Project 6 Microglia in AMD

Role of complement receptors and microglia in age-related macular degeneration

Summary

Age-related macular degeneration (AMD) is a leading cause of blindness in industrialized countries. Early AMD manifests as drusen deposits between the outer retina and the pigment epithelium. AMD can progress to late stage forms including choroidal neovascularization (CNV) and an atrophic form with macular degeneration of photoreceptors and the pigment epithelium. The etiology of AMD is not completely understood. However, genetic association studies in large patient cohorts and in situ analyses in AMD eyes as well as animal models mimicking some features of AMD have identified an important contribution of dysregulated innate immunity in the eye. Our project of the first funding period of FOR 2240 has revealed local dysregulation of complement factors in ocular fluids of AMD patients and found a correlation of disease progression with hyperreflective foci (HF) as potential imaging markers for retinal immune cells. Furthermore, using a light damage paradigm and laser-triggered CNV in mice we established a comprehensive analysis of microglia and macrophages in the retina. Resident microglia-specific targeting of key immune pathways and immunomodulatory compounds could elucidate a vicious cycle of microglia reactivity, retinal degeneration and neoangiogenesis. These findings together provide evidence for an interaction of reactive microglia with aberrant complement proteins in the pathogenesis of AMD. However, the underlying mechanisms that link both systems are only incompletely explored. In this follow-up project we postulate that particularly the anaphylatoxin complement cleavage products C3a and C5a sustain chronic microglia reactivity via receptormediated recruitment and activation. We further postulate that hyperreflective foci could potentially mirror complement-regulated microglia responses and may therefore be used to monitor disease development in AMD patients and animal models. The project is subdivided into four specific aims in which we will determined the impact of C3a/C5a anaphylatoxins on microglia in vitro, test the effects of microglial C3a/C5a receptor deficiency in the laser-CNV and light damage models, evaluate the expression and localization of C3aR/C5aR in human retinal sections, and finally correlate the dynamics of hyperreflective foci with ocular complement factors in AMD patients and controls. The results of these studies will provide new insights into the immune-related etiology of AMD and may foster the development of novel effective treatments.

Age-related macular degeneration (AMD) is a complex disease leading to vision loss in the elderly. The pathogenesis of AMD is multifactorial and involves several genetic risk factors related to the complement system, a key component of the innate immune system, extracellular matrix biology, and age-related oxidative stress. In situ analyses of post mortem retinas from AMD patients and AMD-like animal models demonstrated reactivity of microglial cells, the resident immune cells of the retina. Our previous work contributed to the identification of risk variants in complement genes in AMD and showed aberrant complement distribution in human AMD retinas. Moreover, we have generated Htra1 transgenic mice, which model pathological changes in Bruch´s membrane, and detected prominent subretinal microglial reactivity. In this project, we first plan to comprehensively characterize the immunological retinal phenotype of Htra1 Tg / Cfh-/- mice, which mirror two key components implicated in AMD. We will study local complement factor expression and distribution, investigate the microglial activity of Htra1 Tg / Cfh-/- mice in situ and ex vivo and study their pro-inflammatory potential by transplantation into healthy retinas. These mouse studies will be complemented by studies in human AMD patients and controls, involving a comparison of systemic versus local ocular complement activation and correlation with malondialdehyde (MDA) as marker for oxidative stress. Moreover, we will evaluate whether hyperreflective dots seen with SD-OCT in neovascular AMD patients are a potential indicator for retinal immune cell reactivity and correlate these findings with complement biomarkers and genetic risk factors. We expect from our studies deeper insight into the role of retinal complement factors and microglia as mediators of chronic inflammation in AMD.

Most Recent Publications of Project 6

Akhtar-Schafer I, Wang L, Krohne TU, Xu H, Langmann T (2018) Modulation of three key innate immune pathways for the most common retinal degenerative diseases. EMBO molecular medicine 10

Selected Key Publications of Project 6

Luckoff A, Scholz R, Sennlaub F, Xu H, Langmann T (2017) Comprehensive analysis of mouse retinal mononuclear phagocytes. Nature protocols 12: 1136-1150

Schick T, Steinhauer M, Aslanidis A, Altay L, Karlstetter M, Langmann T, Kirschfink M, Fauser S (2017) Local complement activation in aqueous humor in patients with age-related macular degeneration. Eye (London, England) 31: 810-813

