Significance Dysfunction of the innate immune system is involved in the pathogenesis of Alzheimer’s disease (AD); however, the pathophysiological mechanisms underlying these dysfunctions are unclear. Here we report that stimulation of IL-33/ST2 signaling rescues memory deficits and reduces the accumulation of β-amyloid in APP/PS1 mice that exhibit select pathologies associated with AD. Although impaired IL-33/ST2 signaling is associated with early progression of AD, IL-33 injection rescues contextual memory deficits and reduces the accumulation of β-amyloid in APP/PS1 mice. IL-33 skews the microglia toward an alternative activation state with enhanced Aβ phagocytic capacity and elevated antiinflammatory gene expression, which results in a decreased proinflammatory response in the brain. Thus, this study suggests that IL-33 can be developed as a new therapeutic intervention for AD.

Abstract Alzheimer’s disease (AD) is a devastating condition with no known effective treatment. AD is characterized by memory loss as well as impaired locomotor ability, reasoning, and judgment. Emerging evidence suggests that the innate immune response plays a major role in the pathogenesis of AD. In AD, the accumulation of β-amyloid (Aβ) in the brain perturbs physiological functions of the brain, including synaptic and neuronal dysfunction, microglial activation, and neuronal loss. Serum levels of soluble ST2 (sST2), a decoy receptor for interleukin (IL)-33, increase in patients with mild cognitive impairment, suggesting that impaired IL-33/ST2 signaling may contribute to the pathogenesis of AD. Therefore, we investigated the potential therapeutic role of IL-33 in AD, using transgenic mouse models. Here we report that IL-33 administration reverses synaptic plasticity impairment and memory deficits in APP/PS1 mice. IL-33 administration reduces soluble Aβ levels and amyloid plaque deposition by promoting the recruitment and Aβ phagocytic activity of microglia; this is mediated by ST2/p38 signaling activation. Furthermore, IL-33 injection modulates the innate immune response by polarizing microglia/macrophages toward an antiinflammatory phenotype and reducing the expression of proinflammatory genes, including IL-1β, IL-6, and NLRP3, in the cortices of APP/PS1 mice. Collectively, our results demonstrate a potential therapeutic role for IL-33 in AD.

Alzheimer’s disease (AD) is the most common type of dementia in the elderly population, and its prevalence is increasing with the rapidly growing global elderly population (1). AD is characterized by progressive memory loss and other cognitive dysfunctions, such as impaired locomotor ability, reasoning, and judgment (2, 3). The hallmarks of AD pathology include the presence of extracellular amyloid plaque deposits, composed of Aβ peptides, and the intracellular formation of neurofibrillary tangles in the brain, together with synaptic dysfunction and neuronal loss.

The mechanisms underlying the onset and progression of AD remain unclear. Mutations of amyloid precursor protein and presenilins that result in abnormal production of Aβ peptides are suggested to play a dominant role in the pathogenesis of early-onset familial AD (4). Identification of several innate response genes in genome-wide association studies support the idea that innate immunity is involved in the more common sporadic late-onset form of AD (LOAD) (4).

Studies of transgenic mouse models of AD suggest the impairment of microglial function to reduce Aβ burden as a causal factor in the disease (5). Specifically, in AD, microglial activation attempts to eliminate Aβ accumulation by enhancing Aβ phagocytosis, clearance, and degradation; however, the continuous generation and aberrant accumulation of Aβ in the brain causes microglial dysfunction by reducing Aβ receptors and increasing the generation of inflammatory mediators (5). Chronic neuroinflammation induced by microglia contributes to pathological progression and symptom severity in late stages of the disease. Importantly, although sometimes contradictory, modulation of the immune system by specific interleukin signals such as IL-12/IL-23 and IL-10, as well as the inflammasome pathway, ameliorates AD-like pathology (6⇓⇓–9).

