NEURODEVELOPMENT

Astrocyte-derived interleukin-33promotes microglial synapse engulfmentand neural circuit developmentIlia D. Vainchtein,1* Gregory Chin,1* Frances S. Cho,5,7 Kevin W. Kelley,1

John G. Miller,1 Elliott C. Chien,1 Shane A. Liddelow,8† Phi T. Nguyen,1,6

Hiromi Nakao-Inoue,1 Leah C. Dorman,1,5 Omar Akil,3 Satoru Joshita,9,10

Ben A. Barres,8 Jeanne T. Paz,4,7 Ari B. Molofsky,2‡ Anna V. Molofsky1‡

Neuronal synapse formation and remodeling are essential to central nervous system (CNS)development and are dysfunctional in neurodevelopmental diseases. Innate immune signalsregulate tissue remodeling in the periphery, but how this affects CNS synapses is largelyunknown. Here,we show that the interleukin-1 family cytokine interleukin-33 (IL-33) is producedby developing astrocytes and is developmentally required for normal synapse numbers andneural circuit function in the spinal cord and thalamus.We find that IL-33 signals primarilyto microglia under physiologic conditions, that it promotes microglial synapse engulfment, andthat it can drive microglial-dependent synapse depletion in vivo.These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development.

Neuronal synapse formation depends ona complex interplay between neurons andtheir glial support cells. Astrocytes providestructural, metabolic, and trophic supportfor neurons (1, 2). Gray-matter astrocytes

are in intimate contact with neuronal synapsesand are poised to sense local neuronal cues. Incontrast, microglia are the primary immune cellsof the central nervous system (CNS) parenchyma.Microglia regulate multiple phases of develop-mental circuit refinement (3, 4), both inducingsynapse formation (5, 6) and promoting synapseengulfment (7, 8), in part via complement, aneffector arm of the innate immune system (9).Excess complement activity has been implicatedin schizophrenia, a neurodevelopmental disorderthat includes cortical gray matter thinning andsynapse loss (10), suggesting that microglial synap-se engulfment may have broad implications forneuropsychiatric disease.

Despite the emerging roles of astrocytes andmicroglia in neuronal synapse formation andremodeling, how they coordinate synaptic ho-meostasis in vivo remains obscure. Interleukin-33(IL-33) is an IL-1 family member with well-described roles as a cellular alarmin releasedfrom nuclear stores after tissue damage, in-cluding in spinal cord injury (11, 12), stroke (13),and Alzheimer’s disease (14). Whereas many cy-tokines are primarily defined by their roles ininflammation and disease (e.g., IL-1, tumor ne-crosis factor–a, or IL-6), IL-33 also promoteshomeostatic tissue development and remodeling(15). The CNS undergoes extensive synapse re-modeling during postnatal brain development,but a role for IL-33 or other stromal-derived cy-tokines is unknown. Here, we report that IL-33is produced postnatally by synapse-associatedastrocytes, is required for synaptic developmentin the thalamus and spinal cord, and signals tomicroglia to promote increased synaptic engulf-ment. These findings reveal a physiologic require-ment for cytokine-mediated immune signaling inbrain development.We previously developed methods to identify

functionally heterogeneous astrocytes by expres-sion profiling of distinct CNS regions (16). In anRNA-sequencing screen of developing forebrainastrocytes (P9) (flow sorted using an Aldh1l1eGFP

reporter), we identified the cytokine IL-33 as acandidate that is both astrocyte-enriched andheterogeneously expressed by astrocytes through-out the CNS (fig. S1, A to C). We confirmedastrocyte-specific developmental expressionof IL-33 in spinal cord and thalamus using anuclear-localized IL-33 reporter (Il33mCherry/+)(Fig. 1A) and validated these findings with flowcytometry and protein immunostaining (fig. S2).By adulthood, a subset of oligodendrocytes alsocolabeled with IL-33 (fig. S2, C to E), consistentwith previous reports (11). Thus, astrocytes are

the primary source of IL-33 during postnatalsynapse maturation.Althoughmost IL-33–positive cells were astro-

