Modulation of neuroimmune responses as potential treatment strategies in ischemic stroke

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DISSERTATIONS | NATALIA KOŁOSOWSKA | MODULATION OF NEUROIMMUNE RESPONSES AS... | No 531

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PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

NATALIA KOŁOSOWSKA

MODULATION OF NEUROIMMUNE

Ischemic stroke is a severely disabling condition and its clinical management still remains a major challenge. In this thesis, the

actions of three agents in models of stroke were investigated: miR-669c, interleukin 13 and HX600, an agonist for RXR-NURR1

complex. These studies aimed to explore the mechanisms of central and peripheral immunomodulation, as well to address the therapeutic potential of regulating microglial and macrophage activation in the early phase

after cerebral ischemia.

NATALIA KOŁOSOWSKA

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MODULATION OF NEUROIMMUNE RESPONSES AS POTENTIAL TREATMENT

STRATEGIES IN ISCHEMIC STROKE

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Natalia Kołosowska

MODULATION OF NEUROIMMUNE RESPONSES AS POTENTIAL TREATMENT

STRATEGIES IN ISCHEMIC STROKE

Publications of the University of Eastern Finland Dissertations in Health Sciences

No 531

University of Eastern Finland Kuopio

2019

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Series Editors

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Associate professor (Tenure Track) Tarja Kvist, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Professor Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

Lecturer Veli-Pekka Ranta, Ph.D.

School of Pharmacy Faculty of Health Sciences

Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland

www.uef.fi/kirjasto

GRANO Kuopio, 2019

ISBN: 978-952-61-3189-4 (print/nid.) ISBN: 978-952-61-3190-0 (PDF)

ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)

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Author’s address: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Doctoral programme: Doctoral Programme in Molecular Medicine Supervisors: Professor Jari Koistinaho, M.D., Ph.D.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Professor Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland

KUOPIO FINLAND

Reviewers: Docent Mikko Airavaara, Ph.D.

Institute of Biotechnology HiLIFE Unit

University of Helsinki HELSINKI

FINLAND

Assistant Professor of Neuroscience Karin Hochrainer, Ph.D.

Feil Family Brain & Mind Research Institute Weill Cornell Medicine

NEW YORK UNITED STATES

Opponent: Professor Peter Ponsaerts, Ph.D.

Laboratory of Experimental Hematology

Vaccine and Infectious Disease Institute (VaxInfectio) University of Antwerp

ANTWERP BELGIUM

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Kolosowska, Natalia

Modulation of neuroimmune responses as potential treatment strategies in ischemic stroke

Kuopio: University of Eastern Finland

Publications of the University of Eastern Finland Dissertations in Health Sciences 531. 2019, 112 p.

ISBN: 978-952-61-3189-4 (print) ISSNL: 1798-5706

ISSN: 1798-5706

ISBN: 978-952-61-3190-0 (PDF) ISSN: 1798-5714 (PDF)

ABSTRACT

Cerebral ischemia is one of the main leading causes of profound disabilities and death worldwide, and its effective clinical management still remains a challenge.

Currently available therapeutic interventions include pharmacological thrombolysis with rtPA and mechanical recanalization of blocked arteries, or a combination of both. Although beneficial, these treatments have substantial limitations, such as the risk of hemorrhagic transformation and a narrow therapeutic time window, thus they are administered only to a fraction (~5%) of stroke patients. Therefore novel, more effective and broadly applicable therapeutic approaches are urgently needed to combat this highly debilitating neurological condition.

In this doctoral dissertation, using three different approaches, we studied the actions of miR-669c, interleukin 13 and HX600 in models of ischemic stroke and inflammation. In the first study we evaluated the effects of intracerebral, lentiviral- mediated overexpression of miR-669c in mice exposed to transient ischemic stroke.

We found miR-669c overexpression to be neuroprotective in this model by enhancement of M2a anti-inflammatory microglial activation and concurrent inhibition of proinflammatory signaling. In the second study we tested whether systemic administration of recombinant IL-13 could ameliorate ischemic damage in a mouse model of permanent ischemia. IL-13 delivery indeed reduced ischemic lesion in mice, essentially by augmenting microglial and macrophage alternative polarization, similar immunomodulatory effects as those described in the first study.