IL-33, an alarmin of the IL-1 family, is a crucial mediator of the innate immune response and a regulator of immune cell infiltration and activation (10). IL-33 is expressed in various cell types (10, 11). IL-33 binds to a heterodimeric receptor complex, comprising ST2 and IL-1RAcP, and triggers the intracellular cascade involve myeloid differentiation factor 88 (MyD88) and NF-κB. This signaling selectively activates type 2 T-helper cells, mast cells, neutrophils, and alternatively-activated macrophages (10). In the central nervous system (CNS), IL-33 is constitutively expressed in oligodendrocytes (12), whereas ST2 is expressed mainly in microglia and astrocytes (13). IL-33 is a pleiotropic cytokine with diverse functions in various infectious and inflammatory diseases that may exert beneficial or detrimental effects on the disease (10, 14). Genetic studies have identified three single nucleotide polymorphisms in the IL-33 gene that are associated with a reduced risk of AD development (15). IL-33 transcript levels are decreased in the brains of LOAD cases (15). How IL-33 is involved in the pathogenesis of AD, in particular whether replenishment of IL-33 can alleviate AD pathology, remains unclear, however.

Here we show that IL-33 treatment reverses synaptic plasticity and cognitive deficits in APP/PS1 mice, an AD mouse model that exhibits selected pathologies of AD, including amyloid plaque deposition and cognitive impairment. IL-33 reduces soluble Aβ and amyloid plaque load by promoting the recruitment and phagocytic activity of microglia. Furthermore, IL-33 injection modulates the immune response of APP/PS1 mice and polarizes microglia/macrophages toward an antiinflammatory phenotype. Collectively, our results demonstrate the potential therapeutic role of IL-33 in AD.

Discussion There is no effective therapy for AD, in part because of our limited knowledge of its underlying pathophysiological mechanisms. Nonetheless, manipulation of the innate immune system has been considered a promising approach for the development of an effective therapeutic strategy for AD. The present study demonstrates that peripheral IL-33 administration in AD mouse models alleviates AD-like pathology by enhancing microglial phagocytosis and degradation of Aβ. Furthermore, IL-33 treatment switches the microglia/macrophages to an alternative activation phenotype, resulting in a reduction of the proinflammatory response in the AD mouse brains. Considered along with the beneficial effects of IL-33 on synaptic and memory functions in the AD-like mouse model, our results suggest that IL-33 administration can be developed as a potential therapeutic intervention for AD. IL-33 is a pleiotropic cytokine whose roles depend on inflammation sites and stimulant types. IL-33/ST2 signaling plays a protective role in some diseases caused by sterile inflammation (where the activation of innate immunity is not triggered by infection), such as atherosclerosis and obesity (10, 14). In atherosclerosis, IL-33 reduces atherosclerotic plaque formation by reducing macrophage foam cell formation and increasing production of the antiinflammatory cytokine IL-5 and antioxidized low-density lipoprotein antibody (33, 34). Similarly, Aβ-stimulated sterile inflammation in AD also plays an important role in disease pathogenesis (35). IL-33 expression is reduced in the brains of patients with AD (15), and sST2 is increased in the serum of patients with MCI, as demonstrated in the present study (Fig. 1A). These results raise the intriguing possibility that impaired IL-33/ST2 signaling contributes to the nonresolution of sterile inflammation, resulting in amyloid plaque deposition, chronic neuroinflammation, and increased symptom severity in AD. Indeed, our findings show that replenishment of IL-33 is able to reduce the AD-like pathology in the brains of APP/PS1 mice. What are the underlying mechanisms that mediate the ability of IL-33/ST2 signaling to attenuate AD pathology? Clearance of Aβ is controlled by complex mechanisms, and its dysregulation has been suggested to be a major disease culprit (36). The first responders to Aβ accumulation are the microglia, which are recruited to amyloid plaques. Interestingly, the Aβ-clearing capacity of