Easing Microglial Brakes Alleviates Amyloid Pathology in Mice

Rather than giving them more gas, can microglia be driven to clear amyloid plaques by letting up on their brakes? Yes, say scientists led by Marco Colonna at Washington University in St. Louis. In the April 3 Science Translational Medicine, they describe an inhibitory microglial receptor, LILRB4, that is upregulated around plaques in Alzheimer’s and in mouse models of amyloidosis. An antibody against LILRB4 enabled microglia to curtail amyloid accumulation in mice. How? By preventing the LILRB4 ligand, ApoE, from binding the receptor, the antibody suppressed LILRB4 activation, leaving microglia free to respond to plaques.

  • Microglia expressing LILRB4 surround plaques in AD cortical tissue.
  • LILRB4 dampens microglial response to plaques in mice.
  • Mice given a LILRB4 antibody had more phagocytic microglia, fewer plaques.

“This is an important paper given microglial activity must be carefully controlled, such as by the immune checkpoint inhibitory receptor LILRB4,” Zhiqiang An at the University of Texas Health Science Center, Houston, told Alzforum.

Others praised the research, too. “LILRB4 is a beautiful target specific to microglia engaged with plaques, and this antibody induces a novel immunomodulatory response,” said Oleg Butovsky of Brigham and Women’s Hospital in Boston. “This is a very interesting paper on microglial mechanisms in AD pathogenesis with implications for potential drug development,” Yadong Huang, University of California, San Francisco, told Alzforum.

Leukocyte Ig-like receptors (LILRs) are a group of 11 microglial markers; five activate the cells, five inhibit them. Among the latter are LILRB2 and LILRB4. LILRB2 binds toxic Aβ oligomers, leading to synapse loss (Oct 2018 news; Jun 2021 news). LILRB4 binds ApoE and is highly expressed by microglia surrounding plaques in amyloidosis mice, aka disease-associated microglia (Deng et al., 2018; Yin et al., 2023; Sep 2023 news). Genome-wide association studies pegged the locus containing both LILRB2 and LILRB4 as a risk factor for AD (Sep 2021 news; Apr 2022 news).

To understand how LILRB2 and B4 are expressed in the AD brain, co-first authors Jinchao Hou and Yun Chen ran single-nucleus RNA-Seq on microglia from postmortem cortical tissue. Compared to microglia from healthy people, those from AD cases highly expressed LILRB4 and moderately upregulated LILRB2. Immunohistochemistry revealed more LILRB4-positive microglia in AD cases, especially around amyloid plaques (image below).

Sprouting LILRB4. While ramified microglia (green) hung out in the cortex of a healthy adult (left), microglia expressing LILRB4 (red) surrounded an ApoE-positive (white) amyloid plaque (blue) in a person who had had AD (right). [Courtesy of Hou et al., Science Translational Medicine, 2024.]

The scientists recapitulated these changes in mice by crossing 5xFAD animals with those carrying a 200-kb chunk of the human LILR gene, which encoded LILRB4 and a few other LILRs. Hou and colleagues used this construct to maintain the promotor and enhancer elements driving LILRB4 in human cells. In 6-month-old offspring, microglia expressed more LILRB4 than 5xFAD controls, and the crosses accumulated more plaques.

Intriguingly, fewer LILRB4-positive microglia surrounded amyloid plaques in the offspring, suggesting that human LILRB4 caused the cells to respond poorly to plaques. The data hint that LILRB4-positive microglia near plaques in AD may poorly clear amyloid.

Might blocking the receptor improve clearance? The scientists created an antibody against LILRB4 called ZM3.1, then injected it into the abdomens of 4-month-old 5xFAD/LILRB4 mice weekly for two months. In treated animals, 1.5 times as many Iba1-positive microglia surrounded amyloid plaques in the cortex than in controls, and the microglia upregulated phagocytic genes and engulfed almost twice as many Aβ fibrils. Treated animals also had half as many plaques in the hippocampus, cortex, and amygdala and fewer nearby dystrophic neurites (image below). Taken together, the results suggest that blocking LILRB4 switched microglia into gear to clear plaques.

