oxidative stress re-

sulted from sustained chronic neuroinflammationunderlies the mechanism of sepsis-associated long-termneurotoxicity, we measured changes of protein nitrosyla-tion [68, 69] and α-synuclein phosphorylation at positionSer-129 [70], which are critical for the pathogenesis ofPD [71–73]. Enhanced nitrosylation of proteins in thenigral region was observed in WT, but not in NLRP3−/−

or IL-1R1−/−, mice at 10 months after LPS injections(Fig. 7a and Additional file 4: Figure S4). Besides SN,aggregation of α-synuclein in PD patients can develop indifferent brain regions, such as the hippocampus [74].Prominent increases in the immunoreactivity of Ser-129 phosphorylated α-synuclein in SN and

hippocampus of WT mice at 10 months after LPStreatment was found. By contrast, LPS did not in-crease Ser-129 phosphorylated α-synuclein-ir inNLRP3−/− or IL-1R1−/− mice (Fig. 7b, c).

DiscussionFailure of immune resolution may lead to chronicbrain inflammation and subsequent neurodegenerativediseases, such as Parkinson’s or Alzheimer’s diseases.Elucidation of the molecular mechanism mediatingthe transition from acute to chronic neuroinflamma-tion is paramount to further understanding thepathogenesis of these neurodegenerative diseases.Here, we provide the first evidence indicating thatNLRP3 inflammasome-generated IL-1β from microgliais the key cytokine in governing the transition of neu-roinflammatory phases elicited by severe endotoxemia.NLRP3 gauges the severity of endotoxemia anddetermines the amount of mature IL-1β to producefrom its precursor protein. Excessive release of IL-1βtriggers the transition of acute to chronic neuroin-flammation and eventually results in progressive neu-rodegeneration. Our study not only identified a novelrole of IL-1β in the pathophysiology of endotoxemia-elicited neuroinflammation but also suggested a newstrategy for developing potential therapies for neuro-degenerative diseases. A schematic drawing depictinghow brain IL-1β levels determine the fate of neuroin-flammation induced by endotoxemia is shown inFig. 8.

Roles of IL-1β in neuroinflammationStudies indicate that proinflammatory cytokines/che-mokines, such as TNFα, IL-6, IL-1β, or MCP-1, etc.,released of from activated microglia are importantmediators for sepsis-associated brain inflammation[75]. This study unveiled a distinct role of IL-1β inthe neuroinflammatory process. Unlike TNFα, whichis necessary in triggering acute neuroinflammation[17], IL-1β is dispensable for the initiation of acuteneuroinflammation but is essential for the launch ofchronic neuroinflammation in mice with peripheralLPS administration.Roles of IL-1β in mediating infection-induced acute

sickness behaviors, the pathophysiological interactionsbetween brain and immune systems, are well known[76–79]. However, information is limited regarding in-volvement of IL-1β in the long-term consequences inthe brain after severe systemic infections. Previous re-ports indicate that IL-1β causes neurotoxicity per seor exacerbates pre-existing inflammatory response [80,81]. However, the concentrations of IL-1β used inthese studies were several orders of magnitude higherthan the maximal amount that can be produced from


brain. Thus, the pathophysiological function of IL-1βremains unclear. In this study, we provided strongevidence demonstrating a novel role of this cytokinein sepsis-induced long-term neuropathology. Thisconclusion was mainly based on the results from thefollowing two studies.

High and low doses of peripheral LPS produce similar acuteinflammation, but generate differential outcomes ofsustained chronic inflammation and neurodegenerationA dose-response study of LPS revealed that 1 and5 mg/kg of LPS produced comparable proinflamma-tory cytokine induction and microglial activation in