microglia in APP/PS1 mice is impaired with aging, as revealed by reduced expression of Aβ-binding receptors and Aβ-degrading enzymes (37). In particular, it has been suggested that microglia become inefficient in phagocytosing Aβ during AD progression. IL-33 specifically enhances Aβ uptake by microglia. The increased numbers of CD68+ phagocytic microglial cells around amyloid plaques in APP/PS1 mice after IL-33 treatment suggests that IL-33 is able to restore the phagolysosomal activity of microglia and promote Aβ clearance. Furthermore, treatment of primary adult microglia with IL-33 enhances Aβ uptake, whereas abolishing ST2 expression in microglia reduces this uptake. These findings suggest that IL-33–dependent activation of ST2 signaling in microglia enhances Aβ phagocytosis and clearance. Further phenotypic characterization of this microglial subset that exhibits increased Aβ phagocytic capacity will provide insight into the mechanisms underlying the IL-33–stimulated clearance of Aβ. In particular, recent identification of markers specific for resident microglia may facilitate further analysis of microglial regulation by IL-33 (38). Direct degradation of Aβ by endopeptidases is another important pathway for its clearance (39). Exogenous expression of one of the enzymes, neprilysin, in the brains of AD transgenic mice causes a reduction of Aβ oligomers, the major species causing synaptotoxicity (40), whereas reducing the activity of neprilysin in the APP/neprilysin−/− double transgenic mice results in accumulation of Aβ, specifically the oligomeric form (41). The increase of neprilysin protein in the brains of APP/PS1 mice after IL-33 administration (Fig. 4 A and B) provides insight into the mechanism of the enzymatic degradation of Aβ in the IL-33–treated condition. On exposure to Aβ during the progression of AD, microglia are converted into proinflammatory and activated phenotypes. Activated microglia result in Aβ accumulation by enhancing the production of Aβ peptides and reducing its phagocytosis and degradation (22). Along with recruiting monocytes to lesion sites, IL-33 acts as an alarmin for the polarization of macrophages toward the alternative phenotype after CNS injury (42). Indeed, we show that IL-33 administration causes an increase in the antiinflammatory genes Arg1 and Fizz1 in microglia/macrophages in the brain, suggesting that IL-33 enhances the polarization of microglia/macrophages to an alternative activation phenotype. How does IL-33 modulate the functions of microglia/macrophages? Toll-like receptors (TLRs), including TLR2 and TLR4, have been identified as innate pattern recognition receptors that mediate the proinflammatory response (43, 44). TLR signaling via the adaptor protein MyD88 mediates inflammatory responses, such as inducing microglial phagocytic activity in early stages of inflammation and increasing transcription of genes encoding IL-1 family cytokines (44, 45). Indeed, inhibition of TLR2 or TLR4 reduces Aβ-stimulated microglial production of IL-1β and IL-6 (46, 47). IL-33/ST2 signaling acts as a negative regulator of TLR signaling by competing with MyD88 (48, 49). Specifically, ST2 activation recruits MyD88, which leads to MAPK activation for gene transcription (10). Whether this mechanism mediates the suppression of proinflammatory gene production in IL-33–treated APP/PS1 mice awaits further study. It is tempting to speculate that relatively high levels of IL-33 in the CNS of healthy individuals (50) may be a key innate factor that protects against AD. The breakdown of this fail-safe mechanism owing to genetic disposition or environmental pressure thus may contribute to the onset of AD. Indeed, genetic studies have linked IL-33/ST2 single nucleotide polymorphisms to clinical AD (15, 51), suggesting that our observations in the murine AD model could be extrapolated to humans. Recent clinical trials of AD treatments have been unsuccessful, likely because the regimens aimed to bind monoclonal antibodies to Aβ or to prevent further Aβ accumulation at a late disease stage (52). Our finding that IL-33 mobilizes innate immunity to prevent and clear established Aβ accumulation even at late stages of the disease represents a new treatment paradigm for AD.