Releasing the Brakes. Compared to a 5xFAD mouse expressing human LILRB4 (left), one given a LILRB4 antibody (right) had half as many amyloid plaques (top) and half as many dystrophic neurites as measured by BACE1 levels (red, bottom). [Courtesy of Hou et al., Science Translational Medicine, 2024.]

The immunotherapy’s effects on behavior were less clear. Mice given ZM3.1 spent less time in the open arms of an elevated plus maze, i.e., had less anxiety, than untreated animals. However, they still took just as long as untreated 5xFAD mice to find an underwater platform. Nevertheless, Colonna sees this as a positive. “Changes in behavior reflect the antibody's ability to impact function,” he told Alzforum, “regardless of what that function is.”

How did ZM3.1 reduce pathology? In vitro binding experiments showed the antibody thwarting recombinant LILRB4’s binding to mouse ApoE, whether attached to Aβ fibrils or as a free lipoprotein. ZM3.1 also prevented LILRB4 from binding human ApoE3 or E4. In silico modeling and mutagenesis experiments homed in on a spot where both ApoE and ZM3.1 bind LILRB4: a loop between two extracellular Ig domains of the receptor.

All told, the authors believe that ZM3.1 binds to LILRB4 on microglia, preventing ApoE from accessing and activating the receptor. This keeps the metaphorical foot off the microglial brake, allowing the cells to respond to plaques (image below).

Blocking LILRB4 Unleashes Microglia. In AD (left), ApoE in lipoproteins (yellow disks) or plaques (brown squiggles) binds LILRB4 (purple hook) on microglia (blue cells). This keeps the cells silent, allowing amyloid pathology to ravage nearby neurons (brown cells). Treatment (right) with the antibody ZM3.1 (green Y) prevents ApoE-LILRB4 binding, letting microglia tame amyloid plaques and safeguard neurons. [Courtesy of Yun Chen, WashU.]

An sees therapeutic potential for ZM3.1. “It seems feasible to develop LILRB4 neutralizing antibodies to activate microglia cells to reduce plaque accumulation in AD patients,” he said. Butovsky wondered how the immunotherapy might work in APOE4 carriers, since the isoform prevents expression of disease-associated microglia genes, such as LILRB4 (Oct 2023 news).

Colonna is working with a Japanese pharmaceutical company to bring ZM3.1 into clinical trials for AD, though they do not have a timeline yet. “We still have to find the best drug to activate microglia, so it is important to test more antibodies and small molecules,” Colonna said.—Chelsea Weidman Burke

Comments

  1. This is very interesting work, as it dives into the plethora of receptors that control microglial function. For very good reasons, the field has focused extensively on some specific molecules, such as TREM2 or CD33, but microglia express a vast diversity of activation (ITAMs) and inhibitory (ITIMs) receptors. The functional output of microglia will be the result of a complex computing of the integrated signal through all these receptors. In this manuscript, the authors focus on LILRB4, which is considered an inhibitory receptor.

    The humanization strategy of the LILRB4 receptor is very elegant and adds stronger translational value to the work. There are some intriguing pieces of data, especially regarding the impact of the anti-LILRB4 treatment. The histopathological analysis shows increased accumulation of microglia around amyloid plaques upon treatment. However, the differential expression analysis shows a significant reduction in disease-associated microglia (DAM) genes such as Cd9, Alx, and Cd63. Could the treatment elicit a specific subtype of DAMs with protective function? Could this indicate that DAMs are more complex than we think, and that different subsets will engage in different functions? This could potentially open new and very exciting avenues of research.

    Like every good paper, this thought-provoking piece of work leaves many questions. Is ApoE the only endogenous ligand of LILRB4? What is the nature of the protective microglia subtype elicited by the treatment? How does it compare with those that arise after TREM2 activation of anti-Ab immunotherapy? 