Karlstetter M, Kopatz J, Aslanidis A, Shahraz A, Caramoy A, Linnartz-Gerlach B, Lin Y, Luckoff A, Fauser S, Duker K, Claude J, Wang Y, Ackermann J, Schmidt T, Hornung V, Skerka C, Langmann T*, Neumann H* (2017) Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation, and protects from vascular damage in the retina. EMBO molecular medicine 9: 154-166. *shared senior authors

Altay L, Scholz P, Schick T, Felsch M, Hoyng CB, den Hollander AI, Langmann T*, Fauser S* (2016) Association of Hyperreflective Foci Present in Early Forms of Age-Related Macular Degeneration With Known Age-Related Macular Degeneration Risk Polymorphisms. Investigative ophthalmology & visual science 57: 4315-20. *shared senior authors

Luckoff A, Caramoy A, Scholz R, Prinz M, Kalinke U, Langmann T (2016) Interferon-beta signaling in retinal mononuclear phagocytes attenuates pathological neovascularization. EMBO molecular medicine 8: 670-8

Fritsche LG et al., Ersoy L, Caramoy A, Langmann T et al.,  Fauser S et al., Heid IM (2016) A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nature Genetics 48(2):134-43

Dannhausen K, Karlstetter M, Caramoy A, Volz C, Jagle H, Liebisch G, Utermohlen O, Langmann T (2015) Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function. Biochemical and biophysical research communications 464: 434-40

Karlstetter M, Scholz R, Rutar M, Wong WT, Provis JM, Langmann T (2015) Retinal microglia: just bystander or target for therapy? Progress in retinal and eye research 45: 30-57

Scholz R, Caramoy A, Bhuckory MB, Rashid K, Chen M, Xu H, Grimm C, Langmann T (2015) Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration. Journal of neuroinflammation 12: 201

Scholz R, Sobotka M, Caramoy A, Stempfl T, Moehle C, Langmann T (2015) Minocycline counter-regulates pro-inflammatory microglia responses in the retina and protects from degeneration. Journal of neuroinflammation 12: 209

More Publications of Project 6

Cursiefen C, Bock F, Clahsen T, Regenfuss B, Reis A, Steven P, Heindl LM, Bosch JJ, Hos D, Eming S, Grajewski R, Heiligenhaus A, Fauser S, Austin J, Langmann T. [New Therapeutic Approaches in Inflammatory Diseases of the Eye – Targeting Lymphangiogenesis and Cellular Immunity: Research Unit FOR 2240 Presents Itself]. Klin Monbl Augenheilkd. 2017 May;234(5):679-685.

Langmann T, Fauser S. [Polysialic Acid for Immunomodulation in an Animal Model for Wet Age-Related Macular Degeneration (AMD)]. Klin Monbl Augenheilkd. 2017 May;234(5):657-661.

Martens TF, Peynshaert K, Nascimento TL, Fattal E, Karlstetter M, Langmann T, Picaud S, Demeester J, De Smedt SC, Remaut K, Braeckmans K. Effect of hyaluronic  acid-binding to lipoplexes on intravitreal drug delivery for retinal gene therapy. Eur J Pharm Sci. 2017 May 30;103:27-35.

Grassmann F, Kiel C, Zimmermann ME, Gorski M, Grassmann V, Stark K; International AMD Genomics Consortium (IAMDGC), Heid IM, Weber BH. Genetic pleiotropy between age-related macular degeneration and 16 complex diseases and traits. Genome Med. 2017 Mar 27;9(1):29.

Nebel C, Aslanidis A, Rashid K, Langmann T. Activated microglia trigger inflammasome activation and lysosomal destabilization in human RPE cells. Biochem Biophys Res Commun. 2017 Mar 11;484(3):681-686.

Wolf A, Aslanidis A, Langmann T. Retinal expression and localization of Mef2c support its important role in photoreceptor gene expression. Biochem Biophys Res  Commun. 2017 Jan 29;483(1):346-351.

Micklisch S, Lin Y, Jacob S, Karlstetter M, Dannhausen K, Dasari P, von der Heide M, Dahse HM, Schmölz L, Grassmann F, Alene M, Fauser S, Neumann H, Lorkowski S, Pauly D, Weber BH, Joussen AM, Langmann T, Zipfel PF, Skerka C. Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator. J Neuroinflammation. 2017 Jan 5;14(1):4.

Scholz R, Langmann T. Gut flora connects obesity with pathological angiogenesis in the eye. EMBO Mol Med. 2016 Dec 1;8(12):1361-1363.