In the third study, we determined if HX600, an agonist for the retinoid X receptor- NURR1 complex, could impact outcome of ischemic stroke. We showed that HX600 reduces microglia-expressed proinflammatory mediators and prevents neuronal death in in vitro co-cultures of neurons and microglia, as well as in vivo in permanent ischemia in mice. The three studies presented in this doctoral thesis collectively highlight the prominence of targeted microglial/macrophage immunomodulation after ischemia. In conclusion, our results delineate novel mechanisms and pathways capable of modulating the outcome of ischemic stroke and neuroinflammatory

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conditions, and as such may hold promise as future therapeutic interventions for these burdensome diseases.

National Library of Medicine Classification: QU 55.2, QU 58.7, QV 247, QW 568, WL 356 Medical Subject Headings: Ischemic Stroke; Neuroinflammation; Alternative Microglial Activation; MicroRNAs; Interleukin 13; Nuclear Receptors

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Kolosowska, Natalia

Keskushermoston immuunivasteen muokkaaminen aivohalvauksen hoidossa Kuopio: Itä-Suomen yliopisto

Publications of the University of Eastern Finland Dissertations in Health Sciences 531. 2019, 112 s.

ISBN: 978-952-61-3189-4 (nid.) ISSNL: 1798-5706

ISSN: 1798-5706

ISBN: 978-952-61-3190-0 (PDF) ISSN: 1798-5714 (PDF)

TIIVISTELMÄ

Aivohalvaus on yksi yleisimmistä vammautumisen ja kuoleman syistä ympäri maailmaa. Sen kliininen hoito on yhä haaste. Tällä hetkellä käytössä olevat hoidot ovat tukkeutuneen valtimon farmakologinen liuotushoiuto tai mekaaninen rekanalisaatio, tai näiden yhdistelmä. Näillä hoitomuodoilla on omat rajoitteensa, sillä ne lisäävät aivoverenvuodon riskiä ja niillä on hyvin kapea terapeuttinen aikaikkuna. Tämän vuoksi ne sopivat vain pienelle osalle potilaita. Sen vuoksi uusien, tehokkaampien hoitomuotojen kehittäminen on erittäin tärkeää. Tässä väitöskirjatyössä tutkittiin kolmen uuden potentiaalisen hoitomuodon toimivuutta hiiren prekliinisessä aivohalvausmallissa. Ensimmäisessä osatyössä näytimme, miten lentivirusvälitteisesti annosteltu miR-669c vähentää transientin aivohalvauksen aiheuttamaa kudostuhoa moduloimalla mikrogliasolujen aktivaatiota ja pienentämällä aivohalvauksen aiheuttamaa tulehdusvastetta.

Toisessa osatyössä osoitimme, että systeemisesti annosteltu interleukiini-13 (IL-13) suojaa aivohalvauksen aiheuttamaa kudostuhoa vastaan niin ikään indusoimalla mikrogliojen ja makrofagisolujen polarisaatiota hermosolujen toimintaa tukevaan suuntaan. Kolmannessa osatyössä osoitimme, että NURR1 aktivaattori HX600 suojaa aivohalvauksen aiheuttamaa solutuhoa vastaan vähentämällä mikroglia-välitteistä hermosolujen kuolemaa in vitro sekä in vivo. Tässä väitöskirjatyössä tuotetaan tietoa uusista aivohalvauksen molekyylitason lääkeainekohteista ja osoitetaan, että aivohalvauksen aiheuttaman tulehdusvasteen modulointi on lupaava uusien hoitomuotojen kehityskohde.

Luokitus: QU 55.2, QU 58.7, QV 247, QW 568, WL 356

Yleinen suomalainen asiasanasto: Iskeeminen Aivohalvaus; Neuroinflammaatio;

Vaihtoehtoinen Mikrogliojen Aktivointi; MikroRNA; Interleukiini 13; Tumareseptorit

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ACKNOWLEDGEMENTS

The studies presented in this doctoral dissertation were performed in the Molecular Brain and Neuroinflammation Research Groups, A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, during the years 2014-2019. These studies were supported by Marie Skłodowska-Curie ITN nEUROinflammation, Emil Aaltonen Foundation, Antti and Tyyne Soininen Foundation, Business Finland, EU Joint Programme-Neurodegenerative Disease Research (JPND) project and Academy of Finland.