Methods Reagents. Murine recombinant IL-33 was obtained from Biolegend (580506). sST2-Fc recombinant protein (1004-MR-050) and the ST2/IL-1 R4 Quantikine ELISA Kit (DST200) were purchased from R&D Systems. Antibodies specific for IL-33 (clone Nessy-1; ab54385) and APP (clone DE2B4; ab11132) were purchased from Abcam. Iba1 antibody (019-19741) was purchased from Wako; APC-conjugated antibody against CD11b (clone M1/70) and FITC-conjugated antibody against CD45 (clone 30-F11), from eBioscience; CD68 antibody (FA-11), from AbD Serotec; monoclonal 4G8 antibody (recognizes Aβ 17–24; SIG-39220) for immunohistochemistry and 6E10 antibody (recognizes Aβ 1–16; SIG-39320) for Western blot analysis, from Covance; insulin-degrading enzyme antibody (PC730), from Calbiochem; neprilysin antibody (H-321), from Santa Cruz Biotechnology; P-p38 (4511), p38 (9212), P-ERK1/2 (4377), and ERK1/2 (9102) antibodies, from Cell Signaling Technology; and actin antibody (clone AC-40; A4700), from Sigma-Aldrich. Fluorescein-beta-amyloid (1–42) (A-1119-1) was obtained from rPeptide; methoxy-X04 (4920), from Tocris Bioscience; DAB, from DAKO; Hoechst 34580 (H21486), human Aβ x–40 (KHB3481), and Aβ x–42 (KHB3441) commercial ELISA kits and Alexa Fluor secondary antibodies, from Life Technologies; and the histamine ELISA kit (ENZ-KIT140-0001), from Enzo Life Sciences. Oligomeric Fluorescein-Aβ 1–42 Preparation. Monomeric Aβ 1–42 was dissolved in dry DMSO and diluted in ice-cold phenol red-free F-12 medium to a final concentration of 100 μM. The fluorescein-Aβ 1–42 solution was incubated at 4 °C for 24 h and then centrifuged at 14,000 × g for 10 min. The supernatant was frozen in liquid nitrogen as aliquots and stored at −20 °C for up to 1 mo (18). Human Subjects. All cases involved Han Chinese patients. Clinical diagnosis was established according to criteria published by the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer’s Disease and Related Disorders Association (2). The cases of MCI were gathered from outpatient or inpatient clinics at the Department of Neurology, Second Affiliated Hospital of the Zhejiang University School of Medicine, and the control group without dementia was recruited from the Health Examination Center between 2014 and 2015. Data on age, sex, education, medical history, and family history were recorded as well (SI Appendix, Table S1). The sera of 17 healthy controls (NC) and 18 patients of mixed sex with MCI were collected. All of the NC and patients with MCI recruited were age ≥60 y. Patients with other neurologic or psychiatric disorders or clinically significant medical conditions were excluded. No patient had a history of head trauma or alcohol or drug abuse. This study was approved by the Institutional Ethics Committees of the Second Affiliated Hospital of the Zhejiang University School of Medicine and The Hong Kong University of Science and Technology (HKUST). Informed consent for participation in this study was obtained either directly or from a legal guardian. Mice. The APP/PS1 double-transgenic mice, generated by incorporating a human/murine APP construct bearing the Swedish double mutation and the exon-9–deleted PSEN1 mutation (APPswe + PSEN1/dE9) (16), were obtained from the Jackson Laboratory (B6C3-Tg[APPswe, PSEN1dE9]85Dbo/J). The generation of 5XFAD mice overexpressing the K670N/M671L (Swedish), I716V (Florida), and V717I (London) mutations in human APP (695), as well as M146L and L286V mutations in human PS1, has been described previously (53). The 5XFAD mice were kindly provided by Sookja Kim Chung, The University of Hong Kong, Pokfulam, Hong Kong. The St2−/− mice were obtained from Andrew McKenzie, University of Cambridge, Cambridge, United Kingdom (54). Genotypes were confirmed by PCR analysis of tail or ear biopsy specimens. WT C57BL/6 mice were obtained from Jackson Laboratory. All mice were housed in the HKUST Animal and Plant Care Facility, and all animal experiments were approved by the HKUST Animal Ethics Committee. Mice of the same sex were housed four per cage with a 12-h light/dark cycle and food and water ad libitum. All in vivo experiments were performed on sex- and age-matched groups. Mice of both genders were used for experiments. The mice were assigned at random to the