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◥ cytes under the control of the Gfap promoter. RESEARCH ARTICLE SUMMARY These studies identified several axon guidance molecules as critical mediators of microglia- NEUROSCIENCE astrocyte interactions in the context of in- flammation. By combining RABID-seq with Barcoded viral tracing of single-cell interactions in genetic perturbation studies in vivo, valida- tion with primary mouse and human cells in central nervous system inflammation vitro, and the analysis of MS patient samples by immunostaining and single-cell RNA-seq, Iain C. Clark†, Cristina Gutiérrez-Vázquez†, Michael A. Wheeler†, Zhaorong Li, Veit Rothhammer, we established that microglia-astrocyte inter- Mathias Linnerbauer, Liliana M. Sanmarco, Lydia Guo, Manon Blain, Stephanie E. J. Zandee, actions mediated by Sema4D-PlexinB1, Sema4D- Chun-Cheih Chao, Katelyn V. Batterman, Marius Schwabenland, Peter Lotfy, Amalia Tejeda-Velarde, PlexinB2, and Ephrin-B3–EphB3 promote CNS Patrick Hewson, Carolina Manganeli Polonio, Michael W. Shultis, Yasmin Salem, Emily C. Tjon, pathology in EAE—and potentially MS. Nota- Pedro H. Fonseca-Castro, Davis M. Borucki, Kalil Alves de Lima, Agustin Plasencia, Adam R. Abate, bly, Ephrin-B3–EphB3 participated in forward Douglas L. Rosene, Kevin J. Hodgetts, Marco Prinz, Jack P. Antel, and reverse signaling, which boosted both Alexandre Prat, Francisco J. Quintana* microglia and astrocyte pathogenic activities via the regulation of nuclear factor kBand mammalian target of rapamycin, respectively. INTRODUCTION: Glial cells of the central nervous ing method for the identification of thousands Finally, we demonstrated that a CNS-penetrant system (CNS), including astrocytes and microg- of CNS cell interactions in vivo and the simul- small-molecule inhibitor of the kinase activity lia, play critical roles in development, tissue taneous analysis of the transcriptome of in- of the EphB3 intracellular domain amelio- Downloaded from repair, and homeostasis. However, dysregulated teracting cells with single-cell resolution. We rates EAE in both acute and chronic progres- astrocyte and microglia responses contribute to applied this technique, named rabies barcode sive models. the pathogenesis of neurologic diseases. Indeed, interaction detection followed by sequencing environmental chemicals, microbial metabo- (RABID-seq), to the study of microglia-astrocyte CONCLUSION: We developed RABID-seq, a lites, and cell-cell interactions have been shown communication in the context of CNS inflam- novel approach for the simultaneous investi- to modulate disease-promoting responses in mation in EAE and MS. gation of cell interactions and the transcrip- http://science.sciencemag.org/ astrocytes and microglia in the context of mul- tome of interacting cells in vivo with single- tiple sclerosis (MS) and its model, experimen- RESULTS: To develop RABID-seq, we engineered cell resolution. RABID-seq identified signaling tal autoimmune encephalomyelitis (EAE). In an mRNA-barcoded library in glycoprotein G– pathways controlled by the axon guidance particular, although astrocyte interactions with deficient pseudorabies virus (RabDG-BC), which molecules Sema4D-PlexinB1, Sema4D-Plex- microglia are known to play important roles in spreads between interacting cells but can only inB2, and Ephrin-B3/EphB3 as mediators the pathology of MS and other neurologic dis- replicate in cells that transgenically express viral of microglia-astrocyte interactions that pro- eases, the pathways that facilitate astrocyte- glycoprotein G. We pseudotyped the RabDG-BC mote CNS pathogenesis and also as candidate microglia cross-talk are poorly understood, plasmid library using envelope of sub- targets for therapeutic intervention in neuro- and consequently, few therapeutic inventions group A (EnvA) packaging. Thus, the pseudo- logic disorders.▪

are available to target them. typed virus only infects cells that transgenically express the EnvA receptor, TVA. After its rep- on April 22, 2021 RATIONALE: Understanding the complexity of lication in cells that express TVA and viral glyco- The list of author affiliations is available in the full article online. astrocyte-microglia cross-talk in CNS inflam- protein G, RabDG-BC infects interacting cells, †These authors contributed equally to this work. mation requires the study of precise neuro- labelling them with the virus-encoded bar- *Corresponding author. Email: [email protected] Cite this article as I. C. Clark et al., Science 372, eabf1230 immune interactions in vivo, but methodologies code. To study RABID-seq astrocyte interac- (2021). DOI: 10.1126/science.abf1230 for defining the specific cell types, pathways, tions in vivo during CNS inflammation in the and molecules that mediate these interactions EAE model of MS, we used transgenic mice READ THE FULL ARTICLE AT are limited. We developed a virus-based barcod- expressing glycoprotein G and TVA in astro- https://doi.org/10.1126/science.abf1230

Elucidation of microglia- astrocyte interactions by EAE scRNA-seq rabies barcode interaction detection followed by sequencing (RABID-seq). Pseudotyped rabies virus EAE score expressing barcoded mRNA tar- Time gets Gfap+ astrocytes, where it replicates before infecting Microglia Microglia EPHRINB3 neighboring cells, leaving a bar- Astrocyte coded trace. Single-cell RNA EPHB3 sequencing reads both cellular mRNAs and viral barcodes, allowing for the reconstruction of in vivo cell interactions and the PLXNB1/2 SEMA4D A38 Astrocyte EPHB3 EPHRINB3 Inflammation and transcriptional analysis of interact- neurodegeneration ing cells with single-cell resolution.

Clark et al., Science 372, 360 (2021) 23 April 2021 1of1 RESEARCH

◥ as it spreads between interacting cells, allow- RESEARCH ARTICLE ing the reconstruction of cellular cross-talk in vivo via scRNA-seq. By encoding spatial NEUROSCIENCE relationships directly into the transcriptome, RABID-seq detects cell interactions that other- Barcoded viral tracing of single-cell interactions in wise would not be detected by single-cell pro- filing alone. Using RABID-seq, we identified central nervous system inflammation the axon guidance molecules Sema4D-PlexinB2 and Ephrin-B3–EphB3 as mediators of microglia- Iain C. Clark1,2†, Cristina Gutiérrez-Vázquez1†, Michael A. Wheeler1,3†, Zhaorong Li1,3, astrocyte interactions that promoted CNS Veit Rothhammer1,4, Mathias Linnerbauer1,4, Liliana M. Sanmarco1, Lydia Guo1, Manon Blain5, pathology in EAE. Moreover, we identified a Stephanie E. J. Zandee6, Chun-Cheih Chao1, Katelyn V. Batterman7, Marius Schwabenland8, CNS-penetrant small-molecule inhibitor of Peter Lotfy1,3, Amalia Tejeda-Velarde1‡, Patrick Hewson1, Carolina Manganeli Polonio1, EphB3 signaling that ameliorates acute and Michael W. Shultis1, Yasmin Salem1, Emily C. Tjon1, Pedro H. Fonseca-Castro1, Davis M. Borucki1, chronic progressive EAE. In summary, RABID- Kalil Alves de Lima1, Agustin Plasencia1, Adam R. Abate9,10, Douglas L. Rosene7, Kevin J. Hodgetts1, seq provides an approach for the comprehen- Marco Prinz8,11,12, Jack P. Antel5, Alexandre Prat6, Francisco J. Quintana1,3* sive investigation of cell interactions in the CNS with single-cell resolution, identifying Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study microglia-astrocyte interactions and candi- astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by date targets for therapeutic intervention in sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA- neurologic disorders. seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia- Downloaded from astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis RABID-seq overview (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in RabDG is a powerful tool for studying cell vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D interactions because it can be targeted to and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, specific cell types, including astrocytes and respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia other glia (21–25) (Fig. 1A). To study astrocyte proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia- cell interactions, we engineered the RabDG http://science.sciencemag.org/ astrocyte interactions and candidate therapeutic targets. virus to express barcoded mCherry (RabDG- mCherry-BC). Because barcode sequences are inserted before the transcriptional stop of the strocytes are central nervous system (CNS)– In the context of autoimmune CNS disorders polyadenylated mCherry transcript, the tran- resident glial cells with important roles such as multiple sclerosis (MS) and its pre- scribed mRNA barcode can be analyzed by in health and disease. Astrocyte functions clinical model, experimental autoimmune en- scRNA-seq (Fig. 1B). In addition, mCherry allows A in development, homeostasis, and disease cephalomyelitis (EAE), astrocyte activation the isolation by flow cytometry of fluorescently are controlled by cell interactions (1–10). is modulated by T cells and other peripheral labeled barcoded cells in a RabDG-transduced For example, astrocyte interactions with mi- immune cells recruited to the inflamed CNS cell network (Fig. 1C), enabling the simultaneous – croglia regulate synaptic pruning (11), neuro- (3, 6, 10, 12 16). However, the full extent of analysis of cell transcriptomes and RabDGbar- degeneration (2), and CNS inflammation (12). cell interactions that control astrocyte responses codes by high-throughput droplet-based scRNA- on April 22, 2021 and the molecular mechanisms involved are seq (Fig. 1D). poorly understood. The investigation of those After amplification and sequencing, we de- 1Ann Romney Center for Neurologic Diseases, Brigham and interactions is further complicated by the het- tected ~1.5 million unique sequences in the Women’s Hospital, Harvard Medical School, Boston, MA erogeneity of astrocytes and other cell types, as barcoded RabDG-mCherry-BC plasmid library 2 02115, USA. Department of Bioengineering, University of well as the need to define the specific cell sub- (fig. S1, A to C). We pseudotyped the rabies California, Berkeley, California Institute for Quantitative Biosciences, Berkeley, CA 94720, USA. 3Broad Institute of sets participating in interactions of interest. virus from the barcoded RabDG-mCherry-BC MIT and Harvard, Cambridge, MA 02142, USA. 4Department High-throughput genomic approaches such plasmid library using envelope protein of sub- of Neurology, University Hospital Erlangen, Friedrich– as single-cell RNA sequencing (scRNA-seq) and group A (EnvA) packaging, which only infects Alexander University Erlangen–Nürnberg, 91054 Erlangen, Germany. 5Neuroimmunology Unit, Montreal Neurological spatial transcriptomics can profile thousands cells expressing the EnvA receptor TVA and Institute, Department of Neurology and Neurosurgery, McGill of individual cells, but challenges remain in thereby allows the genetic targeting of cells of University, Montreal, QC H3A 2B4, Canada. applying these approaches to study cell inter- interest in vivo (21, 25, 26). Because the result- 6Neuroimmunology Research Laboratory, Centre de Recherche du Centre Hospitalier de l’Université de Montréal actions. Moreover, although some techniques ing pseudotyped rabies virus library was esti- 4 5 (CRCHUM), Montreal, QC H2X 0A9, Canada. 7Department of can profile immune cell interactions (17, 18) mated to contain 10 to 10 unique barcodes (fig. Anatomy and Neurobiology, Boston University School of and cell networks based on the sequencing of S1D), we predicted that 91 to 99% of infected 8 Medicine, Boston, MA 02118, USA. Institute of microdissected units (19) or the use of photo- cells will be uniquely barcoded if 1000 cells were Neuropathology, University of Freiburg, D-79106 Freiburg, Germany. 9Department of Bioengineering and Therapeutic activatable markers (20), these approaches initially infected with the pseudotyped RabDG- Sciences, University of California, San Francisco, California cannot easily profile cell interactions in the mCherry-BC virus library (Fig. 1, E and F). Institute for Quantitative Biosciences, San Francisco, CA CNS and may fail to detect interactions in- We used an in vitro system to confirm that 94158, USA. 10Chan Zuckerberg Biohub, San Francisco, CA, USA. 11Signaling Research Centres BIOSS and CIBSS, volving only a small subset of cells. infection with pseudotyped RabDG-mCherry- University of Freiburg, D-79106 Freiburg, Germany. 12Center We developed rabies barcode interaction BC virus is restricted to TVA-expressing cells for Basics in NeuroModulation (NeuroModulBasics), Faculty detection followed by sequencing (RABID-seq) (Fig. 1, G to I) and developed a polymerase of Medicine, University of Freiburg, D-79106 Freiburg, Germany. to identify astrocyte cell interactions and chain reaction (PCR)–based strategy for ampli- *Corresponding author. Email: [email protected] the molecular phenotypes of interacting cells fying rabies connection barcodes from cDNA †These authors contributed equally to this work. in vivo. RABID-seq uses glycoprotein G–deficient generated by the inDrop workflow (Fig. 1J and ‡Present address: Immunology Department, Hospital Universitario Ramón y Cajal, Madrid, Spain, Instituto Ramón y Cajal de pseudorabies virus (RabDG) engineered to fig. S1, E and F). Notably, RabDG-mCherry-BC Investigación Sanitaria (IRYCIS), Madrid, Spain. express a fluorescent mRNA-encoded barcode sequencing libraries retain three crucial pieces