I would like to express my deep gratitude to my supervisors, Professors Jari Koistinaho and Tarja Malm, for their guidance, sharing their profound knowledge and providing support of any kind during my studies. I also thank my co-supervisor, Associate Professor Katja Kanninen, for her valuable input to my doctoral thesis projects and support during the final months of my work at AIVI. I am grateful to Assistant Professor Karin Hochrainer and Docent Mikko Airavaara for their excellent reviews of my dissertation. I would like to thank Professor Peter Ponsaerts for acting as an opponent during the thesis defence.

I would like to warmly thank all co-authors who contributed to the research articles presented here: Professor Seppo Ylä-Herttuala, Hiramani Dhungana, Meike Keuters, Sanna Loppi, Paula Korhonen, Maria Gotkiewicz, Mikko Huuskonen, Flavia Scoyni, Tiia Turunen, Mikko Turunen, Sara Wojciechowski, Velta Keksa-Goldsteine, Gundars Goldsteins, Daphne Box, Mika Laine, Olli Kärkkäinen, Alexandra Grubman, Yuriy Pomeshchik, Martina Giordano, Hiroyuki Kagechika, Anthony White, Seppo Auriola, Gary Landreth and Kati Hanhineva.

I want to express my thanks to Mirka Tikkanen and Laila Kaskela for their genuine commitment to ongoing studies and proper organization of the lab facilities.

I would also like to thank all current and past members of Molecular Brain, Neuroinflammation and Neurobiology of Disease Research Groups, who as lab colleagues were not only sharing work space, but also spending their leisure time with me on various occasions.

Finally, I thank my beloved family, dear friends and caring fiancé Konstantin for their invaluable support during the time of my doctoral studies. I am forever grateful to have you in my life. Kochani Rodzice (i Donisiu), dziękuję Wam za Waszą bezwarunkową miłość i wsparcie, które okazaliście mi nie tylko w czasie moich studiów doktoranckich, ale i przez całe życie.

Kuopio, 27 September 2019

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LIST OF ORIGINAL PUBLICATIONS

This dissertation is based on the following original publications:

I Kolosowska N, Gotkiewicz M, Dhungana H, Box D, Huuskonen M T, Korhonen P, Scoyni F, Kanninen K M, Turunen T A, Turunen M P, Ylä- Herttuala S, Koistinaho J, Malm T. Intracerebral overexpression of miR-669c is protective in mouse ischemic stroke model by targeting MyD88 and inducing M2a alternative microglial activation. Submitted manuscript.

II Kolosowska N*, Keuters M H*, Wojciechowski S, Keksa-Goldsteine V, Laine M, Malm T, Goldsteins G, Koistinaho J, Dhungana H. Peripheral administration of IL-13 induces anti-inflammatory microglial/macrophage responses and provides neuroprotection in ischemic stroke. Neurotherapeutics, 2019 Aug 1. doi: 10.1007/s13311-019-00761-0.

III Loppi S, Kolosowska N, Kärkkäinen O, Korhonen P, Huuskonen M T, Grubman A, Dhungana H, Wojciechowski S, Pomeshchik Y, Giordano M, Kagechika H, White A, Auriola S, Koistinaho J, Landreth G, Hanhineva K, Kanninen K M, Malm T. HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage. Brain, Behavior, and Immunity, 2018 Oct;73:670-681. doi: 10.1016/j.bbi.2018.07.021.

Equal contribution*

The publications were adapted with the permission of the copyright owners.

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5.2 Systemic IL-13 treatment promotes anti-inflammatory microglial and macrophage activation, and provides neuroprotection in mouse cerebral ischemia (II) ... 70 5.2.1 IL-13 treatment alleviates ischemic brain damage ... 70 5.2.2 IL-13 treatment reduces leukocyte infiltration to the lesion area without altering peri-ischemic astrogliosis ... 71 5.2.3 IL-13 enhances anti-inflammatory immune responses in the ischemic brain... 71 5.2.4 IL-13 increases expression of M2 markers in the ischemic brain and elevates anti-inflammatory cytokine levels in the plasma ... 71 5.2.5 IL-13 improves somatosensory and locomotor impairments in mice after pMCAo ... 71 5.2.6 IL-13 promotes alternative polarization of primary microglia under