    View all comments by Renzo Mancuso

  2. Microglia with unique transcriptional signatures have been identified in Alzheimer’s disease patients and animal models (Keren-Shaul et al., 2017). Understanding the regulation and impact of these states is essential if we want to know how to modulate microglia for therapeutic purposes. Transcription factors are starting to emerge (Gosselin et al., 2017) and are necessary for understanding the regulation of these complex transcriptional states. However, cell surface receptors such as TREM2, CD33, and here, LILRB4 are particularly appropriate for therapeutic approaches.

    In this manuscript by Hou, Chen et al., the authors describe LILRB4 as a modulator of microglia. They evaluate the impact of LILRB4 on transcriptional states, microglia phagocytosis, and its overall impact on amyloid pathology. The development and characterization of an antibody that crosses the blood-brain barrier is critical here, and well done. This study also highlights the importance of developing the right tools to broadly understand microglia in disease; targeting human genes and assessing their impact on microglia transcription and function. 

    One caveat the authors highlight is the use of male amyloidosis mice only. While they emphasize the importance of testing it in models that recapitulate other hallmarks of diseases, such as tau and cerebral amyloid angiopathy, and in male and female animals, I would also emphasize the importance of testing LILRB4 in xenograft models. It is now possible to evaluate the impact on human iPSC-derived microglia transplanted in the mouse brain (Mancuso et al., 2019; Hasselmann et al., 2019) which would confirm the findings in human microglia.  Overall, the authors define a very clear path for the identification and validation of new regulators of microglia states that could be highly promising therapeutically. 

    References:

    Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, David E, Baruch K, Lara-Astaiso D, Toth B, Itzkovitz S, Colonna M, Schwartz M, Amit I. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017 Jun 15;169(7):1276-1290.e17. Epub 2017 Jun 8 PubMed.

    Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki JC, Sajti E, Jaeger BN, O'Connor C, Fitzpatrick C, Pasillas MP, Pena M, Adair A, Gonda DD, Levy ML, Ransohoff RM, Gage FH, Glass CK. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017 Jun 23;356(6344) Epub 2017 May 25 PubMed.

    Mancuso R, Van Den Daele J, Fattorelli N, Wolfs L, Balusu S, Burton O, Liston A, Sierksma A, Fourne Y, Poovathingal S, Arranz-Mendiguren A, Sala Frigerio C, Claes C, Serneels L, Theys T, Perry VH, Verfaillie C, Fiers M, De Strooper B. Stem-cell-derived human microglia transplanted in mouse brain to study human disease. Nat Neurosci. 2019 Dec;22(12):2111-2116. Epub 2019 Oct 28 PubMed.

    Hasselmann J, Coburn MA, England W, Figueroa Velez DX, Kiani Shabestari S, Tu CH, McQuade A, Kolahdouzan M, Echeverria K, Claes C, Nakayama T, Azevedo R, Coufal NG, Han CZ, Cummings BJ, Davtyan H, Glass CK, Healy LM, Gandhi SP, Spitale RC, Blurton-Jones M. Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo. Neuron. 2019 Sep 25;103(6):1016-1033.e10. Epub 2019 Jul 30 PubMed.

    View all comments by Martine Therrien

  3. This is an exciting study by Hou and colleagues showing that modulating LILRB4 using an antibody likely inhibits LILRB4 binding to APOE, activating microglia and attenuating Aβ-related measures, such as plaque load and neuronal dystrophy. This study follows on from important earlier work by Kim and colleagues (2013) and Zhao and colleagues (2022) showing that the related inhibitory receptor LILRB2 binds Aβ. Additionally, with Valentina Escott-Price we showed gene variation in LILRB4 associated with Alzheimer’s disease risk (2019), and Bellenguez and colleagues identified variation for LILRB2 associated with dementia (2022). 

    This study by Hou et al. is thorough, well-powered, and well-reasoned. In future work, it would be good to explore which sub-population, or state of microglia, express LILRB4, as would be seen by single-cell RNA-Seq, and whether these are homeostatic, amyloid-responsive, disease-associated microglia, or interferon-responsive. It would be useful to know how the anti-LILRB4 antibody shifts microglia between different states. The data showing a positive association of the anti-LILRB4 antibody with phagocytic pathways, and a negative association with interferon pathways is interesting.