Clark et al., Science 372, eabf1230 (2021) 23 April 2021 1of15 RESEARCH | RESEARCH ARTICLE

Fig. 1. Reconstruction A In vivo RabΔG barcoded viral tracing B RabΔG barcode structure of single-cell tran- RabΔG-mCherry-BC scriptomes and con- Barcode 1 nectomes using RABID-seq. (A) Bar- Stop coded RabDG virus is codon delivered via intracranial 5’ mCherry PCR handle Barcode PCR handle pA 3’ injection, and barcodes Barcode 2 V: not T VHDBVHDBATVHDBVHDBATVHDBVHDB transfer to neighboring H: not G D: not C Rabies-encoded barcode cells as RabDG virus Time B: not A 2.8x1011 possible combinations spreads throughout C D E F interacting cells. Flow cytometry isolation Single cell RNA-sequencing n=1000 cells labeled 91-99% CI 1.0 1.0 (B) The RabDG genome 0.9 Cells seeded 0.9 500 5000 0.8 1000 10000 inDrop maximum expresses mCherry, 0.8 (in 12h) 2000 15000 capture efficiency 0.7 3000 20000 Oil 0.7 4000 which enables the inDrop v3 0.6 0.6 0.5 recovery and sequencing 0.5 0.4 of virus-infected cells. 0.4 0.3 mCherry 0.3

Fraction uniquely labeled k=100 The mCherry transcript 0.2 k=1000 0.2

k=10000 Fraction of network captured 0.1 0.1 harbors a unique bar- k=100000 0.0 0.0 FSC RT reagents Oil 1 10 100 1000 10000 100000 1000000 0.5 1 2 4 8 16 32 64 128 256 code with semirandom Cells labeled Connections per cell structure, flanked by Downloaded from Pseudotyped RabΔG-mCherry-BC-EnvA (pseudotyped) constant regions to G HIPseudotyped RabΔG EnvA EnvA facilitate amplification. 40 *** lengths are not WT TVA WT TVA to scale. (C) Flow ) HEK293 HEK293-TVA 30 cytometry recovery of 1.03% 35.8% HEK293

+ http://science.sciencemag.org/ mCherry cells from the HEK293-TVA 20 CNS. FSC, forward scatter. (D) Single-cell RNA- + EnvA EnvA 10 sequencing of mCherry mCherry mCherry+ (percent cells. RT, reverse 0 transcription. (E)Fraction 293 293-TVA of uniquely labeled cells J FSC cDNA whole transcriptome amplification RabΔG barcode recovery as a function of RabDG mCherry PCR handle primer 5+N cycles index primer 10 cycles barcode library diversity inDrop PCR RabΔG PCR Cell priming ... TSO mCherry handle barcode handle UMI barcode site 2 and number of cells RabΔG Cell TTTTTT barcode inDrop forward barcode inDrop transduced. (F)Fraction TTTTTT

cDNA primer index on April 22, 2021 of the in vivo network TTTTTT primer TTTTTT scRNA-seq library prep captured using inDrop TTTTTT cDNA ... (maximum 60% cell inDrop inDrop forward reverse primer cDNA primer Adapter ligation Cell Transcriptome library barcode inDrop capture rate over a index primer index maximum period of primer 12 hours of encapsulation) K Transcriptome analysis Connectome reconstruction Connectome and scRNA linkage Interactome discovery Pro-inflammatory Anti-inflammatory as a function of the num- interactions interactions ber of connections that Cells each cell makes for different numbers of vs transduced cells. (G to I)RabDGpseudo-

typing for cell targeting. tSNE2 Astrocyte T cell Macrophage (G) Schematic of RabDG tSNE1 Networks Single-cell transcriptomes Pro-inflammatory Monocyte Microglia pseudotyping workflow astrocyte and cell infectability. WT, wild type. (H) Fluorescence-activated cell sorting (FACS) analysis showing that WTA material is further amplified using a two-step approach with mCherry-specific pseudotyped virus only infects HEK293-TVA cells in vitro. HEK293, human embryonic primers followed by PCR primers targeting the constant region flanking the barcode. kidney–293 cells. (I) Percent of HEK293 or HEK293-TVA cells infected with (Bottom right) Sequencing libraries are prepared from WTA product to produce pseudotyped RabDGvirus.n = 4 samples per group. Unpaired two-tailed t test. scRNA-seq libraries. TSO, template-switching oligonucleotide. (K)Linkageoftran- (J) Generation of scRNA-seq libraries from inDrop using a SMART-seq approach scriptome and connectome data enables reconstruction of genome-wide transcrip- with template switching and whole-transcriptome amplification (WTA). (Top right) tional signatures of interacting cells in vivo. Data shown as means ± SEM. ***P < 0.001. of information: (i) a scRNA-seq cell barcode to and (iii) a unique molecular identifier (UMI) to scRNA-seq. Interactions between cells were assign RabDG rabies barcodes to single-cell count RabDG barcode transcripts(Fig.1J).After determined by the presence of shared barcodes transcriptome data, (ii) a unique rabies barcode sequencing, barcodes were identified, counted, (fig. S1, G to H), allowing for the reconstruc- structure that allows efficient error correction, and associated with individual cells captured by tion of cellular networks with genome-wide

Clark et al., Science 372, eabf1230 (2021) 23 April 2021 2of15 RESEARCH | RESEARCH ARTICLE transcriptional information in vivo at single- removed oligodendrocytes and neurons to en- natures and chemokine-mediated signaling in cell resolution (Fig. 1K). Thus, RABID-seq able us to focus on astrocytes, microglia, and microglia connected to astrocytes displaying a allows the high-throughput analysis of single- infiltrating immune cells. We built astrocyte- high proinflammatory phenotype (>90th per- cell interactions in vivo. centric cell interaction networks by including centile) (Fig. 2, I and J). Indeed, the analysis only rabies barcodes that were detected in at of ligand-receptor interactions (34)between RABID-seq detects astrocyte cell interactions least one astrocyte, limiting potential confound- these high–proinflammatory phenotype astro- in naïve and EAE mice ing effects that could result from Cre leakiness. cytes (>90th percentile) and microglia in peak We used transgenic mice expressing the ra- In naïve mice, we detected astrocyte-astrocyte EAE recapitulated previous reports (12)ofin- bies glycoprotein G and the EnvA receptor interactions, as well as interactions with microg- creased proinflammatory FLT1 signaling and (TVA) under the control of the Gfap promoter lia and other cells. In EAE mice at the peak of decreased aryl hydrocarbon receptor–driven (GfapTVA/G mice) (27) to target the initial in- disease, astrocyte interaction networks were anti-inflammatory responses in astrocytes trig- fection of pseudotyped RabDG-mCherry-BC more diverse (Fig. 2, D and E) and included gered by microglia-produced VEGF-B (vascular virus to astrocytes and limit the subsequent interactions with peripheral cells, such as endothelial growth factor B) (Fig. 2, K and L) transfer of barcodes (Fig. 2A and fig. S2A). T cells, that were recruited to the inflamed (12). The microglial phagocytosis of cell debris Specifically, the RabDG-mCherry-BC virus CNS (3, 13, 16, 30). containing mCherry protein did not generate a can only initially infect Gfap+ astrocytes ex- We developed an inflammation score based fluorescentsignalstrongenoughtomaskthe pressing TVA, which also express the rabies on the activation of the inflammatory response signal generated by the RabDG-based tracing glycoprotein G. New viral particles produced defined by the Ontology initiative (fig. S8, of cell interactions (35)(fig.S2D). by Gfap+ astrocytes incorporate the rabies A to C, and data S1). We then selected astro- We next used RABID-seq to identify mech- glycoprotein G into their envelopes, thereby cytes with the highest (>90th percentile) and anisms of microglia-astrocyte communication acquiring the ability to infect and barcode lowest (<10th percentile) proinflammatory in EAE mice at the peak of disease, detecting Downloaded from neighboring cells. However, because Gfap− transcriptional phenotypes in EAE that dis- the activation of pathways associated with axon neighboring cells do not express the rabies played interactions with T cells (fig. S8D). guidance molecules (Fig. 3A and fig. S9A). Axon glycoprotein G, they cannot further dissemi- Astrocytes with the highest proinflammatory guidance molecules play important roles dur- nate the virus (28, 29). This approach genet- scores were connected to T cells that ex- ing development but are co-opted by tumors ically targets Gfap+ astrocytes for initial hibited proinflammatory phenotypes and high and inflammatory processes in the periphery barcoding, revealing astrocyte-centric cell tumor necrosis factor–a (TNFa) signaling via (36). Thus, we examined axon guidance pathways http://science.sciencemag.org/ interaction networks. Using this system, we nuclear factor kB(NF-kB) (136 astrocytes, 506 associated with -plexin, ephrin-EPH, detected the spread of RabDG-mCherry-BC by T cells, 3796 connections) (fig. S8, E and F, and netrin, and Slit/Robo signaling in the microglia- flow cytometry, which peaked 7 days after data S2), in agreement with the reported in- astrocyte cell networks that we identified in naïve transduction (fig. S2B). In validation studies, crease in proinflammatory astrocyte responses and peak-EAE mice. The analysis of the single-cell we confirmed the expression of TVA/G in by proinflammatory T cells (3). Conversely, RABID-seq dataset detected the activation of astrocytes in GfapTVA/G mice (fig. S2C), titered T cells connected to astrocytes displaying Sema4D/PlexinB during peak EAE, driven by the RabDG-mCherry-BC virus to seed 1000 cells the lowest proinflammatory phenotype (132 Sema4d expression in microglia and Plxnb2 in GfapTVA/G mice upon infection (fig. S2, astrocytes, 684 T cells, 3847 connections) expression in astrocytes (Fig. 3, B to D, and D and E), and detected RabDG-mCherry-BC showed higher expression of molecules asso- fig. S9B). We made similar observations during