experimental conditions. Sample sizes were chosen primarily on the basis of experience with similar types of experiments. All of the animal experiments were conducted in the light phase. In Vivo Experiments. Murine recombinant IL-33 (200 ng per mouse) was injected i.p. into 6- to 25-mo-old mice. For studies of amyloid plaque load, microglial phagocytosis of Aβ, mRNA expression, and LTP, mice were injected with IL-33 for 2 consecutive days. The field excitatory postsynaptic potentials were recorded using a MED64 multichannel recording system (Panasonic International). For behavioral tests, mice were injected i.p. with IL-33 as indicated in Fig. 1 D and E. The open field test and contextual fear conditioning were conducted as described previously (55, 56). Soluble ST2 (sST2) was delivered into APP/PS1 mice via mini-osmotic pumps (model 1004; Alzet) at 0.11 µL/h. The min-iosmotic pumps were adjusted intracerebroventricularly in the right hemisphere. The pumps were loaded with murine recombinant sST2 protein (240 ng per pump; 10 µg/mL) or with vehicle in artificial cerebrospinal fluid (aCSF; 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl 2 ·2H 2 O, 1 mM NaH 2 PO 4 ·2H 2 O, 1.3 mM MgCl 2 ·6H 2 O, 26.2 mM NaHCO 3 , 11 mM d-glucose). After 2 d of sST2 delivery, IL-33 was administered i.p. to mice for another 2 d. At the end of the treatment, the mice were killed and their brains fixed with 4% paraformaldehyde. LTP. In brief, after the mice were killed, whole brains were immediately resected and soaked in ice-cold aCSF supplemented in 95% O 2 /5% CO 2 . Brain slices (300 μm) were then prepared using a vibratome (HM650V; Thermo Fisher Scientific) and soaked in oxygenated aCSF for 2 h at 32 °C. The mouse hippocampal slices were placed on a precoated polyethylenimine (Sigma-Aldrich) on a MED–P210A probe (Panasonic International) fabricated with 8 × 8 electrode arrays (20 × 20 μm, indium tin oxide and platinum black) with a 100-μm interelectrode distance. Electrodes were manually placed at the CA3–CA1 region under a microscope (MIC-D; Olympus). Each slice was submerged in and superfused with oxygenated aCSF at 1.3–1.5 mL/min. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the dendritic layer of CA1 neurons by selecting an electrode in the Schaffer collateral pathway as the stimulating electrode. Based on the stimulus–response curve, a stimulation intensity that evoked an fEPSP with 30–40% of the maximum response was selected. LTP was induced by three trains of high-frequency stimulation (100 Hz for 1 s, delivered 30 s apart). The field potential response after the tetanus was recorded for 60 min. The magnitude of LTP was quantified as the percentage change in the average slope of the fEPSP over 60 min after LTP induction (18). Open Field Test. The locomotor activity of the mice in the open field test was recorded and tracked using a photobeam activity system and software (San Diego Instruments). In brief, experimental mice were placed in the center of an open-top chamber (16 × 16 × 15 inches) with an array of photobeams around the periphery. The mice were allowed to explore the chamber for 15 min each day for 3 consecutive days. Locomotor activity in the three training trials was recorded by the photobeams, and the distance moved was subsequently determined. The chamber was cleaned with 70% ethanol after each trial. Contextual Fear Conditioning. On the day of training, the mice were allowed to explore in an enclosed training chamber with the floor wired to an electric shock generator for 180 s. The mice were then exposed to a pure tone for 30 s, followed by a 2-s foot shock (0.8 mA). At 60 s after delivery of the second shock, the mice were returned to their home cages. The fear response (freezing) was assessed at 1 d and 7 d after the electric shock. The mice were re-exposed in the original chamber for 5 min, and the time of freezing was measured using the Contextual NIR Video Fear Conditioning System (Med Associates). Immunohistochemistry. Coronal cryosections (20 μm) of perfused mouse brains were used for immunohistochemistry. For 4G8 immunostaining, antigen retrieval was performed by microwave heating of the sections in sodium citrate buffer (10 mM trisodium citrate, 0.5% Tween-20 in H 2 O, pH 