    The authors showed P35, T30, and Y121 residues of LILRB4 contributed to APOE binding. In future work it would be good to see how LILRB4 carrying mutations of these residues affect Aβ load in mice—one would hypothesize reduced Aβ load—then test anti-LILRB4 antibodies in these animals. Investigating which residues this anti-LILRB4 ZM3.1 antibody, and other related antibodies, bind alongside APOE, may help increase clinical utility. Furthermore, it would be useful to understand if the anti-LILRB4 antibody mediates changes in Aβ pathology’s impact upon tau.

    This careful study opens new opportunities to modulate microglial activity in Alzheimer’s disease.

    References:

    Kim T, Vidal GS, Djurisic M, William CM, Birnbaum ME, Garcia KC, Hyman BT, Shatz CJ. Human LilrB2 is a β-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model. Science. 2013 Sep 20;341(6152):1399-404. PubMed.

    Zhao P, Xu Y, Jiang LL, Fan X, Ku Z, Li L, Liu X, Deng M, Arase H, Zhu JJ, Huang TY, Zhao Y, Zhang C, Xu H, Tong Q, Zhang N, An Z. LILRB2-mediated TREM2 signaling inhibition suppresses microglia functions. Mol Neurodegener. 2022 Jun 18;17(1):44. PubMed.

    Salih DA, Bayram S, Guelfi S, Reynolds RH, Shoai M, Ryten M, Brenton JW, Zhang D, Matarin M, Botia JA, Shah R, Brookes KJ, Guetta-Baranes T, Morgan K, Bellou E, Cummings DM, Escott-Price V, Hardy J. Genetic variability in response to amyloid beta deposition influences Alzheimer's disease risk. Brain Commun. 2019;1(1):fcz022. Epub 2019 Oct 10 PubMed.