Fig. 5 Genetic or pharmacological inhibition of NLRP3 or IL-1R1 does not affect TNFα release but protects dopaminergic neurons against LPS-induced toxicity in neuron-glia cultures. Neuron-glial cultures prepared from WT, NLRP3−/−, and IL-1R1−/− mice were treated with LPS (20 ng/ml)or vehicle medium. Data were from three independent experiments. Scale bar = 50 μm. At 7 days after LPS, representative images of THimmunostaining (dopaminergic neurons) (a) and TH-ir neuron number (b) were shown. At 7 days after LPS injection and post-treatment ofMCC950 (10 μM at 6 h) and IL-1Ra (0.5 μg/ml at 9 h), representative images of TH immunostaining (c) and TH-ir neuron number (d) were shown.b, d ****p < 0.0001 and NS compared with respective vehicle medium group. Two-way ANOVA followed by Bonferroni post hoc multiplecomparison test for (b) and one-way ANOVA followed by Bonferroni post hoc multiple comparison test for (d). At 7 days after LPS and post 12 haddition of different amounts of recombinant mouse IL-1β to NLRP3−/− neuron-glial cultures, representative images of TH immunostaining (e)and TH-ir neuron number (f). *p < 0.5 and #p < 0.0001 compared with respective vehicle group. One-way ANOVA followed by Bonferroni post hocmultiple comparison test


acute neuroinflammation (Fig. 1j). Interestingly, weeksor months after LPS injection, sustained chronicinflammation and neuronal loss were observed only inhigh doses of LPS-treated mice (Figs. 3 and 6). Aunique pattern of brain IL-1β expression led us to be-lieve that this cytokine plays a key role in sustaininghigh dose LPS-elicited chronic neuroinflammation.Despite no difference in both mRNA and precursorprotein levels between high and low doses of LPS, wefound that mature IL-1β was the only cytokinedisplaying a dose-dependent increase in the brain(Fig. 1d–i). As will be discussed below, production oflarger amount of brain mature IL-1β is a critical linkbetween higher degree of endotoxemia and chronicneuroinflammation.

Deficiency in NLRP3-IL-1β-IL-1R1 does not prevent theinitiation of sepsis-associated acute neuroinflammation, butimpedes long-term neuroinflammation andneurodegenerationProduction of most cytokines in immune cells istranscriptionally regulated; often translated precur-sor proteins are fully processed and mature cyto-kines are released extracellularly. But the productionof mature IL-1β is an exception. Processing of pre-cursor proteins to mature IL-1β is tightly regulatedby inflammasomes, such as NLRP3 inflammasome;only a portion of the precursor is converted tomature IL-1β. This extra regulatory mechanismserves a critical function in gauging the severity ofassaults.

Fig. 6 Endotoxemia-elicited neurodegeneration is ameliorated in NLRP3−/− or IL-1R1−/− mice. Representative images of TH immunostaining (a) inSN of WT, NLRP3−/−, and IL-1R−/− mice at 10 months after LPS 5 mg/kg or saline i.p injection were shown (n = 3/group). Scale bar = 300 μm. BrainTH-ir neuron number was manually (b) and stereologically (c) counted, respectively, after LPS or vehicle treatment indicated in picture (a). ***p <0.001 and NS compared with respective saline group. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test


We found that injection of LPS (5 mg/kg; i.p.) producedsimilar increases in the expression of several major brainproinflammatory cytokines, such as TNFα, IL-6 (1 h afterLPS injection), and microglial activation (6 h after LPSinjection) during the acute inflammatory phase in WTand mutant (NLRP3−/−, IL-1R1−/−) mice (Fig. 3a and

Additional file 3: Figure S3). By contrast, LPS-inducedchronic inflammatory response and progressive neuronalloss in certain brain regions were found in the WT mice,but not in mutant mice (Figs. 3b–d and 6).Taken together, our findings strongly suggest that the

NLRP3-IL-1β-IL-1R1 signaling does not participate in

Fig. 7 Peripheral LPS injection yields less protein nitrosylation and α-synuclein phosphorylation in NLRP3−/− or IL-1R1−/− mice. At 10 months afterLPS (5 mg/kg, ip) or saline injection, representative images of 3NT immunofluorescence (green) in SN and the intensity of 3NT quantified withImageJ were shown (a). N = 3/group. Bar = 300 μm. Representative images of Ser-129 phosphorylated α-synuclein staining in SN (b) andhippocampus (Hip) (c) and the intensity of Ser-129 phosphorylated α-synuclein staining (brownish staining in the mossy fibers) quantified withImageJ were shown (S129 in the pictures means Ser-129 phosphorylated). N = 3/group. Scale bar = 400 μm (SN) or 300 μm (Hip). ***p < 0.001,****p < 0.001, and NS compared with respective saline group. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test


the initiation of acute neuroinflammation, rather thecritical role of NLPR3-generated IL-1β is to gate thetransition of acute to chronic neuroinflammation.