6.0) for 10 min, followed by incubation with 3% H 2 O 2 in H 2 O for 5 min to inhibit endogenous peroxidase activity. The sections were blocked with 2% goat serum in Tris-buffered saline for 20 min and then labeled with 4G8 antibody (dilution 1:500) in blocking buffer overnight at 4 °C. The sections were then labeled with a Dako HRP-linked goat anti-mouse/rabbit IgG antibody for 50 min at room temperature and developed with a Dako DAB chromogen kit. Imaging was performed using a Leica DM6000 B compound microscope system. The area of Aβ plaques in the cortex of the brain sections was analyzed by the “Analyze Particles” function of ImageJ. The region of the cortex used for amyloid plaque analysis was above the hippocampus. Three brain sections per mouse (∼200–300 μm apart) were analyzed, and the average percentage of cortical area occupied by amyloid plaques was calculated. To examine the colocalization of amyloid plaques and microglia, the brain sections were blocked with 1% BSA, 4% goat serum, and 0.4% Triton X-100 in Dulbecco’s PBS (DPBS) for 30 min at room temperature and then incubated with Iba1 (1:500 dilution) and 4G8 (1:1,000 dilution) antibodies overnight at 4 °C. Imaging was performed using a Leica TCS SP8 confocal system. Quantification of the 3D-colocalization was conducted using the Imaris software (Bitplane). To determine the blood–brain barrier penetrability of IL-33, 11-mo-old APP/PS1 mice were injected i.p. with IL-33 (200 ng) in DPBS or DPBS alone. Mouse cortices and sera were collected for IL-33 ELISA. Aβ Extraction, Western Blot Analysis, and ELISA. Aβ was sequentially extracted from the soluble and insoluble fractions of the mouse cortex (16, 18). Frozen brain tissues were lysed in indicated lysis buffers with various protease inhibitors (18). In brief, the mouse cortex was homogenized in buffer containing 250 mM sucrose, 20 mM Tris⋅HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, and protease inhibitor mixture (Sigma-Aldrich) or in radioimmunoprecipitation assay buffer. Densitometric quantification of protein band intensity from Western blot analysis was performed using ImageJ. Soluble Aβ was sequentially extracted by diethylamine (soluble), followed by formic acid (insoluble). Levels of soluble and insoluble Aβ were determined by Western blot analysis. Soluble Aβ x–40 and Aβ x–42 were analyzed by ELISA. Isolation of Mouse Microglia. Adult mice were anesthetized and perfused with ice-cold DPBS. The isolated forebrain were cut into small pieces and dissociated enzymatically and mechanically using a papain-based neural dissociation kit (130-092-634; Miltenyi Biotec) with a gentleMACS dissociator system (Miltenyi Biotec) according to the manufacturer’s instructions. Percoll gradient (30%; Sigma-Aldrich) was used to remove myelin. The resultant mononuclear cell suspensions were used for flow cytometry analysis or for preparation of microglial cultures (57). The purity of microglia/infiltrating monocyte isolation was routinely >90% as determined by fluorescence staining for CD11b and flow cytometry analysis. Analysis of Microglial Phagocytosis of Aβ. For in vivo analysis, the experiment was performed as described previously (29). APP/PS1 or WT mice (17 mo old) were injected i.p. with IL-33 in PBS or PBS alone daily for 2 consecutive days. The next day, the mice were injected i.p. with methoxy-X04 (10 mg/kg). The mice were deeply anesthetized at 3 h after methoxy-X04 injection and perfused in the left ventricle with ice-cold PBS. Isolated mononuclear cell suspensions from the mouse forebrain were then incubated with a mouse FcR blocking reagent (Miltenyi Biotec) and labeled with APC-conjugated mouse CD11b (1:100 dilution) and FITC-conjugated CD45 (1:100 dilution) antibodies and analyzed using a BD FACSAria IIIu flow cytometer. Myeloid cells were identified as CD11b+ cells and were further classified as microglia or infiltrating monocytes based on low (CD11b+CD45lo) or high (CD11b+CD45hi) CD45 expression, respectively (29). For the in vitro microglial phagocytosis assay, microglia cells were prepared from St2+/+ or St2−/− mice (1.5 to 2 mo old) and cultured for 6–7 d in vitro. The cells were then pretreated with IL-33 for 20 h, followed by fluorescein-Aβ 1–42 (2 μM) for 1 h. The