    Bellenguez C, Küçükali F, Jansen IE, Kleineidam L, Moreno-Grau S, Amin N, Naj AC, Campos-Martin R, Grenier-Boley B, Andrade V, Holmans PA, Boland A, Damotte V, van der Lee SJ, Costa MR, Kuulasmaa T, Yang Q, de Rojas I, Bis JC, Yaqub A, Prokic I, Chapuis J, Ahmad S, Giedraitis V, Aarsland D, Garcia-Gonzalez P, Abdelnour C, Alarcón-Martín E, Alcolea D, Alegret M, Alvarez I, Álvarez V, Armstrong NJ, Tsolaki A, Antúnez C, Appollonio I, Arcaro M, Archetti S, Pastor AA, Arosio B, Athanasiu L, Bailly H, Banaj N, Baquero M, Barral S, Beiser A, Pastor AB, Below JE, Benchek P, Benussi L, Berr C, Besse C, Bessi V, Binetti G, Bizarro A, Blesa R, Boada M, Boerwinkle E, Borroni B, Boschi S, Bossù P, Bråthen G, Bressler J, Bresner C, Brodaty H, Brookes KJ, Brusco LI, Buiza-Rueda D, Bûrger K, Burholt V, Bush WS, Calero M, Cantwell LB, Chene G, Chung J, Cuccaro ML, Carracedo Á, Cecchetti R, Cervera-Carles L, Charbonnier C, Chen HH, Chillotti C, Ciccone S, Claassen JA, Clark C, Conti E, Corma-Gómez A, Costantini E, Custodero C, Daian D, Dalmasso MC, Daniele A, Dardiotis E, Dartigues JF, de Deyn PP, de Paiva Lopes K, de Witte LD, Debette S, Deckert J, Del Ser T, Denning N, DeStefano A, Dichgans M, Diehl-Schmid J, Diez-Fairen M, Rossi PD, Djurovic S, Duron E, Düzel E, Dufouil C, Eiriksdottir G, Engelborghs S, Escott-Price V, Espinosa A, Ewers M, Faber KM, Fabrizio T, Nielsen SF, Fardo DW, Farotti L, Fenoglio C, Fernández-Fuertes M, Ferrari R, Ferreira CB, Ferri E, Fin B, Fischer P, Fladby T, Fließbach K, Fongang B, Fornage M, Fortea J, Foroud TM, Fostinelli S, Fox NC, Franco-Macías E, Bullido MJ, Frank-García A, Froelich L, Fulton-Howard B, Galimberti D, García-Alberca JM, García-González P, Garcia-Madrona S, Garcia-Ribas G, Ghidoni R, Giegling I, Giorgio G, Goate AM, Goldhardt O, Gomez-Fonseca D, González-Pérez A, Graff C, Grande G, Green E, Grimmer T, Grünblatt E, Grunin M, Gudnason V, Guetta-Baranes T, Haapasalo A, Hadjigeorgiou G, Haines JL, Hamilton-Nelson KL, Hampel H, Hanon O, Hardy J, Hartmann AM, Hausner L, Harwood J, Heilmann-Heimbach S, Helisalmi S, Heneka MT, Hernández I, Herrmann MJ, Hoffmann P, Holmes C, Holstege H, Vilas RH, Hulsman M, Humphrey J, Biessels GJ, Jian X, Johansson C, Jun GR, Kastumata Y, Kauwe J, Kehoe PG, Kilander L, Ståhlbom AK, Kivipelto M, Koivisto A, Kornhuber J, Kosmidis MH, Kukull WA, Kuksa PP, Kunkle BW, Kuzma AB, Lage C, Laukka EJ, Launer L, Lauria A, Lee CY, Lehtisalo J, Lerch O, Lleó A, Longstreth W Jr, Lopez O, de Munain AL, Love S, Löwemark M, Luckcuck L, Lunetta KL, Ma Y, Macías J, MacLeod CA, Maier W, Mangialasche F, Spallazzi M, Marquié M, Marshall R, Martin ER, Montes AM, Rodríguez CM, Masullo C, Mayeux R, Mead S, Mecocci P, Medina M, Meggy A, Mehrabian S, Mendoza S, Menéndez-González M, Mir P, Moebus S, Mol M, Molina-Porcel L, Montrreal L, Morelli L, Moreno F, Morgan K, Mosley T, Nöthen MM, Muchnik C, Mukherjee S, Nacmias B, Ngandu T, Nicolas G, Nordestgaard BG, Olaso R, Orellana A, Orsini M, Ortega G, Padovani A, Paolo C, Papenberg G, Parnetti L, Pasquier F, Pastor P, Peloso G, Pérez-Cordón A, Pérez-Tur J, Pericard P, Peters O, Pijnenburg YA, Pineda JA, Piñol-Ripoll G, Pisanu C, Polak T, Popp J, Posthuma D, Priller J, Puerta R, Quenez O, Quintela I, Thomassen JQ, Rábano A, Rainero I, Rajabli F, Ramakers I, Real LM, Reinders MJ, Reitz C, Reyes-Dumeyer D, Ridge P, Riedel-Heller S, Riederer P, Roberto N, Rodriguez-Rodriguez E, Rongve A, Allende IR, Rosende-Roca M, Royo JL, Rubino E, Rujescu D, Sáez ME, Sakka P, Saltvedt I, Sanabria Á, Sánchez-Arjona MB, Sanchez-Garcia F, Juan PS, Sánchez-Valle R, Sando SB, Sarnowski C, Satizabal CL, Scamosci M, Scarmeas N, Scarpini E, Scheltens P, Scherbaum N, Scherer M, Schmid M, Schneider A, Schott JM, Selbæk G, Seripa D, Serrano M, Sha J, Shadrin AA, Skrobot O, Slifer S, Snijders GJ, Soininen H, Solfrizzi V, Solomon A, Song Y, Sorbi S, Sotolongo-Grau O, Spalletta G, Spottke A, Squassina A, Stordal E, Tartan JP, Tárraga L, Tesí N, Thalamuthu A, Thomas T, Tosto G, Traykov L, Tremolizzo L, Tybjærg-Hansen A, Uitterlinden A, Ullgren A, Ulstein I, Valero S, Valladares O, Broeckhoven CV, Vance J, Vardarajan BN, van der Lugt A, Dongen JV, van Rooij J, van Swieten J, Vandenberghe R, Verhey F, Vidal JS, Vogelgsang J, Vyhnalek M, Wagner M, Wallon D, Wang LS, Wang R, Weinhold L, Wiltfang J, Windle G, Woods B, Yannakoulia M, Zare H, Zhao Y, Zhang X, Zhu C, Zulaica M, EADB, GR@ACE, DEGESCO, EADI, GERAD, Demgene, FinnGen, ADGC, CHARGE, Farrer LA, Psaty BM, Ghanbari M, Raj T, Sachdev P, Mather K, Jessen F, Ikram MA, de Mendonça A, Hort J, Tsolaki M, Pericak-Vance MA, Amouyel P, Williams J, Frikke-Schmidt R, Clarimon J, Deleuze JF, Rossi G, Seshadri S, Andreassen OA, Ingelsson M, Hiltunen M, Sleegers K, Schellenberg GD, van Duijn CM, Sims R, van der Flier WM, Ruiz A, Ramirez A, Lambert JC. New insights into the genetic etiology of Alzheimer's disease and related dementias. Nat Genet. 2022 Apr;54(4):412-436. Epub 2022 Apr 4 PubMed.