cytes, not all developing astrocytes expressedIL-33, and this number increased in the earlypostnatal period (fig. S3) (17). In fact, IL-33 wasdetected only in gray matter, where most synaps-es are located (Fig. 1B and figs. S2H and S3D).In the thalamus, which receives regionally dis-tinct sensory synaptic inputs, IL-33 expressionin the visual nucleus (dLGN) increased coincidentwith eye opening [postnatal days 12 to 14 (P12to P14)] (Fig. 1, C and D). Removal of afferentsensory synapses by enucleation at birth pre-vented this developmental increase in IL-33 ex-pression (Fig. 1, E and F), whereas dark rearing,in which synapse maturation is largely preserved(18), had no effect. Molecular profiling of IL-33–positive astrocytes in both thalamus and spinalcord (Fig. 1, G and H) revealed a negative cor-relationwithwhite-matter astrocytemarkers (Gfapand Vimentin), enrichment for genes involved inastrocyte synaptic functions (connexin-30/Gjb6)(19), and enrichment in G protein–coupled andneurotransmitter receptors (e.g., Adora2b andAdra2a) (tables S1 to S3 and data S1). Together,these data demonstrate that IL-33 expression iscorrelated with synaptic maturation and marks asubset of astrocytes potentially sensitive to syn-aptic cues, raising the question of whether IL-33plays a role in synapse development.To determine whether IL-33 regulates neu-

ral circuit development and function, we ex-amined the effect of IL-33 deletion on synapsenumbers and circuit activity. In the thalamus,a region with high IL-33 expression, an intra-thalamic circuit between the ventrobasal nucleus(VB) and the reticular nucleus of the thalamus(RT) displays spontaneous oscillatory activity thatcan also be evoked by stimulating the internalcapsule that contains cortical afferents (20, 21).We quantified this oscillatory activity in slicesfrom young adult mice (P30 to P40), whichrevealed enhanced evoked activity in responseto stimulation (Fig. 2, A and B, and fig. S4, Aand B), as well as elevated spontaneous firingin the absence of IL-33 (Fig. 2C and fig. S4C).This increase could result at least in part fromenhanced numbers of glutamatergic synapses.To investigate this hypothesis, we performedwhole-cell patch-clamp recordings of VB neuronsto quantify miniature excitatory postsynapticcurrents (mEPSCs) (Fig. 2D). We found thatthe frequency of mEPSCs was enhanced inVB neurons from IL-33–deficient mice, where-as the amplitude and the kinetics were un-changed (fig. S4D). Together, these results suggestthat IL-33 deficiency leads to excess excitatorysynapses and a hyperexcitable intrathalamiccircuit.In the spinal cord, a-motor neurons (a-MN)

are the primary outputs of the sensorimotor cir-cuit and receive inputs from excitatory (VGLUT2+)and inhibitory (VGAT+) interneurons (Fig. 2E)(22). We conditionally deleted IL-33 from astro-cytes (hGFAPcre) (16) (fig. S5A) and found in-creased numbers of excitatory and inhibitory

RESEARCH

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1Department of Psychiatry/Weill Institute for Neurosciences,University of California, San Francisco, San Francisco, CA, USA.2Department of Laboratory Medicine, University of California,San Francisco, San Francisco, CA, USA. 3Department ofOtolaryngology, University of California, San Francisco, SanFrancisco, CA, USA. 4Department of Neurology, University ofCalifornia, San Francisco, San Francisco, CA, USA.5Neuroscience Graduate Program, University of California, SanFrancisco, San Francisco, CA, USA. 6Biomedical SciencesGraduate Program, University of California, San Francisco, SanFrancisco, CA, USA. 7Gladstone Institute of NeurologicalDisease, San Francisco, CA 94158, USA. 8Department ofNeurobiology, Stanford University, Palo Alto, CA, USA.9Department of Medicine, Division of Gastroenterology andHepatology, Shinshu University School of Medicine, Matsumoto,Japan. 10Research Center for Next Generation Medicine, ShinshuUniversity, Matsumoto, Japan.*These authors contributed equally to this work.†Present address: Neuroscience Institute and Department ofNeuroscience and Physiology, New York University, LangoneMedical School, New York, NY, USA.‡Corresponding author. Email: anna.molofsky@ucsf.edu (A.V.M.);ari.molofsky@ucsf.edu (A.B.M.)