spread to neighboring cells, such as microglia ciated with the suppression of inflammation the priming phase of EAE (fig. S9, C and D) (fig. S2F). Next, we transduced the forebrain (e.g., Ctla4, Ikzf4, Il2ra,andIl10) (fig. S8G). and when studying Plxnb2+ astrocyte inter- on April 22, 2021 of naïve GfapTVA/G mice or EAE GfapTVA/G Indeed, when we analyzed subnetworks of actions with Sema4d+ monocytes during peak mice with RabDG-mCherry-BC virus 7 or Il10ra+ astrocytes and Il10+ T cells (fig. S8H), EAE(fig.S9,FtoH).Wealsodetectedsig- 12 days after immunization with MOG35-55 we detected interleukin-2 (IL-2)–signal trans- nificant activation of Ephrin-B–mediated sig- to target priming and peak EAE, respectively ducerandactivatoroftranscription5(STAT5) naling (Fig. 3, B and C). (Fig. 2B). At 7.5 days after transduction, we signaling pathways that have been associated sorted mCherry+ cells by flow cytometry (fig. with regulatory T cells (fig. S8I) (31), recapit- Microglia-astrocyte Sema4D-PlexinB2 S3A) and analyzed them by scRNA-seq (figs. ulating IL-10–driven anti-inflammatory effects signaling promotes CNS inflammation in EAE S3, B to F; S4; and S5). Cell types were evenly of T cells on astrocytes (32, 33). Thus, RABID- Sema4D signals through the PlexinB1 and distributed across each sample analyzed by seq can be used to simultaneously identify PlexinB2 receptors (36, 37), but the analysis of RABID-seq in naïve and EAE mice (figs. S3 to astrocyte cell interactions and the transcrip- the RABID-seq dataset detected higher Plxnb2 S5). We detected RabDG-mCherry–encoded tional features of interacting cells at the single- expression in astrocytes during EAE (Fig. 3, D barcodes in all samples analyzed and con- cell level. and E, and fig. S9B). Thus, to investigate the firmed sufficient sequencing depth for all role of Sema4D-PlexinB2 signaling during EAE, samples (fig. S6 and S7, A to C). Specifically, Identification of microglia-astrocyte signaling we analyzed the single-cell transcriptional sig- we detected an average of 1000 barcodes per via axon guidance molecules by RABID-seq natures of interacting Sema4d+ microglia and mouse across the eight mice analyzed (fig. S6, Microglia-astrocyte interactions play important Plxnb2+ astrocytes detected by RABID-seq. We D to F), consistent with our target seeding roles during CNS development, homeostasis, subdivided our RABID-seq data into nonover- rate (fig. S2E), previously set to minimize and disease (2, 11, 12). However, a comprehen- lapping networks of Plxnb2+ or Plxnb2− astro- barcode collisions (Fig. 1, E and F). In vitro sive understanding of these interactions and cytes, connected to Sema4d+ or Sema4d− studies suggested that early- versus late- how they shift during inflammation is still microglia (Fig. 3, F to H). Plxnb2+ astrocytes seeded cells could be distinguished on the missing (Fig. 2F). RABID-seq detected microg- interacted preferentially with Sema4d+ microg- basis of barcode UMI abundance (figs. S7D). lial control of astrocytes mediated by IL-1, TNF, lia (Fig. 3, F and G), exhibiting increased We used scRNA-seq to analyze 32,280 RabDG- and C1q in EAE mice relative to naïve mice, activation of semaphorin-plexin signaling con- barcoded cells, including astrocytes, microglia, consistent with previous reports (2)(Fig.2,G comitant with increased activation of proin- monocytes, and T cells, from naïve or peak- and H, and data S3 to S5). Moreover, we de- flammatory responses (Fig. 3, H to J, and data EAE mice (Fig. 2C). Our cell isolation method tected the activation of proinflammatory sig- S6). In addition, the analysis of a published

Clark et al., Science 372, eabf1230 (2021) 23 April 2021 3of15 RESEARCH | RESEARCH ARTICLE

Fig. 2. RABID-seq A Mouse transgenic B Gfap(Cre/+); C D schematic Gfap(Cre/+);gp4-IRES-TVA(f/+) mice analysis of astro- gp4-IRES-TVA(f/+) mice 7.5dpi Astrocyte-connected cell types Astrocyte connectivity Gfap promoter Cre recombinase 4 cyte cell interac- peak Cell types: Naive Cell types: Peak EAE Naive transduced sac tions in naïve Cre loxP loxP 3 priming T cells, 0.2% (n=19) and EAE mice. STOP CAG G-IRES-TVA transduced sac Astrocytes 20% (A) Transgenic loxP 2 (n=1,961) CAG G-IRES-TVA Endothelial/ mouse line gener- EAE score Epithelial cells, ( 4.8%46) n=1,465) 3 TVA 1 ated to target (n=6, 1 Microglia, 6 Peak EAE Gfap-expressing Gfap+ 5% tSNE2 0 tSNE2 cells with the 51015 10,409 cells 21,871 cells Endothelial/ Epithelial cells, Day tSNE1 tSNE1 21.2% (n EnvA-TVA system. =4,239) Astrocytes 8.3% Endothelial/epithelial cells Fibroblast-like (n=1,652) Astrocytes Macrophages (B) EAE disease ) D endritic cells, 23.3 Dendritic cells Microglia (n ls, 4.1% (n=829)875 = Granulocytes Monocytes T cel n= 4, % ( 662)

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were harvested c F 7.5 days after Ma Fibroblast-like infection for Macrophages Microglia scRNA-seq. Monocytes T cells Downloaded from Error bars indi- Endothelial/epithelial cells Astrocytes Dendritic cells cate mean ± SEM. Granulocytes (C) t-distributed stochastic neigh- bor embedding FGAstrocyte-microglia peak EAE networks HAstrocyte-MG peak EAE networks vs. other cells: (tSNE) plots of Astrocytes (n=1,652) Microglia (n=6,891) TNF, IL-1β, IL-1α, C1q single-cell RABID- Naive http://science.sciencemag.org/ seq data from Regulators naïve and peak- EAE mice. The number of cells that passed bio- informatic filters is EAE displayed near the TNF: p-value: 6.96e-39, z-score: 3.803 IL-1β: p-value: 2.15e-21, z-score: 1.879 origin. (D)Circos IL-1α: p-value: 2.54e-08, z-score: 0.149 plots of astrocyte Network heterogeneity C1q: p-value: 3.41e-05, z-score: 1.453

cell interactions in IJKAstrocyte-microglia peak EAE networks Up in Down in Up in microglia connected to >90th on April 22, 2021 naïve and peak- connected connected >90th percentile inflamed astrocytes to top 10% to top 10% percentile inflamed astrocytes EAE mice. Percent- inflamed inflamed Astrocytes (n=163) Microglia (n=3,799) ages are shown Hallmark: Inflammatory response GO: Molecular function ID: M5932 Chemokine receptor binding (GO:0042379) relative to the total Cytokine activity (GO:0005125) Phospholipase activator activity (GO:0016004) number of connec- Lipase activator activity (GO:0060229) NES: 2.08 Collagen binding (GO:0005518) tions. n is the FDR: 0.008 Endopeptidase activity (GO:0004175)

Microglia 0123 number of cells -log10(p-value) of each cell type. GO: Cellular response to cytokine stimulus L (E) Network rep- GO: 0071345 Vegfb Flt1 resentation of CellPhoneDB: astrocyte cell NES: 2.04 FDR: 0.006 p-value: 0.032 interactions. To Microglia Microglia Astrocyte provide a sense of scale, increasingly smaller portions of the network are selected and enlarged. Statistical analysis: right-tailed Fisher’s exact test. (I) Visualization of >90th Cells are colored by cell type, as determined using scRNA-seq data. (F to percentile proinflammatory astrocyte-microglia subnetworks. (J) GSEA (gene set K) Analysis of astrocyte-microglia interactions during peak EAE by RABID-seq. enrichment analysis) preranked analysis of scRNA-seq data comparing microglia (F) Schematic of heterogeneous interactions between astrocytes and microglia connected to >90th percentile proinflammatory astrocytes versus microglia during EAE. (G) Network representation of astrocyte-microglia interactions connected to <10th percentile proinflammatory astrocytes. (K) Analysis by gene detected by RABID-seq. (H) IPA (ingenuity pathway analysis) network analysis of ontology: molecular function of microglia connected to >90th percentile single-cell RABID-seq data showing predicted upstream regulators in astrocytes proinflammatory astrocytes. (L) CellPhoneDB identification of VEGFB-FLT1 connected to microglia (MG) versus astrocytes connected to other cells. signaling between microglia and >90th percentile proinflammatory astrocytes. scRNA-seq dataset of 48 MS patients and to C), similar to observations made in astro- S10, D and E). Thus, microglia-astrocyte in- matched controls (3) detected increased in- cytes and microglia when we merged our teractions mediated by Sema4D-PlexinB2 pro- teractions between microglial SEMA4D and RABID-seq data with previously published mote CNS inflammation during EAE—and astrocytic PLXNB2 in MS patients (fig. S10, A scRNA-seq data from MS patients (3)(fig. potentially MS.