Coppieters F, Ascari G, Dannhausen K, Nikopoulos K, Peelman F, Karlstetter M, Xu M, Brachet C, Meunier I, Tsilimbaris MK, Tsika C, Blazaki SV, Vergult S, Farinelli P, Van Laethem T, Bauwens M, De Bruyne M, Chen R, Langmann T, Sui R, Meire F, Rivolta C, Hamel CP, Leroy BP, De Baere E. Isolated and Syndromic Retinal Dystrophy Caused by Biallelic Mutations in RCBTB1, a Gene Implicated in Ubiquitination. Am J Hum Genet. 2016 Aug 4;99(2):470-80.

Zeilbeck LF, Müller BB, Leopold SA, Senturk B, Langmann T, Tamm ER, Ohlmann A. Norrin mediates angiogenic properties via the induction of insulin-like growth factor-1. Exp Eye Res. 2016 Apr;145:317-26.

Van Schil K, Karlstetter M, Aslanidis A, Dannhausen K, Azam M, Qamar R, Leroy BP, Depasse F, Langmann T, De Baere E. Autosomal recessive retinitis pigmentosa with homozygous rhodopsin mutation E150K and non-coding cis-regulatory variants in CRX-binding regions of SAMD7. Sci Rep. 2016 Feb 18;6:21307.

Scholz R, Sobotka M, Caramoy A, Stempfl T, Moehle C, Langmann T. Minocycline counter-regulates pro-inflammatory microglia responses in the retina and protects from degeneration. J Neuroinflammation. 2015 Nov 17;12:209.

Scholz R, Caramoy A, Bhuckory MB, Rashid K, Chen M, Xu H, Grimm C, Langmann T. Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration. J Neuroinflammation. 2015 Nov 2;12:201.

Langmann T. Intracellular Toll-Like Receptors Help Retinal Microglia Sense Corneal Infections. Invest Ophthalmol Vis Sci. 2015 Nov;56(12):7387.

Dannhausen K, Karlstetter M, Caramoy A, Volz C, Jägle H, Liebisch G, Utermöhlen O, Langmann T. Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function. Biochem Biophys Res Commun. 2015 Aug 21;464(2):434-40.

Breukink MB, Schellevis RL, Boon CJ, Fauser S, Hoyng CB, den Hollander AI, de Jong EK. Genomic Copy Number Variations of the Complement Component C4B Gene Are Associated With Chronic Central Serous Chorioretinopathy. Invest Ophthalmol Vis Sci. 2015 Aug 1;56(9):5608-13.

Grassmann F, Friedrich U, Fauser S, Schick T, Milenkovic A, Schulz HL, von Strachwitz CN, Bettecken T, Lichtner P, Meitinger T, Arend N, Wolf A, Haritoglou C, Rudolph G, Chakravarthy U, Silvestri G, McKay GJ, Freitag-Wolf S, Krawczak M, Smith RT, Merriam JC, Merriam JE, Allikmets R, Heid IM, Weber BH. A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD). Neuromolecular Med. 2015 Jun;17(2):111-20.

Van Schil K, Meire F, Karlstetter M, Bauwens M, Verdin H, Coppieters F, Scheiffert E, Van Nechel C, Langmann T, Deconinck N, De Baere E. Early-onset autosomal recessive cerebellar ataxia associated with retinal dystrophy: new human hotfoot phenotype caused by homozygous GRID2 deletion. Genet Med. 2015 Apr;17(4):291-9.

Enders P, Muether PS, Hermann M, Ristau T, Fauser S. Long-term alterations of systemic vascular endothelial growth factor levels in patients treated with ranibizumab for age-related macular degeneration. Retina. 2015 Mar;35(3):454-8.

Aslanidis A, Karlstetter M, Scholz R, Fauser S, Neumann H, Fried C, Pietsch M, Langmann T. Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia. J Neuroinflammation. 2015 Apr 19;12:77.

Szulzewsky F, Pelz A, Feng X, Synowitz M, Markovic D, Langmann T, Holtman IR, Wang X, Eggen BJ, Boddeke HW, Hambardzumyan D, Wolf SA, Kettenmann H. Glioma-associated microglia/macrophages display an expression profile different from M1 and M2 polarization and highly express Gpnmb and Spp1. PLoS One. 2015 Feb 6;10(2):e0116644.