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inputs onto a-MN at P30; global deletion ofIl1rl1 (ST2) (Fig. 2, F to I) or Il33 (fig. S5, B andC) phenocopied this finding. Neuronal somasize, interneuron numbers, and oligodendro-cyte numbers were unchanged (fig. S5, D to F).However, by adulthood, IL-33 deficiency ledto increased gray-matter expression of glialfibrillary acidic protein (GFAP) (fig. S5, G andH), a marker of tissue stress. We also foundthat Il33−/− animals had deficits in acousticstartle response, a sensorimotor reflex mediatedby motor neurons in the brainstem and spinalcord (Fig. 2, J and K) (23). Auditory acuity andgross motor performance were normal (fig. S5,I and J). Taken together, these data demon-strate that IL-33 is required for normal synapsenumbers and circuit function in the thalamusand spinal cord.

To determine the cellular targets of IL-33 sig-naling, we first quantified expression of its ob-ligate co-receptor IL1RL1 (ST2) (15). We detectedIl1rl1 in microglia by RNA sequencing (7.1 ± 2.1fragments per kilobase of transcript per millionmapped reads) and by quantitative polymerasechain reaction (qPCR), in contrast to astrocytes,neurons, or the lineage-negative fraction (Fig. 3A).The transcriptome of acutely isolated microg-lia from Il33−/− animals revealed 483 signifi-cantly altered transcripts, including reducedexpression of nuclear factor kB (NF-kB) targets(e.g., Tnf, Nfkbia, Nfkbiz, and Tnfaip3) (Fig. 3,B and C; fig. S6A; and data S2), consistent withdiminished NF-kB signaling (24). The transcrip-tome of Il33−/− astrocytes was unchanged (fig.S6B), arguing against cell-autonomous rolesof IL-33 in this context. These data demon-

strate physiologic signaling by IL-33 tomicrogliaduring brain development, raising the ques-tion of whether it promotes physiologic microg-lial functions.Given the increased synapse numbers in IL-33–

deficient animals, we investigated whether IL-33is required for microglial synapse engulfment.We detected engulfed PSD-95+ synaptic punctawithin spinal cord microglia throughout devel-opment, as in other CNS regions (7, 8), and founddecreased engulfment in microglia from Il33−/−

animals (P15) (Fig. 3D). This was further validatedby dye labeling of spinal cord motor neurons,which revealed fewer dye-filled microglia inIl33−/− (fig. S7). Conversely, local injection ofIL-33 increased PSD-95 within microglia inboth spinal cord (Fig. 3E) and thalamus (fig.S8, A and B) and altered markers consistent

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astrocytes and oligodendrocyte marker CC1 in spinal cord ventral horn(scale bar, 50 mm). (B) Gray matter restricted expression of Il33lacZ in thespinal cord at P30 (scale bar, 0.5 mm). (C and D) Il33lacZ increases inthe visual thalamus (dLGN) during eye opening, normalized to sensorimotorthalamus (VB) (scale bar, 0.5 mm). (E) Representative images of Il33lacZ inP21 thalamus in littermate controls and after perinatal enucleation (scale bar,

0.5 mm). (F) Il33lacZ mean pixel intensity in dLGN. (G) Representative flowplot of spinal cord from Il33mCherry/Aldh1l1GFP mice at P15 with sortinggates indicated. (H) Heat map of the top 444 differentially expressedgenes in Il33-mCherry+ versus mCherry- astrocytes in spinal cord andthalamus (fold change > 2; adjusted P value < 0.05), select candidateshighlighted. One-way analysis of variance (ANOVA) with Tukey’s post hoccomparison or Student’s t test. All points represent independent biologicalreplicates. *P < 0.05, ****P < 0.0001.

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with microglial activation, including IL1RL1-dependent down-regulation of P2Y12 (fig. S9,A to C) (25). In vitro, IL-33 promoted synapto-some engulfment by purified microglia, whereasthe canonical IL-1 family member IL-1b had noeffect (fig. S9, D and E). In vivo, injection of IL-33 into the developing spinal cord led to twofolddepletion of excitatory synapses (colocalized

VGLUT2/PSD-95), whereas conditional deletionof IL1RL1 from microglia partly reversed thiseffect (Cx3cr1cre:Il1rl1fl/fl) (Fig. 3, F and G). Incomparison, global loss of Il1rl1 completelyreversed IL-33–dependent synapse depletion inspinal cord (fig. S10) and thalamus (fig. S8, C andD), suggesting that nonmicroglial sources of ST2could also contribute. These data indicate that