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Fig. 3. RABID-seq A Astrocyte-microglia networks: Peak EAE vs. naive B Astrocyte-microglia networks: Peak EAE vs. naive identifies a role Axon guidance pathways - p-value: 1.05e-6 Reactome: axon guidance pathways Extracellular space for Sema4D- Axon guidance PlexinB2 signaling Plasma membrane EPH-Ephrin signaling Semaphorin interactions in microglia- Sema4D migration & growth-cone collapse astrocyte commu- Sema4D in semaphorin signaling EPHB-mediated forward signaling nication. (A) IPA EPHA-mediated growth cone collapse Cytoplasm EPH-ephrin mediated repulsion of cells analysis of axon Other semaphorin interactions guidance pathway Signaling by Robo receptor Netrin mediated repulsion signals genes activated in Ephrin signaling astrocyte-microglia Netrin-1 signaling Axonal growth inhibition (RHOA activation) networks in peak- Sema3A PAK dependent Axon repulsion EAE versus naïve SEMA3A-Plexin repulsion signaling mice. (B) Differen- 02468 Nucleus tially regulated -log10(FDR) axon guidance path- CDMicroglia connected to astrocytes Astrocyte-microglia connections E Peak EAE vs. naive Plxnb1 ways in astrocyte- Peak EAE vs. naive Plxnb2 Up in peak EAE astrocytes Up in peak EAE microglia p=0.0255 microglia networks 300 * Srgap2 n=27 n=240 Ephrin receptor signaling Cdc42 Dpysl2 in peak-EAE versus Sema4d Nfat5 30 Ppp3ca Pak2 naïve mice. FDR, Rock1 Itgb1 Ephrin B signaling Rock2 Downloaded from 200 Rhoa 2 false discovery rate. Gsk3b Lrrc4c Sema3e Fyn 20 (C) Differentially Epha6 Axonal guidance signaling Nfatc2 Arhgef12 Gnai3 regulated axon Mapk1 Sema4d Abl1 Gnai2 Nras Cdc42

100 Dcc −log10(p.adj) −log10(p.adj) Itgb1 Semaphorin signaling Cxcr4 Dpysl2 10 1 guidance pathways Plxnc1 Pak2 Chp1 Chp1 Efna5 Epha6 Srgap2 Plxnb2 Normalized counts in microglia Kras Nfat5 Rac2 Gsk3b Fyn Ephrin A signaling Mapk3 Plxnc1 connected to astro- Fes Sema3e Rac1 Rock2 0 0 Pak1 Sema7a Nfatc1

0 http://science.sciencemag.org/ cytes in peak-EAE −2 −1 0 1 2 3 3 2 1 0 -1 -2 -3 0 5 10 15 Peak EAE versus naïve mice. -log10(p-value) log2(fold change) log2(fold change) astrocytes (D) Differential gene Astrocytes Plxnb2+/- astrocyte expression analysis F Plxnb2+ Plxnb2- H Sema4d+/- microglia networks J Astrocytes connected to MG of astrocytes Astrocyte Plxnb2 Microglia Sema4d + Plxnb2+ A Plxnb2- A connected to Sema4d+ MG Sema4d- MG A+ MG+ microglia in peak- A+ MG- GO: Innate immune response A- MG+

Density GO: 0045087 EAE versus naïve Sema4d A- MG- 0123 0123 mice. Differentially Normalized expression Normalized expression expressed genes A+ NES: 1.75 A- A- A+ Microglia I MG+ FDR: 0.016 [adjusted P value MG+ MG- MG- on April 22, 2021 - (P.adj) < 0.05] from Plxnb2 axon guidance path- ways in astrocytes Sema4d (left) and microglia Astrocytes connected to MG (right) are colored G Astrocytes Microglia and labeled by gene Plxnb2+ A Plxnb2- A Plxnb2+ astrocytes connected to Sema4d+ microglia Dusp15 Sema4d+ MG Sema4d- MG 1.5 Plxnb2- astrocytes connected to Sema4d- microglia name on volcano Gng4 GO: Response to cytokine plots of −log10(P.adj) 1.0 Dnajc19 GO: 0034097 0.5 Rela versus fold change. Density 0 (E) Expression of 0 2 4 Plxnb1 and Plxnb2 in Log10(Number of connections) NES: 1.62 0.9 FDR: 0.019 peak-EAE astro- Expression 0.6 2 cytes. Two-tailed 1 0.3 0 Density −1 paired t test on 0 0 2 4 −2 percent per mouse. Log10(Strength of connections) (F) Subnetworks of − Plxnb2+/− astrocytes connected to Sema4d+/− microglia. (G)Densityplotsofthe MG+: Sema4d+ microglia; MG-: Sema4d microglia. (I) Differential number of interactions between Plxnb2+ astrocytes connected to Sema4d+ between astrocytes in the Plxnb2-Sema4d subnetworks determined by RABID-seq. microglia and Plxnb2− astrocytes connected to Sema4d− microglia. (H) Normalized (J) GSEA preranked analysis of single-cell RABID-seq data comparing Plxnb2+ − single-cell expression of Plxnb2 in astrocytes and Sema4d in microglia within astrocytes connected to Sema4d+ microglia (A+ MG+) to Plxnb2 astrocytes − the subnetworks shown in (F). A+: Plxnb2+ astrocytes; A-: Plxnb2− astrocytes; connected to Sema4d microglia (A- MG-). NES, normalized enrichment score.

We next investigated the role of microglia- in microglia and PlexinB2 in astrocytes during in culture with TNFa/IL-1b proinflammatory astrocyte interactions mediated by Sema4D- EAE (Fig. 4A) and in MS patient samples (Fig. cytokines known to contribute to the patho- PlexinB2 signaling in EAE pathogenesis. By 4B), validating our RABID-seq findings. More- genesis of EAE and MS (3)increasedSema4d immunostaining, we detected increased Sema4D over, the activation of primary mouse microglia expression(Fig.4C).Inaddition,thetreatment

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A GFAP PlexinB2 PlexinB2 Iba1 Sema4D Iba1 Overlay GFAP Sema4D Naive

100 µm Spinal cord

Inset EAE

B GFAP PLEXINB2 TMEM119 SEMA4D Overlay C Sema4d *** 2.0 100 75 * control

Healthy * 10 µm *** 1.5 80 ns * 50 60 1.0 **

NAWM 40

25 Downloaded from 0.5 20 Relative expression (fold) MS patient SEMA4D+ microglia (%) 0 0 0.0 PLEXINB2+ astrocytes (%) Vehicle IL-1β/TNF Lesion Control NAWM Lesion F D Nos2 Il1b E NOS2 IL1B Astrocyte 10 2.5 30 *** 1500 *** Il1b * Vehicle * Nos2 http://science.sciencemag.org/ Sema4D 8 2.0 20 *** 1000 *** 6 1.5 PlexinB2

4 1.0 10 500 Sema4D Relative expression (fold) 2 0.5 Relative expression (fold) 0 0 Relative expression (fold) Relative expression (fold) IL-1β/TNF - - + + - - + + Microglia 0 0.0 SEMA4D - + - + - + - + Astrocytes G sgRNA Cas9-2A-EGFP H Astrocytes J sgRNA Cas9-2A-EGFP K Gfap:: Gfap:: Itgam:: Itgam:: 5 LTR U6 Itgam pAWPRE 5 LTR U6 GfaABC1D pA WPRE sgScrmbl sgPlxnb2 sgScrmbl sgSema4d on April 22, 2021 Edem1 Pcdh7 Ccl4

4 Cd274 4 Ccl8 sgScrmbl Ccl9 sgScrmbl Itgam::sgSema4d Gfap::sgPlxnb2 3 Ccl1 3 *** Slc10a6 Ntrk2 ** 2 2 Slc6a13 EAE score EAE score Npy Kcnj13 1 1

Kcnj12 0 Slc1a2 0 Irs1 5101520 5101520 Day Day I Astrocytes L Nos2, Il1b regulatory network Up in Itgam:: Up in Itgam:: Up in Itgam:: Up in Itgam:: sgPlxnb2 vs. sgScrmbl sgSema4d sgScrmbl sgSema4d sgScrmbl GO: NOS regulator activity Hallmark: Inflammatory response GO: 0030235 ID: M5932 Nos2 p-value: 3.26e-4 z-score: -0.965

NES: -1.62 NES: -2.36 Il1b FDR: 0.021 FDR: 0.009 p-value: 3.59e-7 z-score: -2.189

Fig. 4. Microglia-astrocyte Sema4D-PlexinB2 signaling promotes CNS representative of n = 3 mice per group. (B) Immunostaining of MS patient and inflammation in EAE. (A) Immunostaining analysis of PlexinB2, GFAP, Iba1, healthy control CNS tissue. n = 6 images from N = 3 patients per region. and Sema4D in the spinal cords of naïve and peak-EAE mice. Images are Statistical analysis: unpaired two-tailed t test. NAWM, normally appearing white

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matter. (C) Sema4d expression determined by quantitative PCR (qPCR) in analysis of variance (ANOVA). (Top) Schematic of lentiviral vector. Lentiviral primary mouse microglia treated with IL-1b/TNF versus vehicle. n = 6 vehicle, transduction occurred 7 days before EAE induction to avoid targeting recruited n = 5 IL-1b/TNF. Statistical analysis: Kolmogorov-Smirnov t test. (D) Nos2 and myeloid cells. (H and I) RNA-seq analysis of gene expression (H) and GSEA Il1b expression determined by qPCR in primary mouse astrocytes treated with a preranked (I) of astrocytes isolated from mice transduced with Itgam::sgSema4d recombinant Sema4D fragment with agonistic activity. n = 9 samples per or Itgam::sgScrmbl. (J) EAE disease course in mice transduced with Gfap::Cas9 group, n = 8 samples for Nos2 vehicle. Statistical analysis: Kolmogorov- coexpressing sgPlxnb2 or sgScrmbl. n =10sgScrmbl, n =5sgPlxnb2 mice. Smirnov t test per group. (E) Nos2 and Il1b expression determined by qPCR in Statistical analysis: two-way repeated measures ANOVA. (Top) Schematic primary human fetal astrocytes treated with the indicated compounds. n =5 of lentiviral vector. (K) Differential gene expression determined by RNA-seq in vehicle Nos2, n = 4 vehicle Il1b, n = 3 otherwise. Statistical analysis: unpaired astrocytes from mice transduced with Gfap::sgPlxnb2 versus Gfap::sgScrmbl. two-tailed t test. (F) Schematic depicting microglial Sema4D binding PlexinB2 (L) Upstream regulator analysis by IPA of Gfap::sgPlxnb2 astrocytes relative to expressed in astrocytes. (G) EAE disease course in mice transduced with Gfap::sgScrmbl shows down-regulation of Nos2- and Il1b-driven proinflammatory Itgam::Cas9 coexpressing sgSema4d-orsgScrmbl-targeting lentiviruses. n =10 pathways. Data shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, sgScrmbl, n =5sgSema4d mice. Statistical analysis: two-way repeated measures not significant.

of primary mouse or human astrocytes with Sema4D-PlexinB1) interactions promote CNS cytes in culture with plate-bound Ephrin-B3– a recombinant Sema4D fragment with plexin inflammation during EAE. Fc chimera and activated them with TNFa and agonist activity (38–40) increased the expres- IL-1b. EphB3 activation boosted the expression sion of the proinflammatory genes Nos2 and EphB3 receptor signaling boosts astrocyte of genes associated with astrocyte proinflam- Il1b (Fig. 4, D and E, fig. S11, A and B), sug- proinflammatory activities matory activities such as Il6, Nos2, Csf2,and Downloaded from gesting that Sema4D-triggered Plexin signaling Our RABID-seq studies of astrocyte interac- Tnfa (Fig. 5G); EphB3 activation also boosted boosts astrocyte proinflammatory responses. tions in EAE also detected microglia-astrocyte IL-6 and CCL2 production (Fig. 5H). We ob- To determine whether microglia-astrocyte signaling mediated by erythropoietin-producing tained similar results when we analyzed the interactions mediated by Sema4D-PlexinB2 human hepatocellular B (EphB) receptors effects of EphB3 activation with plate-bound promote CNS inflammation during EAE (Fig. (Fig.3,AandB),whichbelongtoafamilyof Ephrin-B3–Fc chimera on primary human