    View all comments by Dervis Salih

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Crystal Structure of Aβ and Proposed Receptor Solved
  2. Aβ 'Receptors' Retard Fibrillization, Enhancing Toxicity
  3. PLCγ2 Variants Toggle Microglial Plaque Compactors
  4. From a Million Samples, GWAS Squeezes Out Seven New Alzheimer's Spots
  5. Paper Alert: Massive GWAS Meta-Analysis Published
  6. In Amyloid and Tangle Models, APOE4 Paralyzes Microglia

Research Models Citations

  1. 5xFAD (C57BL6)

Paper Citations

  1. Deng M, Gui X, Kim J, Xie L, Chen W, Li Z, He L, Chen Y, Chen H, Luo W, Lu Z, Xie J, Churchill H, Xu Y, Zhou Z, Wu G, Yu C, John S, Hirayasu K, Nguyen N, Liu X, Huang F, Li L, Deng H, Tang H, Sadek AH, Zhang L, Huang T, Zou Y, Chen B, Zhu H, Arase H, Xia N, Jiang Y, Collins R, You MJ, Homsi J, Unni N, Lewis C, Chen GQ, Fu YX, Liao XC, An Z, Zheng J, Zhang N, Zhang CC. LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration. Nature. 2018 Oct;562(7728):605-609. Epub 2018 Oct 17 PubMed.
  2. Yin Z, Rosenzweig N, Kleemann KL, Zhang X, Brandão W, Margeta MA, Schroeder C, Sivanathan KN, Silveira S, Gauthier C, Mallah D, Pitts KM, Durao A, Herron S, Shorey H, Cheng Y, Barry JL, Krishnan RK, Wakelin S, Rhee J, Yung A, Aronchik M, Wang C, Jain N, Bao X, Gerrits E, Brouwer N, Deik A, Tenen DG, Ikezu T, Santander NG, McKinsey GL, Baufeld C, Sheppard D, Krasemann S, Nowarski R, Eggen BJ, Clish C, Tanzi RE, Madore C, Arnold TD, Holtzman DM, Butovsky O. APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFβ-mediated checkpoints. Nat Immunol. 2023 Nov;24(11):1839-1853. Epub 2023 Sep 25 PubMed.

Further Reading

Primary Papers

  1. Hou J, Chen Y, Cai Z, Heo GS, Yuede CM, Wang Z, Lin K, Saadi F, Trsan T, Nguyen AT, Constantopoulos E, Larsen RA, Zhu Y, Wagner ND, McLaughlin N, Kuang XC, Barrow AD, Li D, Zhou Y, Wang S, Gilfillan S, Gross ML, Brioschi S, Liu Y, Holtzman DM, Colonna M. Antibody-mediated targeting of human microglial leukocyte Ig-like receptor B4 attenuates amyloid pathology in a mouse model. Sci Transl Med. 2024 Apr 3;16(741):eadj9052. PubMed.

Annotate

To make an annotation you must Login or Register.