IL-33 regulates synapse numbers in vivo at leastin part via IL1RL1 receptor–mediated signalingin microglia.Our data reveal a mechanism of astrocyte-

microglial communication that is required forsynapse homeostasis during CNS development.We propose that astrocyte-derived IL-33 servesas a rheostat, helping to tune microglial synapse

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Fig. 2. IL-33 deficiencyleads to excess synapsesand abnormal thalamicand sensorimotor cir-cuit function. (A) Sche-matic of extracellularrecording setup tomeasure circuit activitybetween ventrobasal(VB) and reticular tha-lamic (RT) nuclei, withrepresentative recordingshowing activity in fivechannels after stimula-tion of the internalcapsule (i.c.) thatcontains cortical affer-ents. Red arrows indicatereciprocal VB-RT connec-tions. (B) Average tracesand quantification ofmean firing rates revealhigher evoked firing inIl33−/−. (C) Quantificationof mean firing rates in theabsence of stimulationreveals increasedspontaneous firing inIl33−/−. (D) Representativetraces and quantificationof intracellular patch-clamp recordingsfrom neurons in theVB show increased mini-ature excitatory post-synaptic currents(mEPSCs) in Il33−/−.(E) Schematic of motorneuron synaptic afferents.(F and G) Representativeimage and quantificationof excitatory inputsper motor neuron afterconditional deletionof Il33 (hGFAPcre) orglobal deletion of Il1rl1.(H and I) Inhibitory (VGAT+)inputs in the same mice(scale bar, 25 mm).(J) Schematic of startlepathway. (K) Impairedsensorimotor startle in Il33−/− animals. Data in (B) and (C) from wild type: n = 6 to 8 slices, 2 mice. Knockout (KO): n = 14 to 15 slices, 3 mice; pointsare individual recordings. Data in (B) analyzed by Mann-Whitney and (C) with Student’s t test. Data in (D) from n = 23 to 25 cells and 3 to 4 mice pergroup, analyzed by Kolmogorov-Smirnov test. Data in (F) to (I) from n = 3 animals, >75 neurons per genotype, Student’s t test; points are individualneurons. (K) is n = 12/group, two-way ANOVA with Sidak’s multiple comparisons. *P < 0.05, **P < 0.01, ****P < 0.0001. (B) to (I) are mean ± SD; (K) ismean ± SEM.

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engulfment during neural circuit maturation andremodeling (fig. S11). Key unanswered questionsinclude the nature of the cues that induce astro-cyte Il33 expression, the mechanism of IL-33release, and the signals downstreamof IL-33 thatpromote microglial function. These data alsoraise the broader question of how this processaffects neural circuit function. Synapses are themost tightly regulated variable in the develop-

ing CNS (26) and are a primary locus of dys-function in neurodevelopmental diseases. Il33is one of five genes that molecularly distinguishastrocytes from neural progenitors in the devel-oping human forebrain (27), suggesting possiblyconserved roles in the human CNS. Definingwhether signals like IL-33 are permissive orinstructive, promiscuous or synapse specific,is a first step toward understanding how neural

circuits remodel during development and understress.

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Cxcl10Cxcl10Cxcl10Cxcl2Cxcl2Cxcl2NfkbiaNfkbiaNfkbia

Cd83Cd83Cd83

Gadd45bGadd45bGadd45b S100a8S100a8S100a8

PD

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+/V

GLU

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PD

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IL-33IL-33vehiclecrepos:Il1rl1fl/flcreneg creneg

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Fig. 3. IL-33 drives microglial synapse engulfment during develop-ment. (A) Expression of Il1rl1 by qPCR of flow-sorted populations(S, spinal cord; T, thalamus). (B) A total of 484 differentially expres-sed genes in spinal cord microglia at adjusted P value < 0.05.(C) Functionally associated gene clustering (STRING) identifiesimmune genes enriched in wild-type versus Il33−/− microglia.(D) PSD-95 puncta within microglia (yellow arrows) after IL-33deletion (scale bar, 4 mM). (E) Representative image and quantifica-tion of engulfed PSD-95 in vehicle or IL-33 injected spinal cord

(scale bar, 20 mm). (F and G) Colocalized pre- and postsynapticpuncta in spinal cord ventral horn at P14 (yellow arrows) afterIL-33 injection into control mice or in littermates with conditionaldeletion of Il1rl1 (Cx3cr1cre) (scale bar, 3 mm). Points in (A) representmice; in (D) to (G), individual microglia from n = 3 to 5 animalsper group; in (G), images from n = 5 mice per group. In (D) and(E), Student’s t test and in (G), a one-way ANOVA with Tukey’s posthoc comparison. *P < 0.05, ***P < 0.001, ****P < 0.0001; all dataare mean ± SD.