4F), we developed CRISPR-Cas9 lentiviruses transmembrane receptor tyrosine kinases that astrocytes in culture (fig. S12C). Thus, EphB3 http://science.sciencemag.org/ to inactivate Sema4d in microglia and Plxnb2 have important roles in axon guidance, among signaling may boost astrocyte proinflamma- in astrocytes using Itgam-orGfap-driven Cas9 other biological processes (43). EphB recep- tory activities. and targeting single guide RNA, respectively tors are activated by interactions with their (fig. S11, C to F). The inactivation of Sema4d in membrane-bound ligands Ephrin-B1, Ephrin- Microglial Ephrin-B3 and astrocyte EphB3 microglia ameliorated EAE (Fig. 4G). Moreover, B2, and Ephrin-B3, encoded by Efnb1, Efnb2, promote CNS pathology in EAE astrocytes isolated from sgSema4d-targeting and Efnb3, respectively (43). Efnb3 is mostly To study the role of Ephrin-B3 and EphB3 in knockdown mice coexpressing Itgam-driven expressed in the CNS, whereas Efnb1 and Efnb2 the regulation of microglial and astrocyte re- Cas9 showed decreased activation of Nos2 and show a broader expression pattern (44). We sponses in the context of CNS inflammation, proinflammatory signaling, supporting a role detected increased expression of Efnb3,butnot we knocked down Ephb3 in astrocytes and

for microglia-derived Sema4D in promoting Efnb1 or Efnb2, in microglia during EAE (fig. Efnb3 in microglia during EAE using lentivirus- astrocyte pathogenic activities (Fig. 4, H and I). S12A). Efnb3 expression levels in monocytes delivered short hairpin RNAs (shRNAs) ex- on April 22, 2021 Indeed, Plxnb2 inactivation in astrocytes also recruited to the CNS during EAE were similar pressed under the control of Gfap or Itgam resulted in EAE amelioration (Fig. 4J), con- to the expression levels detected in microglia promoters, respectively (12) (fig. S13, A and comitant with the down-regulation of proin- in naïve mice (fig. S12A), suggesting that the B). EAE mice were injected intracerebroven- flammatory pathways in astrocytes associated Ephrin-B3 in monocytes does not play a ma- tricularly at day 7 after immunization, before with NOS2 and IL-1b (Fig.4,KandL,andfig. jor role in the control of EphB3 receptor– disease onset, to target CNS resident cells S11, C and D). dependent astrocyte proinflammatory activities as previously described (3, 9, 12, 13). We did PlexinB1 and PlexinB2 show redundancy in during EAE. Moreover, flow cytometry analysis not detect reduced Efnb3 expression in CNS- some biological systems (41, 42). The analysis of neonatal mouse microglia-astrocyte cocul- infiltrating monocytes, confirming that these of our RABID-seq dataset suggested that Plxnb1 tures detected higher EphB3 expression in cells were not affected by the knockdown (fig. and Plxnb2 are expressed by different astrocyte astrocytes than in microglia (fig. S12B). Finally, S13B). The lentiviruses reached the spinal cord, subpopulations and that Plxnb2+ astrocytes are the analysis of CNS human samples detected and the knockdown of Ephb3 in astrocytes or more abundant than Plxnb1+ astrocytes (Fig. 3E an increase in EPHRINB3+ microglia and Efnb3 in microglia resulted in a comparable and fig. S11G). Thus, we investigated whether EPHB3+ astrocytes in MS lesions (Fig. 5, A to amelioration of EAE (Fig. 6A and fig. S13, C to Sema4D signaling via PlexinB1 might also C). Thus, signaling between microglial Ephrin- G) but did not affect T cell responses (fig. S13, contribute to EAE pathogenesis. Knockdown B3 and EphB3 receptors in astrocytes may play HtoK).Ephb3 and Efnb3 knockdown led to a via Gfap-driven Cas9 coexpressing sgPlxnb1 in a role in CNS inflammation in EAE and, po- reduction in proinflammatory Ly6CHi mono- astrocytes also ameliorated EAE via IL-1b and tentially, MS. cytes recruited to the CNS during EAE (45) NOS2, although to a lesser extent than Plxnb2 Both EphB3 and its ligand Ephrin-B3 are (Fig. 6B). The simultaneous knockdown of (fig.S11,HtoK).Indeed,wealsodetected plasma membrane (43). We used Ephb3 in astrocytes and Efnb3 in microglia increased interactions between microglial transmission electron microscopy to detect did not ameliorate the disease more than SEMA4D and PLXNB1 expressed in astrocytes increased microglia-astrocyte contacts during single knockdowns did (fig. S13L). Moreover, in MS patients (3), although the detected in- EAE (Fig. 5, D to F), suggesting a potential rea- no EAE amelioration was detected after Efnb3 crease in this interaction in MS was lower than son for increased Eph signaling mediated by knockdown in astrocytes (fig. S13M). theincreasedetectedforSEMA4D-PLXNB2 microglia Ephrin-B3 and astrocyte EphB3 inter- Analysis of the transcriptional response of (fig. S10, A and C). Thus, microglia-astrocyte actions. To study the effects of EphB3 signaling astrocytes from EAE mice after the knockdown Sema4D-PlexinB2 (and, to a lesser extent, in astrocytes, we treated neonatal mouse astro- of Ephb3 in astrocytes or Efnb3 in microglia

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Fig. 5. EphB3 receptor * A GFAP EPHB3 Overlay+ B TMEM119 EPHRINB3 Overlay+ C 100 *** signaling boosts astro- DAPI DAPI 80 cyte proinflammatory P) FA 60 HC responses. (A to HC

EPHB3+ 40 10 μm 10 μm C) Immunostaining anal- (% of G ysis of MS and healthy 20 control (HC) CNS tissue 0 HC WM samples for the colocal- Lesion NA ** NAWM NAWM ization of EPHB3 and 80 *** GFAP in astrocytes (A) 60 or EPHRINB3 and NB3+ I MS patient TMEM119 in microglia MS patient 40

(B). (C) Quantification of EPHR Lesion Lesion (% of TMEM119+) 20 immunostaining data. n = 6 or 16 images from 0 WM HC N = 3 patients per DENaive EAE FLesion NA region. Statistical analy- 40 sis: one-way ANOVA and *** Dunnett post-test. AS MG 30 (D and E) Representa- (µm) Downloaded from tive electron micro- 20 graphs of naïve (D) and AS EAE (E) spinal cords. distance 10 Microglia-astrocyte Microglia (MG) cells MG exhibit elongated and 0 Naive EAE dark nuclei with clumped chromatin and dark http://science.sciencemag.org/ cytoplasm, and astro- GH120000 Csf2 Nos2 Il6 Tnfa 300 *** *** cytic cells (AS) are 150 50 400 *** 20 *** *** *** n 90000 characterized by pale 40 300 15 200 nuclei that are usually 100 *** pg/mL) 30 *** 60000 *** 200 10 regular in shape with a *** ** * IL-6 (pg/mL)

20 CCL2 ( thin rim of hetero- 50 100 100 5 30000 chromatin beneath the 10 Relative expression Relative expression Relative expression Relative expressio nuclear membrane. 0 0 0 0 0 0 Fc-EphrinB3: - + - + - + - + - + - + - + - + Fc-EphrinB3: - + - + - + - + Green arrow, intact TNFα + IL-1β TNFα + IL-1β TNFα + IL-1β TNFα + IL-1β TNFα + IL-1β TNFα + IL-1β

myelin; red arrow, myelin on April 22, 2021 destruction; blue arrow, remyelination.Theblackspaceinthetopright one-way ANOVA and Dunnett post-test. Data are representative of two corner of (E) indicates the edge of the tissue section. (F) Quantification of independent experiments. (H) IL-6 and CCL2 concentration in supernatants of microglia-astrocyte distance in electron microscopy images. n =63naïve neonatal mouse astrocytes stimulated with TNFa and IL-1b inthepresenceof mice, n = 167 EAE mice. Statistical analysis: unpaired two-tailed t test. plate-bound Ephrin-B3–Fc chimera. n = 6 samples per group. Statistical (G) Csf2, Nos2, Il6,andTnfa expression determined by qPCR in neonatal analysis: one-way ANOVA and Dunnett post-test. Data are representative of mouse astrocytes stimulated with TNFa and IL-1b in the presence of plate- two independent experiments. Data shown as mean ± SEM. *P <0.05, bound Ephrin-B3–Fc chimera. n = 6 samples per group. Statistical analysis: **P <0.01,***P <0.001. revealed decreased expression of genes asso- ment with decreased microglial proinflam- Ephb3 knockdown in astrocytes (Fig. 6K), sug- ciated with inflammation and neurodegenera- matory transcriptional responses detected by gesting that EphB3-induced reverse signaling tion (Fig. 6, C to E). Thus, microglial Ephrin-B3 RNA-seq, we detected decreased NF-kBacti- contributes to the control of microglia activation and astrocyte EphB3 receptors participate in vation after the knockdown of Efnb3 in mi- by astrocytes. Indeed, plate-bound EphB3-Fc the control of astrocyte proinflammatory activ- croglia or Ephb3 in astrocytes, suggesting that chimera boosted Il1b, Il6, and Nos2 expres- ities during EAE. astrocyte Ephrin-B3 signaling boosts NF-kB– sion as well as CCL2 and IL-6 secretion by driven proinflammatory transcriptional pro- mouse primary microglia activated in culture Reverse Ephrin-B3 signaling boosts NF-kB grams in microglia (Fig. 6, I and J). with lipopolysaccharide (LPS) (Fig. 6, L driven responses in microglia during EAE Ephrin-B3–EphB3 interactions may modu- and M). Thus, reverse Ephrin-B3 signaling In addition to forward signaling in Ephb- late microglial responses via reverse signaling boosts microglial proinflammatory activities expressing cells, the interaction between EphB through Ephrin-B3 in microglia and also in- during EAE. receptors and their membrane-bound Ephrin- directly via EphB3-controlled astrocyte secreted B ligands triggers reverse signaling in Efnb- factors. To study the role of reverse Ephrin-B3 Pharmacologic inhibition of EphB3 receptor expressing cells (46–50). In support of a role of signaling in the control of microglial responses, kinase ameliorates EAE reverse Ephrin-B3 signaling in the control of we cocultured mouse neonatal microglia in vitro Multiple signaling events are triggered by EphB3 microglial responses during EAE, the knock- with mouse neonatal astrocytes prestimulated receptor activation; one of these signaling mech- down of Efnb3 in microglia or Ephb3 in astro- with TNFa and IL-1b. Coculture with prestimu- anisms is EphB3 kinase activity (43). A38 is a cytes decreased proinflammatory gene expression lated astrocytes increased microglial Nos2 ex- CNS-penetrant small molecule that inhibits in microglia (Fig. 6, F to H). Moreover, in agree- pression, and this increase was diminished by EphB3 kinase activity (Fig. 7A) (51). We detected