A permissive role of IL-1β in switching acute self-limitingneuroinflammation to chronic self-propellingneuroinflammationAs mentioned above, the pathophysiological function ofIL-1β during the process of infection-related inflamma-tion in the brain remains unclear. Several observationsfrom this study suggest a permissive role for IL-1β inswitching acute self-limiting neuroinflammation tochronic self-propelling neuroinflammation. Although IL-1β is considered as a proinflammatory cytokine, ourstudy suggests that it is unlikely that IL-1β directly par-ticipates in the formation of acute inflammation. First,blockade of NLRP3-IL-1β-IL-1R1 signaling does not pre-vent the initiation of LPS-induced acute inflammation(Additional file 3: Figure S3 and Fig. 3a). Second, theamount of IL-1β produced by LPS either in cell culturesor in mice brain is several orders of magnitude lowerthan that of most proinflammatory immune factors, suchas TNFα, IL-6, MCP-1, etc. Our study showed that add-ing 80 pg/ml of recombinant IL-1β alone, which is two

times higher than what is measured in vitro, to neuron-glial cultures failed to produce neuronal damage.Instead, adding 40 pg/ml (2.2 × 10−13 M) of IL-1β toLPS-treated neuron-glial cultures from NLRP3-deficientmice fully reinstated LPS-induced progressive dopamin-ergic neuron toxicity (Fig. 5). Third, time course studiesshowed that the release of IL-1β in LPS-simulated cellcultures was behind the other proinflammatory immunefactors and the period of enhanced release of IL-1β wasshort-lived (Fig. 1). Thus, our study strongly suggeststhat depending on the severity of challenge, a corre-sponding amount of IL-1β was strategically released at acritical time point to orchestrate the transition of acuteto chronic phase during the process of infection-relatedinflammation in the brain. Additionally, the permissiverole for IL-1β in switching neuroinflammatory phases issupported by the repression of LPS-induced upregula-tion of late-expressed genes, such as Mac1, NOX2, andMHC-II, in the absence of NLRP3-IL-1β signal (Fig. 4).Aggregated α-synuclein, one of PD hall markers,

triggers microglial NLRP3 inflammasome and the down-stream effector caspase-1 to release mature IL-1β [82].In turn, activated caspase-1 causes truncation andaggregation of α-synuclein [83]. Moreover, inhibition of

Fig. 8 Schematic drawing depicting how brain IL-1β levels determine the fate of neuroinflammation induced by endotoxemia. Initial acuteproinflammatory responses in the brain are similar between mild and severe endotoxemia; however, long-term outcomes are different. Thecurves of microglial activation are represented by TNFα concentrations in brain, because our previous study demonstrated that brain TNFαpeaked at 1 h after LPS i.p. injection and then lasted at low levels in the endotoxemia-elicited neurodegeneration mouse model [17]. The NLRP3-IL-1β pathway gauges the severity of endotoxemia and generates dose-dependent increases of brain mature IL-1β around 7–11 h after LPSinjection. Low levels of brain IL-1β permit the resolution of acute neuroinflammation as occurred in mild endotoxemia. In contrast, high levels ofbrain IL-1β during severe endotoxemia lead to chronic self-propelling neuroinflammation. This figure also suggests that in severe endotoxemia,reducing the production or hampering the function of brain IL-1β during the systemic acute inflammatory stage may provide a therapeuticwindow by preventing the transition of acute to chronic neuroinflammation and lowering the risk of resultant progressive neurodegeneration


NLRP3 inflammasome was found to prevent α-synucleinpathology [84]. It seems that the interplay between ag-gregated α-synuclein and NLRP3 inflammasome is acomponent of the vicious cycle between damagedneuron and reactivated microglia, which is necessary formaintaining chronic self-propelling neuroinflammation.The finding that α-synuclein pathology was greatly di-minished in LPS-injected NLRP3 or IL-1R1 KO mice inthe present study (Fig. 7) adds further evidence indicat-ing the interaction between α-synuclein and NLRP3inflammasome.