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ACKNOWLEDGMENTS

We are grateful to the Molofsky laboratories, the Poskanzerlaboratory, D. H. Rowitch, R. M. Locksley, and J. R. Chan forhelpful comments on the manuscript. Thanks to theJ. Huguenard laboratory for the thalamic network analysiscode, J. P. Girard for Il33lacZ mice, R. T. Lee for Il33fl/fl andIl1rl1fl/fl, M. Colonna and the Mucosal Immunology Studies Team(MIST) for Il33H2B-mCherry, D. Julius for the P2Y12 antibody,and the Gladstone Genomics and Behavioral Cores(P30NS065780) for technical contributions. Funding: A.V.M.is supported by a Pew Scholars Award, NIMH (K08MH104417),the Brain and Behavior Research Foundation, and the BurroughsWellcome Fund. A.B.M. is supported by the National Instituteof Diabetes and Digestive and Kidney Diseases (K08DK101604)and the Larry L. Hillblom Foundation. J.T.P. is supportedby the National Institute of Neurological Disorders and Stroke(R01NS096369). S.A.L. is supported by the AustralianNational Health and Medical Research Council (GNT1052961)and the Glenn Foundation Glenn Award. F.S.C. (NSF 1144247)and P.T.N. (NSF 1650113) were supported by GraduateStudent Fellowships from the National Science Foundation.Author contributions: I.D.V., G.C., A.V.M., and J.G.M. designed,performed, and analyzed most experiments. E.C.C., H.N.-I.,

P.T.N., and L.C.D. contributed to experiments and data analysis.F.S.C. and J.T.P. designed, performed, and analyzed theelectrophysiology experiments. K.W.K. and I.D.V. designedand performed bioinformatics analyses. S.A.L. performedand analyzed culture experiments under the supervision ofB.A.B. O.A. performed and analyzed auditory testing.S.J. generated Il33H2B-mCherry mice. A.V.M. and A.B.M. designedexperiments and wrote the manuscript, together with I.D.V.,G.C., and other authors. Competing interests: None declared.Data and materials availability: Supplementary materialscontain additional data. All data needed to evaluate theconclusions in the paper are present in the paper or thesupplementary materials. RNA-sequencing data are availablethrough GEO no. GSE109354.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/359/6381/1269/suppl/DC1Materials and MethodsFigs. S1 to S11Tables S1 to S3Data S1 and S2References (28–49)

8 November 2016; resubmitted 4 December 2017Accepted 17 January 2018Published online 1 February 201810.1126/science.aal3589

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developmentAstrocyte-derived interleukin-33 promotes microglial synapse engulfment and neural circuit

Anna V. MolofskyNguyen, Hiromi Nakao-Inoue, Leah C. Dorman, Omar Akil, Satoru Joshita, Ben A. Barres, Jeanne T. Paz, Ari B. Molofsky and Ilia D. Vainchtein, Gregory Chin, Frances S. Cho, Kevin W. Kelley, John G. Miller, Elliott C. Chien, Shane A. Liddelow, Phi T.

originally published online February 1, 2018DOI: 10.1126/science.aal3589 (6381), 1269-1273.359Science

, this issue p. 1269Sciencebrain circuitry.mice, disruptions in this process, as caused by deficiency in IL-33, led to too many excitatory synapses and overactiveAstrocytes near a redundant synapse release the cytokine interleukin-33 (IL-33), which recruits microglia to the site. In

found that the microglia are called into action by astrocytes, supportive cells on which neurons rely.et al.Vainchtein are pruned away, leaving mature circuits. Synapses can be eliminated by microglia, which engulf and destroy them.

The developing brain initially makes more synapses than it needs. With further development, excess synapsesCall to action

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