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Fig. 6. Microglia-astrocyte ADBC Astrocytes EAstrocytes Ingenuity pathway analysis: pGfap:: pItgam:: Ephrin-B3–EphB3 shCtrl vs pItgam::shEfnB3 shCtrl shEphB3 shCtrl shEfnB3 8000 B2m 0.0 2 2 3 Ctrl *** signaling promotes CNS *** pGfap::shEphB3 -0.5 1 1 6000 pathology in EAE. (A) EAE pItgam::shEfnB3 Cdc25b -1.0 Ifi27 0 0 z-score 2 Prdx5 z-score in knockdown mice trans- -1.5 −1 −1 4000 z score Csf1r

-2.0 −2 −2 duced with lentiviral in CNS (number) Hi 2000 -2.5 constructs targeting Ephb3 EAE score 1 ***

Ly6C g *** ng li in astrocytes (red), Efnb3 in Ephb3 0 Pathway Signa K g in Regulationne RespoP nse microglia (blue), or nontar- T 0 shCtrl A F 0 5 10 15 20 25 30 Interferon SignalingN ERK/MA geting control (black). Time (days) of the Immu Role of Ctrl (n = 24 mice), pGfap::shEphB3pItgam::shEfnB3 Ccl5 Leukocyte Extravasation Signalin pGfap::shEphb3 (n =17 Neuroinflammation Signalin Notch1 mice), pItgam::shEfnb3 (n = Cxcr6 FG H 22 mice). Statistical analysis: Microglia Microglia pGfap::shEphB3 pItgam::shEfnB3 Foxo3 two-way ANOVA. (B)Quanti- pGfap:: pItgam:: 0.0 0 shCtrl shEphB3 shCtrl shEfnB3 fication of proinflammatory 2 2 1 1 -0.5 -1

monocytes in the CNS of 0 0 e Mt2 z-score z-score -1.0 -2 −1 −1 Il1r1 z-scor EAE mice after the Adamts4 z-score −2 −2 -1.5 -3 knockdown of Ephb3 in Adrb2 astrocytes (red), Efnb3 in -2.0 -4 microglia (blue), or nontar- Downloaded from T3 Pathway A IL-6 Signaling geting control (black). n =5 ST κ NF- B Signaling Hmox1 Glul Chemokine Signaling p38 MAPK Signaling Agt Maturation Dendritic Cell Maturation mice per group. Statistical Manf Death Receptor Signaling Smad3 analysis: one-way ANOVA Osmr Neuroinflammation Signaling Pathway Neuroinflammation Signaling Pathway Hspa1a and Dunnett post-test. (C to Hspa1b B2m E) RNA-seq analysis of Apoe http://science.sciencemag.org/ astrocytes. (C) Differentially Ly9 regulated pathways in Ccl5 I J astrocytes after Efnb3 Iba1 ac-p65 DAPI Overlay 80 *** * knockdown in microglia ) + analyzed by ingenuity path- shCtrl 60 100 μm B2m ways analysis. (D and E) Tlr12 40

Heatmap of differentially (% of Iba1 + expressed genes in astro- Il12rb1 Cxcl13 20 pItgam:: Itgal Itgal shEfnB3 cytes after the knockdown ac-p65 Il6st Tnfrsf14 of Ephb3 in astrocytes (D) or Tnf 0

Ccl12 Efnb3 in microglia (E). Ccrl2 shCtrl on April 22, 2021

(F to H) RNA-seq analysis of pGfap:: Tgif1 shEphB3 Apod microglia isolated from EAE pItgam::shEfnB3pGfap::shEphB3 mice transduced with lenti- Cxcl13 viral vectors targeting Ephb3 KLNos2 Il1b Il6 Nos2 M 50 1500 *** 1000 in astrocytes, Efnb3 in 20 *** 250 * 500 * *** *** microglia, or non-targeting 40 200 n 400 800

15 ) control. (F and G) Heatmap 1000 30 150 300 600 of differentially expressed 10 genes in microglia after 20 100 200 400 500 IL-6 (pg/mL)

5 CCL2 (pg/mL knockdown of Ephb3 in 10 50 100 200 Relative expression Relative expressio astrocytes (F) or Efnb3 in Relative expression Microglia (relative expression) 0 0 0 0 0 0 microglia (G). (H) Relevant Astrocytes: l 3 Fc-EphB3: - + - + - + Fc-EphB3: - + - + iCtrl hb3 s p siCtr phb iE LPS LPS LPS LPS LPS pathways selected from s siE ingenuity pathway analysis TNFα +IL1β of the genes differentially expressed in microglia after Ephb3 knockdown in astrocytes (left) and Efnb3 samples per group. Statistical analysis: one-way ANOVA and Tukey post-test. knockdown in microglia (right). (I) Immunostaining analysis of acetylated p65 (ac- (L and M) Neonatal mouse microglia cultured in plates precoated with EphB3- p65) and Iba1 in the CNS of EAE knockdown mice. (J ) Quantification of ac-p65+ FcChimera and stimulated with LPS. (L) Il1b, Il6,andNos2 expression determined Iba1+ cells. n = 6 mice per group. Statistical analysis: one-way ANOVA and by qPCR and (M) CCL2 and IL-6 production quantified by enzyme-linked Tukey post-test. (K) Astrocytes treated with control or Ephb3-targeting small immunosorbent assay (ELISA) in supernatants. n = 4 samples per group. Statistical interfering RNA and pretreated with TNFa and IL-1b were cocultured overnight analysis: unpaired two-tailed t test. Data are representative of two independent with microglia, and microglial Nos2 expression was determined by qPCR. n =6 experiments.Datashownasmean±SEM.*P < 0.05, ***P <0.001. a dose-dependent inhibition of EphB3 kinase signaling in astrocytes, we pretreated neonatal dependent manner (Fig. 7B); it also decreased activity by A38 in a cell-free assay (fig. S14A). mouse astrocytes in culture with A38 and the production of IL-6, CCL2, and TNFa (Fig. A38 did not induce cytotoxicity or apoptosis (fig. activated them with TNFa and IL-1b.A38 7C). Similar results were obtained when we S14, B and C). To study the effects of EphB3 reduced Il6 expression in astrocytes in a dose- used C9, an additional inhibitor of EphB3

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A BCIl6 * * 2500 *** *** 10000 * 4000 A38 500 *** ** * *** *** *** *** 2000 8000 n 400 3000 1500 6000 300

pg/mL) 2000 ( 1000 4000 α (pg/mL) 200 IL-6 CCL2 (pg/mL) TNF 1000 100 500 2000 Relative expressio

0 0 0 0 0 1 0 1 A38 (µM): 0 0 0 0 0 0 0.1 10 0.1 10 50 0.1 10 0.1 10 0.1 10 0.1 10 A38 (µM): 0.01 0.01 0.1 0.1 0.001 0.001 TNFα + IL-1β TNFα + IL-1β TNFα + IL-1β TNFα + IL-1β

DFNos2 Tnfa E GH ns ns 50 ns * 3e4 30 ns 7e3

*** s) * *** 5 *** *** Ctrl 3 ** *** 40 6e3 ns A38

s 4 2e4 20 30 5e3 2 3 20 4e3 2

EAE score * 1e4 10 1 Number of cell 10 3e3 1 Relative expression Relative expression in CNS (% of infiltrating cell Neurotoxicity (% of max) A38 Treatment: Hi 0 2e3 0 0 Downloaded from 0 4 8 1216202428 0 0 0 - - - - Ly6C C9 ACM: A38 A38 A38 Time (days) Ctrl A38 A38: - + - + A38: - + - + Naive TNFα + IL-1β LPS LPS no ACM anti-TNFα: - - - - + +

IJAstrocytes K Ingenuity pathway analysis EAE-Ctrl EAE-A38 Naive 0 2

1 Up in Up in Up in Up in -1 Il1b A38 Ctrl A38 Ctrl http://science.sciencemag.org/ 0 Cxcl10

z-score Activation of Regulation of -2 −1 Cd14 Cxcl9 innate immune response leukocyte mediated immunity z-score −2 Ccl2 GO: 0002218 GO: 0002703 Cxcl1 -3

Tlr2 NES: -2.000 NES: -1.972 -4 FDR<0.001 FDR<0.001 g y n ling aling aling ation nali thwa g a Signa P Signaling Signaling Production e B Signaling -6 Signalingtor Si m Positive Regulation κ IL IL-1 roinflamm iNOS regulation of NF-κB activity of IL-6 production NF- aso GM-CSF Signaling nterferon Sign K/MAPK Neu PI3K/AKTI Signaling R GO: 0051092 GO: 0032635 NO/ROS p38 MAPKE Sign nflamm ll-like Recep I L To NES: -1.899 NES: -1.783 FDR: 3.11E-04 FDR: 8.40E-03 Neuroinflammation signaling pathway IL-6 signaling iNOS signaling Nfkb2 Ctrl A38 Ctrl A38 Ctrl A38 2 Regulation Positive regulation

of inflammatory response of cytokine production 1 on April 22, 2021 Nos2 GO: 0050727 GO: 0001819 0 z-score -1 NES: -1.923 NES: -2.062 FDR: 1.09E-04 FDR<0.001 -2

NF-κB signaling GM-CSF signaling PI3K/AKT signaling Ctrl A38 Ctrl A38 Ctrl A38 Cellular glucose Postive regulation homeostasis of ROS metabolic process GO: 0001678 GO: 2000379

NES: -1.616 NES: -1.677 FDR: 0.092 FDR: 0.053

Fig. 7. Pharmacologic inhibition of EphB3 receptor kinase ameliorates EAE. 24 hours; TNF blocking antibody was added where indicated. n = 8 samples per (A) A38 structure. (B) Il6 mRNA expression determined by qPCR in neonatal group but n = 4 samples for anti-TNFa groups. Statistical analysis: one-way ANOVA mouse astrocytes stimulated with TNFa and IL-1b in the presence of the and Tukey post-test. Data are representative of two independent experiments. indicated concentrations of A38. n = 3 samples per group. Statistical analysis: (F) Migration assay of splenic CD11b+ monocytes performed using ACM. n =3 one-way ANOVA and Sidak post-test. (C) IL-6, CCL2, and TNFa concentration samples (- and A38), n = 3 samples (C9). Statistical analysis: one way ANOVA measured by ELISA in supernatants of neonatal mouse astrocytes stimulated as and Dunnett post-test. Data are representative of two independent experiments. in (B) with the indicated concentrations of A38. n = 4 and 2 samples (0.1 and (G) EAE in C57Bl/6J mice treated twice a day with vehicle or 20 mg/kg A38 10 groups). Statistical analysis: one-way ANOVA and Dunnett post-test. Data are injected intraperitoneally, starting at the peak of the disease. n = 5 mice per representative of three independent experiments. (D) Nos2 and Tnfa mRNA group. Statistical analysis: two-way ANOVA. Data are representative of two expression determined by qPCR in neonatal mouse microglia stimulated with LPS independent experiments. (H) Quantification of monocytes in the CNS of C57Bl/ in the presence of A38. n = 4 samples. Statistical analysis: one-way ANOVA and 6J mice treated as in (A). n = 5 Ctrl mice, n = 6 A38 mice, and n = 2 naïve mice. Tukey post-test. Data are representative of two independent experiments. (E and Statistical analysis: one-way ANOVA and Tukey post-test. (I to L) RNA-seq F) Primary mouse astrocytes were activated with TNFa and IL-1b, and treated analysis of astrocytes from naïve or EAE mice treated with A38 or vehicle. with A38 or C9. Media was replaced, cells were extensively washed, and new (I) Heatmap of differentially expressed genes in astrocytes. (J) GSEA of astrocytes. medium was added 24 hours later; ACM was collected 48 hours later. (E) ACM (K and L) Ingenuity pathway analysis of genes differentially expressed in was added to the mouse neuron cell line N2A preactivated with IFNg, and astrocytes from A38-treated mice compared with vehicle-treated mice. Data cytotoxicity was determined by quantifying lactate dehydrogenase release after shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