The NLRP3-IL-1β-IL-1R1 axis as a potential target for newdrug therapyExperimental or clinical studies show promising thera-peutic results by targeting IL-1β signaling for a broadspectrum of disorders including cardiovascular diseaseand cancer treatment-associated life-threatening cyto-kine release syndrome [85–88]. From a clinical view-point, our study also provides valuable insightsidentifying the NLRP3-IL-1β-IL-1R1 axis as potentialtargets for developing new drug therapies for preventingsevere infection-elicited chronic inflammation and neu-rodegeneration. In line with this possibility, MCC950, asmall molecule of NLRP3 inflammasome inhibitor, cap-able of gaining access to the brain, was reported to exertneuronal protective effects in several animal PD models[84]. It is notable that, besides neurotoxicity, IL-1β alsoplays neuroprotective function under certain experimen-tal conditions. For example, IL-1β was found to partici-pate in both the classical and alternative activation ofmicroglia in a in vivo spinal cord injury mouse model[89]. In vitro, expression of the alternative activationmarkers arginase-1 and Ym1 was increased in primarymicroglial cultures after IL-4 stimulation and further in-creased after co-treatment with IL-4 and IL-1β. There-fore, cautions must be contextually exercised whenconsidering the application of therapeutic strategies totarget IL-1β.Moreover, astroglia can express NLRP3 inflammasome

and IL-1β under certain conditions, such as in SOD1mouse model of amyotrophic lateral sclerosis (ALS) andhuman sporadic ALS patients, and amyloid β1–42-stimu-lated murine astrocytes [90, 91]. We could not rule outthe role of astroglia in LPS-induced sepsis-associatedneurodegeneration. MCC950 also displayed the potencyto repress astroglial NLRP3 inflammasome [91]. Thesefindings highlight the promising application of NLRP3-IL-1β signaling inhibition in various neurodegenerativedisorders.

ConclusionsThis study uncovers a novel role of the NLRP3-IL-1βsignaling pathway in gauging the severity of sepsis-

associated inflammation and determining whether acuteneuroinflammation will resolve or transition to lowgrade chronic neuroinflammation. These findings alsoprovide novel targets for developing therapy for severesystemic infection-related neurodegeneration.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s12974-020-1728-5.

Additional file 1: Figure S1. Lack of dose-response of LPS-elicited pro-duction of IL-1β precursor in the brain. Representative images of westernblot analysis of IL-1β precursor in C57BL/6 J mice brain tissue at indicatedtime after LPS 1 or 5 mg/kg ip injection. Quantification of the westernblot was presented in Fig. 1e.

Additional file 2: Figure S2. ER stress mediates LPS-induced processingof microglial precursor to mature IL-1β. (a) NLRP3 mRNA was measuredby qPCR at indicated time points in mix-glial cultures after LPS 103 ng/mltreatment. Results were from 3 independent experiments performed induplicate. (b) and (c) YVAD, TUDC and 4μ8C were administrated at indi-cated concentrations in mix-glial cultures at 6 h after LPS 103 ng/ml treat-ment. Culture supernatant levels of IL-1β were measured at 24 h. Resultswere from 3 independent experiments. ****p < 0.0001 compared to ve-hicle group and #p < 0.0001 compared to 103ng/ml group. One-wayANOVA followed by Bonferroni post hoc multiple comparison test.

Additional file 3: Figure S3. Deficiency in NLRP3 or IL-1R1 does notprevent brain initial acute inflammatory response. At 1 h after injection ofLPS (5mg/kg, ip) or saline vehicle in C57BL/6 J mice, brain mRNA levels ofTNFα (a), IL-6 (b), MCP-1 (c), and IL-1β (d) were measured by qPCR (n = 4/group). ***p < 0.001 compared with respective saline vehicle group. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test.

Additional file 4: Figure S4. Peripheral LPS injection enhances proteinnitrosylation in WT but not mutant mice. Representative images of TH(red) and 3-NT/TH double (yellow) immunofluorescence double stainingin SN of WT, NLRP3−/−, and IL-1R1−/− mice at 10 months after LPS 5 mg/kg or saline i.p injection (n = 3/group). Bar = 300 μm. The 3-NT stainingpictures and the quantification of 3-NT intensity were shown in Fig. 7.