proinflammatory conditions ... 72 5.2.7 IL-13 protects N2a cells from inflammation-induced death in co-culture with RAW 264.7 macrophages ... 73 5.3 HX600, a synthetic agonist for RXR-NURR1 heterodimer complex, is neuroprotective in a mouse model of ischemic stroke (III) ... 73 5.3.1 HX600 treatment decreases expression of inflammatory mediators in primary microglia and protects neurons against inflammation-induced cell death when co-cultured with microglia ... 73 5.3.2 HX600 treatment alleviates brain damage and provides functional

improvement after pMCAo ... 74 5.3.3 HX600 administration decreases Iba1, phospho-p38 and TREM-2 levels in the infarcted brain ... 74 5.3.4 HX600 normalizes the proportion of CD45^Hi CD11b^Low and Ly6C^Hi

CD45^Low cells in the ischemic brain ... 74 5.3.5 HX600 treatment normalizes the metabolic profile in the peri-ischemic area ... 74 6 DISCUSSION ... 75 6.1 Overexpression of miR-669c in the brain protects mice against ischemic stroke by enhancing microglial anti-inflammatory responses ... 75 6.2 IL-13 is protective in cerebral ischemia by augmenting microglial/macrophage alternative polarization ... 78 6.3 HX600 treatment reduces ischemic damage by inhibiting microglial proinflammatory activation ... 80 6.4 Study significance in the context of ischemic stroke and neuroinflammation ... 83 7 CONCLUSIONS ... 85 REFERENCES ... 87 ORIGINAL PUBLICATIONS (I – III) ...

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ABBREVIATIONS

2-VO – Bilateral carotid artery ligation model

4-VO – Four Vessel occlusion model ALOX5 – Arachidonate 5-lipoxygenase AMPA – -amino-3-hydroxy-5- methyl-4-isoxazole propionic acid ASC – Apoptosis speck-like adaptor ATG – Autophagy-related protein ATP – Adenosine triphosphate

APAF-1 – Apoptotic protease activating factor 1

ASTIN – Acute Stroke Therapy by Inhibition of Neutrophils

BBB – Blood brain barrier Bcl-2 – B cell lymphoma 2

BDNF – Brain-derived neurotrophic factor

BL – Baseline

C2MC – Chromosome 2 microRNA cluster

CASP3 – Caspase 3

CB2 – Cannabinoid receptor 2 CBA – Cytometric bead array CBF – Cerebral blood flow

CCL2 – Chemokine C-C motif ligand 2 (known also as MCP-1)

CCL3 Chemokine (C-C motif) ligand 3 (known also as MIP-1α)

CCR2 – C-C chemokine receptor type 2 CD – Cluster of differentiation

CDK6 – Cyclin-dependent kinase 6 CNS – Central nervous system COX2 – Cyclo-oxygenase 2 CR – Complement receptor

CXCL10 – Chemokine C-X-C motif ligand 10

CX3CL1 – C-X3-C motif chemokine ligand 1 (known also as fractalkine)

CX3CR1 – C-X3-C motif chemokine receptor 1 (known also as fractalkine receptor)

CT – Computed tomography

CTDSP1 – Carboxy terminal domain small phosphatase 1

DAMP – Damage-associated molecular pattern

DIV – Day in vitro

DMEM – Dulbecco’s modified Eagle’s medium

DPI – Days post injury

EAST – Enlimomab Acute Stroke Trial

ELISA – Enzyme-linked

immunosorbent assay

FACS – Fluorescence-activated cell sorting

FAST – mnemonic acronym standing for Facial drooping, Arm weakness, Speech difficulties and Time to call emergency services

FBS – Fetal bovine serum Fcrls – Fc receptor-like S

FDA – Food and Drug Administration GDNF – Glial cell line-derived neurotrophic factor

GFAP – Glial fibrillary acidic protein GFP – Green Fluorescent Protein HALT – Hu23F2G Phase 3 Stroke Trial HIF-1α – Hypoxia-inducible factor-1α HMGB1 – High-mobility group protein 1

HT – High-throughput

HX600 – 4-[5H-2,3-(2,5-Dimethyl-2,5- hexano)-5-

methyldibenzo[b,e][1,4]diazepin-11- yl]benzoic acid

Iba1 – Ionized calcium-binding adapter molecule 1

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ICAM-1 – Intercellular cell adhesion molecule-1