Langmann T, Ueffing M, Kirchhof B, Holz FG, Pauleikhoff D. [Meeting report — retinal research 2013 in Germany]. Klin Monbl Augenheilkd. 2014 Mar;231(3):266-8.

Karlstetter M, Nothdurfter C, Aslanidis A, Moeller K, Horn F, Scholz R, Neumann H, Weber BH, Rupprecht R, Langmann T. Translocator protein (18 kDa) (TSPO) is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis. J Neuroinflammation. 2014 Jan 8;11:3.

Volz C, Mirza M, Langmann T, Jägle H. Retinal function in aging homozygous Cln3 (?ex7/8) knock-in mice. Adv Exp Med Biol. 2014;801:495-501.
Karlstetter M, Langmann T. Microglia in the aging retina. Adv Exp Med Biol. 2014;801:207-12.

Ristau T, Ersoy L, Lechanteur Y, den Hollander AI, Daha MR, Hahn M, Hoyng CB, Fauser S (2014) Allergy is a protective factor against age-related macular degeneration. Invest Ophthalmol Vis Sci. 55:210-4.

Karlstetter M, Sorusch N, Caramoy A, Dannhausen K, Aslanidis A, Fauser S, Boesl MR, Nagel-Wolfrum K, Tamm ER, Jägle H, Stoehr H, Wolfrum U, Langmann T (2014) Disruption of the retinitis pigmentosa 28 gene Fam161a in mice affects photoreceptor ciliary structure and leads to progressive retinal degeneration. Hum Mol Genet. 23(19):5197-210

Ristau T, Paun C, Ersoy L, Hahn M, Lechanteur Y, Hoyng C, de Jong EK, Daha MR, Kirchhof B, den Hollander AI, Fauser S (2014) Impact of the common genetic associations of age-related macular degeneration upon systemic complement component C3d levels. PLoS One 9(3):e93459.

Helgason H, Sulem P, Duvvari MR, Luo H, Thorleifsson G, Stefansson H, Jonsdottir I, Masson G, Gudbjartsson DF, Walters GB, Magnusson OT, Kong A, Rafnar T, Kiemeney LA, Schoenmaker-Koller FE, Zhao L, Boon CJ, Song Y, Fauser S, Pei M, Ristau T, Patel S, Liakopoulos S, van de Ven JP, Hoyng CB, Ferreyra H, Duan Y, Bernstein PS, Geirsdottir A, Helgadottir G, Stefansson E, den Hollander AI, Zhang K, Jonasson F, Sigurdsson H, Thorsteinsdottir U, Stefansson K (2013) A rare nonsynonymous sequence variant in C3 is associated with high risk of age-related macular degeneration. Nat Genet. 45:1371-4.

van de Ven JP, Nilsson SC, Tan PL, Buitendijk GH, Ristau T, Mohlin FC, Nabuurs SB, Schoenmaker-Koller FE, Smailhodzic D, Campochiaro PA, Zack DJ, Duvvari MR, Bakker B, Paun CC, Boon CJ, Uitterlinden AG, Liakopoulos S, Klevering BJ, Fauser S, Daha MR, Katsanis N, Klaver CC, Blom AM, Hoyng CB, den Hollander AI (2013) A functional variant in the CFI gene confers a high risk of age-related macular degeneration. Nat Genet. 45:813-7.

Vierkotten S, Muether PS, Fauser S (2011) Overexpression of HTRA1 leads to ultrastructural changes in the elastic layer of Bruch’s membrane via cleavage of extracellular matrix components. PLoS One 6:e22959.

Karlstetter M, Walczak Y, Weigelt K, Ebert S, Van den Brulle J, Schwer H, Fuchshofer R, Langmann T (2010) The novel activated microglia/macrophage WAP domain protein, AMWAP, acts as a counter-regulator of proinflammatory response. J Immunol 185:3379-3390.

Langmann T, Di Gioia SA, Rau I, Stohr H, Maksimovic NS, Corbo JC, Renner AB, Zrenner E, Kumaramanickavel G, Karlstetter M, Arsenijevic Y, Weber BH, Gal A, Rivolta C (2010) Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa. Am J Hum Genet 87:376-381.

Corbo JC, Lawrence KA, Karlstetter M, Myers CA, Abdelaziz M, Dirkes W, Weigelt K, Seifert M, Benes V, Fritsche LG, Weber BH, Langmann T (2010) CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors. Genome Res 20:1512-1525.