cells were then fixed with paraformaldehyde and immunostained with Iba1 antibody and Hoechst 34580. Wide-field fluorescence microscopy was performed using an IN Cell Analyzer 6000 system (GE Healthcare). Images were acquired at 16 corresponding positions assigned by the software in each well. Intensity segmentation was applied to each channel using the same thresholding criteria across different samples. The boundaries of microglia were determined according to Iba1+ labeling intensity, and fluorescein-Aβ 1–42 in each Iba1+ cell was quantified. The area of phagocytosed fluorescein-Aβ 1–42 was divided by the corresponding cell area to obtain the relative area of fluorescein-Aβ 1–42 in a given cell. Droplet Digital PCR and qRT-PCR. For droplet digital PCR (ddPCR), RNA from mouse cortices was extracted using TRIzol (Invitrogen) and the RNeasy Mini Kit (Qiagen) and quantified using a BioDrop μLITE microvolume spectrophotometer. Equivalent amounts of RNA were reverse-transcribed using the PrimeScript RT-PCR Kit (TaKaRa). ddPCR was performed according to the manufacturer’s protocol (Bio-Rad). The copy numbers for samples were averaged across duplicates. The copy numbers of target genes were normalized to those of GAPDH or β-actin. For qRT-PCR, cDNA after reverse transcription was preamplified using TaqMan PreAmp Master Mix (Invitrogen). The PCR amplification and real-time detection of PCR products were performed using TaqMan gene expression assay (Applied Biosystems) and Premix Ex Taq qPCR assay (TaKaRa). PCR analyses were conducted in a total volume of 20 µL containing 2 µL of preamplified product. The mRNA expression values were normalized to the level of β-actin, GAPDH, or CD11b as indicated. The following TaqMan probes were used: NLRP3 (Mm00840904_m1), IL-1β (Mm01336189_m1), IL-6 (Mm00446190_m1), IL-13 (Mm00434204_m1), TGFβ (Mm01178820_m1), GAPDH (Mm99999915_g1), β-actin (Mm02619580_g1), ST2 total (Mm00516117_m1), Fizz1 (Mm00445109_m1), Arg1 (Mm00475988_m1), and CD11b (Mm00434455_m1). Statistical Analysis. The investigators who performed the electrophysiological, immunohistochemical, RNA expression, and flow cytometry analyses and the behavioral tests were blinded to the genotypes of the mice and treatment conditions. All data are expressed as arithmetic mean ± SEM, except for human sST2 serum level. All statistical analyses were performed using GraphPad Prism version 6.0. The significance of differences was assessed by the unpaired Student’s t test or one- or two-way ANOVA followed by the Bonferroni post hoc test as indicated. The level of significance was set at P < 0.05. Soluble ST2 levels were recorded for NC subjects and patients with MCI, with data analyses performed using R version 3.2.2. The Shapiro–Wilk test was used to assess the normality of the continuous measurements. Statistical comparisons of ST2 levels in the two subject groups were compared using a two-sample t test.

Acknowledgments We thank Dr. Yu Chen, Dr. Yang Shen, Dr. Fanny C. F. Ip, Dr. Terry C. T. Or, Estella P. S. Tong, Elaine Y. L. Cheng, Minty M. Tian, Kit Cheung, Fion C. Y. Chuang, and William Chau for their excellent technical assistance; Dr. Maggie M. K. Chu for statistical analysis; and members of the N.Y.I. laboratory for many helpful discussions. We also thank Dr. Sookja Kim Chung for the 5XFAD mice, Dr. Richard Ransohoff for expert advice on the preparation and isolation of microglia, and Drs. Karl Herrup and Gary Landreth for helpful discussions. This study was supported in part by the Research Grants Council of Hong Kong SAR (C6003-14G), the National Key Basic Research Program of China (2013CB530900), a Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), and the S. H. Ho Foundation.

Footnotes Author contributions: A.K.Y.F., K.-W.H., M.Y.F.Y., W.-Y.F., F.Y.L., and N.Y.I. designed research; K.-W.H., M.Y.F.Y., X.Z., D.S.Y.M., and I.C.W.C. performed research; T.H.C., B.Z., F.Y.L., and N.Y.I. contributed new reagents/analytic tools; A.K.Y.F., K.-W.H., M.Y.F.Y., W.-Y.F., F.Y.L., and N.Y.I. analyzed data; and A.K.Y.F., W.-Y.F., F.Y.L., and N.Y.I. wrote the paper.

Reviewers: D.M., University of South Florida; and L.A.J.O., Trinity College.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1604032113/-/DCSupplemental.