IFN-γ – Interferon-gamma

IGF-1 – Insulin like growth factor 1 iNOS – Inducible nitric oxide synthase IL – Interleukin

IL-1RAcP – IL-1R accessory protein IL-4Rα – IL-4 receptor α

IL-13Rα1 – IL-13 receptor α 1 IP – Intraperitoneal

IRF8 – Interferon regulatory factor 8 ITGAM – Integrin alpha M (known also as CD11b)

IV – Intravenous

LDH – Lactate dehydrogenase

LFA-1 – Lymphocyte function- associated antigen 1

Lgals3 – Galectin-3 (referred also as Gal3)

LPS – Lipopolysaccharide LTM – Latency to move LV – Lentivirus

Ly6C – Lymphocyte antigen 6 complex, locus C

Ly6G – Lymphocyte antigen 6 complex, locus G6D

Mac-1 – Macrophage integrin (known also as CR3)

MAP2 – Microtubule-associated protein 2

MERTK – Tyrosine-protein kinase Mer precursor

MCP-1 – Monocyte chemoattractant protein-1 (known also as CCL2) M-CSF – Macrophage-colony stimulating factor

MFG-E8 – Milk fat globule EGF-like factor 8

MHC – Major histocompatibility complex

MIP-1α – Macrophage inflammatory protein 1α (known also as CCL3) MMPs – Matrix metalloproteinases

MOI – Multiplicity of infection MRI – Magnetic resonance imaging MSC – Mesenchymal stem cells

mrIL-13 – Mouse recombinant interleukin 13

mTOR – Mechanistic target of rapamycin

MTT – 3-(4, 5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide

NADH – Nicotinamide adenine dinucleotide

NGF – Nerve growth factor NGS – Normal goat serum

NMDA – N-methyl-D-aspartic acid NO – Nitric oxide

NOX – Nicotinamide adenine dinucleotide phosphate oxidase nNOS – Neuronal nitric oxide synthase NR4A – Nuclear receptor 4A (known also as NURR1)

NRS – Normal rabbit serum NT – Neurotrophin

NURR1 – Nuclear receptor related 1 protein (known also as NR4A2)

NURR77 – Nerve growth factor IB (known also as NR4A1)

OGD/R – Oxygen and glucose deprivation with reoxygenation P2X7 – P2X purinoceptor 7

P2Y – Family of purinergic G protein- coupled receptors

P2Y12 – Platelet adenosine diphosphate receptor

PAMP – Pathogen-associated molecular patterns

PARP-1 – Poly (ADP-ribose) polymerase 1

PI – Peri-ischemic

pMCAo – permanent Middle Cerebral Artery occlusion

PPAR-γ – Peroxisome proliferator- activated receptor gamma

PBS – Phosphate buffered saline

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PD – Parkinson´s disease PFA – Paraformaldehyde PO – Per os

PRRs – Pattern Recognition Receptors qPCR – Real-time quantitative polymerase chain reaction

RAGE – Receptor for advanced glycosylation end products

RNS – Reactive nitrogen species ROS – Reactive oxygen species

RPMI – Roswell Park Memorial Institute (medium)

RXR – Retinoid X-receptor

rtPA – Recombinant tissue plasminogen activator

SAP – Stroke-associated pneumonia SC – Subcutaneous

SIGIRR – Single Ig IL-1R-related molecule

SOCS – Suppressor of cytokine signaling

SSV – Standard suspension vehicle SVZ – Subventricular zone

TGF-β – Transforming growth factor β Th2 – T-helper type 2 (lymphocyte) TLR – Toll like receptor

tMCAo – transient Middle Cerebral Artery occlusion

TNF-α – Tumor necrosis factor alpha TNFR – TNF receptor subfamily member receptor

Th2 – Type 2 T helper cell

TIGGIR – three Ig domain-containing IL-1R related

TRAILRs – TNF-related apoptosis- inducing ligand receptors

TREM2 – Triggering receptor expressed on myeloid cells 2

TU – Transducing units

UHPLC – Ultra-high performance liquid chromatography

VCAM-1 – Vascular cell adhesion molecule 1

VEGF – Type A vascular endothelial growth factor

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1 INTRODUCTION

Cerebral ischemia is a result of blood supply disruption to the brain, which in turn causes tissue damage due to oxygen and nutrient starvation. In Europe and Northern America ischemic attack accounts for 85% of brain stroke cases, while the remaining 15% are of hemorrhagic type, caused by blood vessel rupture[1]. World Health Organization reports show that worldwide around 15 million people suffer from stroke each year, with over one third of them dying. This makes cerebral stroke one of the leading causes of long-term disability and the second most frequent cause of death after coronary artery disease. Surviving stroke patients oftentimes face many additional health risks including secondary infections, cardiac events, depression, cognitive decline, dementia and generally poor quality of life.