Abbreviations3-NT: 3-Nitrotyrosine; ER: Endoplasmic reticulum; Iba-1: Ionized calciumbinding adaptor molecule 1; IL-1R1: IL-1 receptor 1; IL-1Ra: Interleukin-1receptor antagonist; IL-1α/1β/6: Interleukin-1α/1β/6; ip: Intraperitoneal;IRE1α: Inositol-requiring enzyme 1α; LPS: Lipopolysaccharide; MCP-1: Monocyte chemoattractant protein-1; MHC-II: Major histocompatibilitycomplex II; NLRP3: Nod-like receptor protein 3; NO: Nitric oxide;PD: Parkinson’s disease; ROS: Reactive oxygen species; TNFα: Tumor necrosisfactor alpha

AcknowledgmentsWe thank Drs Shi-Heng Chen and Lu Zhang for technical supports. We thankDrs Hong Li and Shi-Heng Chen for critical reading of the manuscript. Wethank the Comparative Medicine Branch (CMB) and the Fluorescence Micros-copy and Imaging Center at NIEHS.

Authors’ contributionsJH, JY, YW, and JF conceived and designed the experiments. ZZ, YW, RZ, YL,WB, and SS performed the experiments and analyzed the data. ZZ, YW, andJH wrote the paper, and JY revised it. YG and DT discussed the data andrevised the figures. All authors read and approved the final paper.

FundingThis research was supported in part through the Intramural ResearchProgram at the National Institute of Environmental Health Sciences in theNational Institutes of Health, USA (ZIA ES090082-22); as well as grants fromthe National Natural Science Foundation of China (81270144, 81570084,81970083 and 30800507 to J.F.).


https://doi.org/10.1186/s12974-020-1728-5https://doi.org/10.1186/s12974-020-1728-5

Availability of data and materialsAll data generated or analyzed during this study are included in this article.

Ethics approval and consent to participateAll the use of the animals in this study has been treated humanely and wasperformed in strict accordance with the National Institutes of Healthguidelines.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Respiratory Department, Tianjin Medical University General Hospital, Tianjin300052, China. 2Neurobiology Laboratory, National Institute of EnvironmentalHealth Sciences, National Institutes of Health, Research Triangle Park, NC27709, USA. 3Institute of Infectious Diseases, The Second Hospital of TianjinMedical University, Tianjin 300211, China.

Received: 5 September 2019 Accepted: 28 January 2020

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AbstractBackgroundMethodsResultsConclusions

BackgroundMethodsAnimalsReagentsAnimal treatmentPrimary mouse mesencephalic neuron-glial culturesPrimary mouse mixed glial culturesImmunohistochemistry and double-labeling immunofluorescence of brain slicesImmunocytochemistry of cell culturesReal-time RT-PCR analysisPro-IL-1β Western blot analysisStereological and manual counting assessments of neurodegenerationStatistical analysis

ResultsBrain-delayed mature IL-1β levels, but not initial acute proinflammatory response, are peripheral LPS dose-dependentHigh dose, but not low dose, of peripheral LPS causes long-lasting nigral microglial activationDeficiency of NLRP3 reduces LPS-elicited increase in IL-1β, but not IL-1α, levelsER stress mediates LPS alone-induced NLRP3-dependent IL-1β productionThe NLRP3- IL-1R1 pathway dictates peripheral LPS-elicited transition from acute to chronic neuroinflammationGenetic/pharmacological inhibition of NLRP3 or IL-1R1 represses LPS-elicited production of chronic inflammatory mediators in neuron-glial culturesGenetic mutation or pharmacological inhibition of NLRP3 or IL-1R1 prevents LPS-elicited dopaminergic neurons death

DiscussionRoles of IL-1β in neuroinflammationHigh and low doses of peripheral LPS produce similar acute inflammation, but generate differential outcomes of sustained chronic inflammation and neurodegenerationDeficiency in NLRP3-IL-1β-IL-1R1 does not prevent the initiation of sepsis-associated acute neuroinflammation, but impedes long-term neuroinflammation and neurodegeneration

A permissive role of IL-1β in switching acute self-limiting neuroinflammation to chronic self-propelling neuroinflammationThe NLRP3-IL-1β-IL-1R1 axis as a potential target for new drug therapy

ConclusionsSupplementary informationAbbreviationsAcknowledgmentsAuthors’ contributionsFundingAvailability of data and materialsEthics approval and consent to participateConsent for publicationCompeting interestsAuthor detailsReferencesPublisher’s Note

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