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kinase activity (52)(fig.S14,DandE),or duced by immunization with MOG35-55,which mTOR activation (Fig. 8D). Indeed, we detected when A38 was tested on adult rodent astro- recapitulates aspects of secondary progres- reduced S6 phosphorylation when we used cytes (fig. S14, F and G) or primary human sive MS, including the progressive and ir- flow cytometry to analyze the effects of the astrocytes cultured in the presence of serum or reversible accumulation of neurologic disability pharmacological inhibition of EphB3 ki- inserum-freemedium(fig.S14,HtoJ).Con- (5, 9, 58, 59). Specifically, we evaluated the ef- nase or PI3K in primary astrocytes activated sistent with the higher levels of EphB3 de- fects of A38 administration or Ephb3 knock- in vitro with TNFa and IL-1b (Fig. 8E). Thus, tected in astrocytes compared with microglia down in astrocytes during the progressive phase through its effects on the PI3K-AKT axis, (fig. S12B), A38 showed no effect on the mi- of NOD EAE (fig. S16A). Both the pharmaco- EphB3 kinase activity promotes mTOR acti- croglial expression of the proinflammatory logical inactivation of EphB3 kinase activity vation in astrocytes. genes Nos2 and Tnfa induced by LPS stimula- with A38 and the knockdown of Ephb3 in To investigate the role of mTOR on astro- tion (Fig. 7D), confirming that EphB3 intra- astrocytes by lentivirus-delivered shRNAs cyte responses, we used the mTOR inhibitor cellular signaling is important for the regulation ameliorated NOD EAE, as indicated by the rapamycin (62). Rapamycin treatment sup- of astrocyte responses but not microglial reduced clinical scores and the decreased pressed the expression of proinflammatory responses. recruitment of proinflammatory monocytes genes in astrocytes stimulated in vitro with TNFa (53, 54) and nitric oxide (13, 55)have to the CNS; we did not detect changes in the TNFa and IL-1b (Fig. 8F), recapitulating our been linked to astrocyte pathogenic activities. peripheral T cell response (fig. S16, A to C). previous observations on the effects of A38 Moreover, CCL2 produced by astrocytes pro- Additionally, A38 administration and Ephb3 on astrocytes. Consistent with its inhibitory motes monocyte recruitment to the CNS (5, 56). knockdown in astrocytes suppressed the astro- effects on mTOR, rapamycin suppressed S6 Thus, based on the effects of A38 and C9 on cyte and microglial expression of transcriptional phosphorylation but did not suppress the TNFa, Nos2, and CCL2 expression, we evaluated modules associated with CNS inflammation and phosphorylation of AKT, p85, or NF-kBsub- neurotoxic and chemotactic activities of astrocyte- neurodegeneration (fig. S16, D to G). Thus, unit p65 (Fig. 8D). Downloaded from conditioned medium (ACM) collected from EphB3 signaling in astrocytes promotes CNS mTOR controls mitochondrial function (63), astrocytes pretreated with A38 or C9. The inflammation and is a candidate target for which has been linked to pathogenic activities pharmacological inhibition of EphB3 in astro- therapeutic intervention. of astrocytes and microglia in neurologic dis- cytes reduced ACM neurotoxic and chemo- orders (9 , 64). Indeed, mTOR-driven mitochon- attractant activity (Fig. 7, E and F). The neurotoxic EphB3 kinase activates mTOR and boosts drial respiration produces reactive oxygen species activity was TNF independent (Fig. 7E and fig. mitochondrial ROS production in astrocytes (ROS), which promote proinflammatory gene http://science.sciencemag.org/ S14K), suggesting that its inhibition by A38 Finally, we investigated the mechanisms involved expression and contribute to neurodegenera- involved the regulation of additional neuro- in the control of astrocyte proinflammatory ac- tion (65–67). Thus, we investigated the effects toxicity mechanisms. tivities by EphB3 signaling. We established a of A38 on mitochondrial function and ROS To evaluate the potential of the EphB3 kinase signature score for the activation of the Ephrin production. The inhibition of EphB3 kinase as a therapeutic target during CNS inflamma- pathway in astrocytes and analyzed microglia- activity by A38 decreased basal and maximal tion, we induced EAE in B6 wild-type mice astrocyte subnetworks that contained astro- mitochondrial respiration, as well as adeno- by immunization with MOG35-55 and initiated cytes with high (>90th percentile) and low sine triphosphate (ATP)–linked respiration in treatment with A38 (20 mg/kg body weight (<10th percentile) scores (Fig. 8A). Differen- primary astrocytes (Fig. 8, G and H); similar twice a day by intraperitoneal injection) at tial expression analysis of astrocytes in these results were obtained with rapamycin. Con-

the peak of the disease, using vehicle as a con- subnetworks identified PI3K-AKT-MTOR sig- sistent with its suppressive effects on mito- trol. A38 administration ameliorated EAE, as naling as a potential pathway involved in chondrial respiration, A38 and rapamycin also on April 22, 2021 indicated by the reduction in EAE scores (Fig. Ephrin receptor signaling (Fig. 8B). Further decreased the mitochondrial production of 7G) and the reduced demyelination and axonal analysis of protein interaction networks mod- ROS induced in astrocytes by stimulation with loss detected in histopathological analyses (figs. ulated by A38 treatment identified Pik3r1 as TNFa and IL-1b (Fig. 8I). Accordingly, we de- S13, D to G). A38 also reduced the recruitment of a candidate mediator of the effects of EphB3 tected a decrease in the expression of genes Ly6CHi proinflammatory monocytes to the CNS signaling (Fig. 8C). PIK3R1 encodes the reg- related to ROS metabolic processes in astro- (Fig. 7H) but did not affect T cell responses (fig. ulatory subunits (p85a,p55a,andp50a)of cytes isolated from A38-treated EAE mice (Fig. S15, A to C). A38 did not affect the glia limitans class I PI3Ka, which is associated with the 7J). Moreover, treatment with rotenone and when compared with the vehicle-treated group control of innate immunity (60). Supporting antimycin A, which increase mitochondrial (fig. S15D), as determined by the analysis of CLDN5 a role for PIK3R1 in EphB3 signaling in astro- ROS levels (68–70) (fig. S17B), boosted the ex- as described (57). Moreover, A38 administration cytes, A38 suppressed the phosphorylation of pression of proinflammatory genes in astrocytes decreased astrocyte and microglial expression p85a,p55a, and their downstream signaling (fig. S17C). Thus, the production of mitochon- of transcriptional modules associated with CNS molecule AKT in primary mouse astrocytes in drial ROS driven by PI3K-AKT-mTOR signaling inflammation and neurodegeneration, as deter- culture (Fig. 8D). Similarly, the pharmaco- contributes to the proinflammatory effects of mined by RNA-seq (Fig. 7, I to L, and fig. S15, logical inhibition of class I PI3Ka by ZSTK474 EphB3 kinase activation in astrocytes. E to H). A38 administration concomitant with suppressed the phosphorylation of its target Ephb3 knockdown in astrocytes did not fur- AKT (Fig. 8D). Discussion ther increase the therapeutic effects achieved AKT is reported to activate the transcription Several techniques have been developed to infer by these interventions alone (figs. S14, D to G, factor NF-kB and the mammalian target of cell interactions on the basis of spatial tran- and S15I), suggesting that the amelioration of rapamycin (mTOR) (61). The inhibition of AKT scriptomics (71–77), physical interactions (17, 18) EAE by A38 involves the inhibition of EphB3 phosphorylation by A38 did not suppress NF- or colocalization in tissue (19, 20). However, kinase activity in astrocytes. kB activation, as determined by the analysis of these approaches remain difficult to apply to To further validate the potential of EphB3 its phosphorylation and nuclear translocation the study of cells in the CNS, owing to technical kinase inhibition for the therapeutic modula- in primary astrocytes stimulated with TNFa complexity, throughput limitations, or a lack of tion of CNS inflammation and neurodegener- and IL-1b (Fig. 8D and fig. S17A). However, A38 single-cell resolution with respect to transcrip- ation, we used the nonobese diabetic (NOD) suppressed S6 phosphorylation downstream of tional or interaction information. To overcome mouse model of chronic progressive EAE in- mTORC1, suggesting that A38 interferes with these limitations, we developed RABID-seq,

Clark et al., Science 372, eabf1230 (2021) 23 April 2021 11 of 15 RESEARCH | RESEARCH ARTICLE

A B Hallmark: PI3K AKT D Astrocyte-microglia networks mTOR signaling TNFα + IL-1β Astro >90th %ile Astro <10th %ile - - A38 A38 Z74 Rapa <10th percentile Ephrin >90th percentile Ephrin Ephrin pathway score Ephrin pathway score p-p85α receptor pathway scores receptor pathway scores connected to MG connected to MG ID: M5923 p-p55α

p85α NES: 1.66 p-value: 0.003 p-AKT (Ser473)

p-AKT (Thr308)

MMP2 SMARCA4 ITGAV ITGB1 C PSMB5 ACTN1 SP1 HSP90AA1 PTPN1 AKT MAP3K1 RND1 ILK

ESR1 MAP3K3 EIF1B ITGA5 MYH10 SUMO2 ITGB3 p-S6

HCLS1 CSNK2B CXCR4 ACTR3 MYL12A USP45 FN1 JUN LYN SHC1

GNB2L1 S6 163 astrocytes, 5,109 microglia 164 astrocytes, 5,082 microglia ARPC3 SET ACTR2 YWHAQ TNFRSF1A CBL RALY 21,115 connections 36,917 connections CD44 ACTB WAS YWHAG PIK3CG ARPC1B PIK3R2 p-p65 PIK3CB KAT5 ARPC5 PIK3CD UBC CIB1 ARPC2 RAC1 SYK MAPK8IP1 LCK GAPDH EGFR GO: Ephrin receptor signaling pathway score TRAF6 PIK3R1 SRI XRCC6 APP Microglia Astrocytes TIAM1 HRAS SLA BCL2L1 VAV1

Low High PSEN2 PRKCA SDCBP ERBB2 FYN APH1A CTNNB1 GRIN1 EPHB3 LNX1 EFNB3 Downloaded from EFIl6 Ccl2 Tnfa Csf2 Nos2 *** 80 *** 2.5 200 60 *** 100 - *** *** 25 * *** 70000 * ** *** A38 *** *** 60000 20 2.0 80 Z74 60 150 50000 40 1.5 60 15 40000 40 100 1.0 40 30000 10

Counts 20 20000 20 50 Relative expression (norm. to mode) Relative expression Relative expression Relative expression 0.5 20 Relative expression 5 10000 http://science.sciencemag.org/ pS6 fluorecence intensity 0 0 0 0.0 0 0 0 - - - - - 3 4 5 010 10 10 - A38 Z74 A38 A38 A38 A38 A38

Rapamycin pS6 Rapamycin Rapamycin Rapamycin Rapamycin G H ATP-linked I Rotenone/ Basal respiration Maximal respiration respiration ** 2000 Oligomycin FCCP Antimycin ** 1.2 *** 1000 2000 *** * 800 *** 1600 *** *** 1.1 d d - 800 d

1500 600 cells) cells) cells)

cells) A38 1.0 5 5 5 5 1200 0 Rapa 600 1000 400 0.9 800 400 OCR norm 0.8 OCR normalize OCR normalize 500 OCR normalize 200 (pmol/min/1 (pmol/min/10 (pmol/min/10 400 200 (pmol/min/10 0.7 Relative MitoSOX levels

0 0 0 0 0.6

0306090120 - - - - on April 22, 2021 Time (minutes) A38 A38 A38 A38

Rapamycin Rapamycin Rapamycin Rapamycin

Fig. 8. EphB3 kinase activates mTOR and boosts mitochondrial ROS and Nos2 expression determined by qPCR in neonatal mouse astrocytes stimulated production in astrocytes. (A) Subnetworks of astrocytes interacting with with TNFa and IL-1b inthepresenceofA38orrapamycin.n = 4 samples per group. microglia, as determined by RABID-seq and binned (<10th versus >90th Statistical analysis: one way ANOVA and Dunnett post-test. Data are representative percentile) based on their expression of Ephrin receptor pathway genes (MSigDB of three independent experiments. (G) Seahorse mitochondrial stress test performed ID: M5923). (B) GSEA preranked analysis of RABID-seq data comparing on astrocytes pretreated overnight with A38 or rapamycin. n =2samples.OCR, >90th percentile astrocytes to <10th percentile astrocytes. (C) Protein-protein oxygen consumption rate; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone. interaction analysis of the effects of A38 on astrocytes. (D) Western blot analysis (H) Quantification of basal mitochondrial respiration, maximal mitochondrial of phosphorylated (p-) or total protein for p85a PIK3R1, p55a PIK3R1, AKT, S6, respiration, and ATP-linked respiration calculated from mitochondrial stress assay p65, and GAPDH in primary neonatal mouse astrocytes activated for 30 min from (E). n = 2 samples per group. Statistical analysis: one-way ANOVA and Tukey with TNFa and IL-1b in the presence of the indicated compounds. Z74, ZSTK74 post-test. Data are representative of three independent experiments. (I)Mitochondrial (class I PI3K isoforms inhibitor); Rapa, rapamycin. Blots are representative of three ROS measured by MitoSOX staining after overnight treatment with IL-1b/TNFa and independent experiments. (E) Analysis of S6 phosphorylation determined by A38 or rapamycin. n = 6 samples per group. Statistical analysis: one-way ANOVA and intracellular staining and flow cytometry of astrocytes stimulated as in (D). Tukey post-test. Data are representative of three independent experiments. Data Statistical analysis: one way ANOVA and Tukey post-test. (F) Il6, Ccl2, Tnfa, Csf2, shown as mean ± SEM. *P <0.05,**P <0.01,***P < 0.001.

an unbiased, high-throughput, and acces- sequencing (78) and lowering the barrier shown to be co-opted in the context of cancer sible technology for the study of cell in- to technology adoption. and inflammation (80, 81), but their partici- teractions. RABID-seq uses glycoprotein Microglia-astrocyte communication plays pation in microglia-astrocyte interactions was G–deficient pseudotypedrabiesvirusengi- a central role in CNS physiology (79). Using unknown. We identified a role for Sema4D- neered to express a unique mCherry mRNA RABID-seq, we identified axon guidance mol- PlexinB1 and Sema4D-PlexinB2 interactions barcode to capture interactions directly from ecules as participants in microglia-astrocyte in the microglial control of astrocytes during transcriptomic data, thereby exploiting the communicationinthecontextofCNSinflam- EAE and MS. Sema4D is described to par- maturity and ubiquity of next-generation mation. Axon guidance molecules have been ticipate in neurodevelopment (82, 83), T cell

Clark et al., Science 372, eabf1230 (2021) 23 April 2021 12 of 15 RESEARCH | RESEARCH ARTICLE activation (36, 84), and T cell–driven microg- blockade of Ephrin-B3–EphB3 signaling may ular biology to enable reverse transcription lial activation in EAE (36, 85). Our findings interfere with disease-promoting responses with template switching in drops. Illumina suggest that developing MS therapies that in astrocytes and microglia, as well as addi- sequencing libraries for transcriptome se- target SEMA4D could be improved if the tional mechanisms associated with MS pathol- quencing were prepared from purified whole- therapeutic agent reaches the CNS (86). ogy and linked to this pathway, including the transcriptome–amplified product using an Most microglia-astrocyte interactions known disruption of the blood-brain barrier (109)and adapter ligation approach with the NEBNext to participate in the control of CNS inflam- the inhibition of remyelination (110). In this Ultra II FS Kit (NEB, no. E7805). Illumina mation are mediated by soluble factors context, CNS-penetrant small molecules, such sequencing libraries for rabies barcode se- (2, 5, 6, 11, 12, 58, 87). We identified Ephrin- as A38 described in this work, provide bet- quencing were prepared from the same ma- B3–EphB3 signaling as a participant in microglia- ter therapeutic approaches than other Eph- terial using a two-step nested PCR protocol. astrocyte contact–dependent cross-talk. Eph targeting therapies (111) that have limited InDrops scRNA-seq data were processed using receptor signaling plays important roles in access to the CNS. However, potential off- thepubliclyavailablebioinformaticspipeline development (43); immune regulation (88); target effects are possible as a result of the (https://github.com/indrops/indrops)(117). maintenance of epithelial architecture (89); multiple cell- and context-specific signaling Connectome data were analyzed using scripts and control of neural progenitor prolifera- mechanisms that have been linked to Eph developed for this work that are publicly avail- tion, axonal guidance, and synapse formation receptors (43, 112, 113). able on GitHub at https://github.com/Zha0rong/ (90–92). Eph receptors participate in astrocyte- In summary, we developed RABID-seq, an RABID-seq. neuron communication (93–95), which sug- approach for the high-throughput identifica- gests that, in the context of inflammation, tion of cell interactions and the molecular Ephrin-B3–EphB3 signaling may mediate phenotype of interacting cells with single-cell REFERENCES AND NOTES interactions of astrocytes with cell types other resolution. RABID-seq enabled the identifica- 1. M. A. Anderson et al., Astrocyte scar formation aids Downloaded from central nervous system axon regeneration. Nature 532, than microglia. Notably, EphA receptor up- tion of microglia-astrocyte interactions mediated 195–200 (2016). doi: 10.1038/nature17623; regulation in astrocytes has been reported in by the axon guidance molecules Sema4D- pmid: 27027288 MS patients (96), and EphB2 (1) and EphB3 PlexinB1, Sema4D-PlexinB2, and Ephrin-B3– 2. S. A. Liddelow et al., Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017). (97) are increased in astrocytes after spinal EphB3, which represent candidate targets for doi: 10.1038/nature21029; pmid: 28099414 cord injury. Eph receptor signaling has also therapeutic intervention in MS and other 3. M. A. Wheeler et al., MAFG-driven astrocytes promote CNS inflammation. Nature 578, 593–599 (2020). doi: 10.1038/ been linked to the pathology of Alzheimer’s neurologic disorders. http://science.sciencemag.org/ ’ s41586-020-1999-0; pmid: 32051591 disease (98), Parkinson s disease (99), amy- 4. A. D. Greenhalgh, S. David, F. C. Bennett, Immune cell otrophic lateral sclerosis (100), and schizophrenia Materials and methods summary regulation of glia during CNS injury and disease. Nat. Rev. (101). In this sense, Eph receptor signaling The supplementary materials provide a de- Neurosci. 21, 139–152 (2020). doi: 10.1038/s41583-020- 0263-9; pmid: 32042145 resembles IL-33 (11, 102) and complement tailed description of our materials and methods. 5. L. Mayo et al., Regulation of astrocyte activation by (103–610 ), which participate in CNS develop- AdultC57BL/6J(no.000664)andNOD/ShiLtJ glycolipids drives chronic CNS inflammation. Nat. Med. 20, ment but are also reactivated in neurologic (NOD mice) (no. 001976) were obtained from 1147–1156 (2014). doi: 10.1038/nm.3681; pmid: 25216636 6. V. Rothhammer et al., Type I interferons and microbial diseases. However, a distinguishing feature The Jackson Laboratory. 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expressing cells (46 50), which amplifies homozygous B6;129P2-Gt(ROSA)26Sortm1(CAG-

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Clark et al., Science 372, eabf1230 (2021) 23 April 2021 15 of 15 Barcoded viral tracing of single-cell interactions in central nervous system inflammation Iain C. Clark, Cristina Gutiérrez-Vázquez, Michael A. Wheeler, Zhaorong Li, Veit Rothhammer, Mathias Linnerbauer, Liliana M. Sanmarco, Lydia Guo, Manon Blain, Stephanie E. J. Zandee, Chun-Cheih Chao, Katelyn V. Batterman, Marius Schwabenland, Peter Lotfy, Amalia Tejeda-Velarde, Patrick Hewson, Carolina Manganeli Polonio, Michael W. Shultis, Yasmin Salem, Emily C. Tjon, Pedro H. Fonseca-Castro, Davis M. Borucki, Kalil Alves de Lima, Agustin Plasencia, Adam R. Abate, Douglas L. Rosene, Kevin J. Hodgetts, Marco Prinz, Jack P. Antel, Alexandre Prat and Francisco J. Quintana

Science 372 (6540), eabf1230. DOI: 10.1126/science.abf1230

Single-cell analysis of CNS interactions Despite their importance in the physiology and pathology of the central nervous system (CNS), few methods are Downloaded from available for the unbiased, systematic investigation of cell-to-cell interactions at single-cell resolution. Clark et al. developed RABID-seq, a method that combines barcoded viral tracing with single-cell RNA sequencing (see the Perspective by Silvin and Ginhoux). RABID-seq identified the axon guidance molecules Sema4D-PlexinB2 and EphrinB3-EphB3 as mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis and, potentially, multiple sclerosis. These studies also identified candidate therapeutic molecules for the modulation of microglia-astrocyte interactions in multiple sclerosis. Science, this issue p. eabf1230; see also p. 342 http://science.sciencemag.org/

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