Currently the only clinically approved drug treatment for ischemic stroke is IV thrombolysis with recombinant tissue plasminogen activator (rtPA, Alteplase), which was first approved by the FDA in 1996. While rtPA has been proven beneficial in clinical studies, the vast majority of patients cannot benefit from this treatment, as it has a very narrow therapeutic time window, increases the risk of hemorrhagic complications and can have some other adverse effects such as excitotoxic necrosis[2]. The therapeutic time window for this drug is 4.5 h after stroke onset[3,4].

Administration of rtPA past that time window usually results in a hemorrhagic transformation[5] and increased mortality[6] or insufficient neuroprotection[7,8].

Given within 3-4.5 h of stroke onset rtPA poses a higher risk of intracerebral hemorrhage, which has been found in 6% of patients[6]. Other treatment strategies for cerebral ischemia include endovascular procedures like mechanical thrombectomy with or without thrombolysis, and hemicraniectomy in the case of most severe brain infarcts[9]. These procedures are however not widely used due to limited expertise and lack of equipment in many hospitals. Altogether, ischemic stroke still remains untreatable for the majority of patients, and more effective and broader applicable therapeutics for this highly debilitating neurological condition are urgently needed.

In recent years immunomodulation has been highlighted as a promising approach for cerebral ischemia treatment. After stroke onset the brain communicates with the peripheral immune system via autonomous nervous system circuits and the hypothalamic–pituitary–adrenal axis, which play important functions in modulation of peripheral immune responses[10]. There is a clear evidence that immune system activation protects brain tissue from necrotic burden, is critical for long-term recovery and supports brain regenerative capacity after ischemic damage[11].

Conversely, activation of certain innate immunity pathways may lead to exacerbation of stroke-associated deficits[12]. In recent years, modulation of microglial and macrophage polarization during different phases of cerebral ischemia has been increasingly gaining interest and is now considered a key factor contributing to the outcome of stroke[13,14]. This doctoral thesis focuses on several

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experimental approaches targeting ischemia-driven immune responses, which are aimed at controlling excessive proinflammatory reactions and enhancing the beneficial effects of alternative polarization of microglia and macrophages. In the presented research we evaluated the therapeutic potential of miR-669c, IL-13 and nuclear receptor X ligand HX600, which are novel mediators of neuroinflammation in the context of cerebral infarction. In the first study we assessed the capacity of intracerebrally overexpressed miR-669c to improve stroke outcome by enhancing microglial/macrophage alternative polarization in transient and permanent ischemia. In the second study we investigated the efficacy of systemic IL-13 administration to achieve cytoprotection in permanent ischemia, again by inducing microglial/macrophage alternative activation in the brain. In the third study, the therapeutic potential of peripherally administered HX600, an agonist of retinoid X receptor and nuclear receptor related 1 protein (RXR-NURR1) heterodimer complex, was examined in permanent ischemic stroke. HX600 treatment provided neuroprotection primarily through alleviating proinflammatory microglial activation. Taken together, our data give new insights into neuroimmune responses in cerebral ischemic stroke, and further confirm that immunomodulation may represent an effective and promising approach for ischemic stroke treatment in the future.

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2 LITERATURE REVIEW

2.1 ISCHEMIC STROKE 2.1.1 Etiology and risk factors

Ischemic stroke is caused by a cessation of cerebral blood flow due to arterial occlusion or stenosis, and can be classified into several subtypes, depending on location, cause and size of infarct[15]. Cardio-embolic (CEI) strokes, which usually occur without underlying cerebrovascular pathology, are induced by traveling emboli resulting from atrial fibrillation, presence of mechanical valve or endocarditis.

On the other hand, stroke induced by large-artery atherosclerosis (LAAS) is the result of atherosclerotic plaque buildup in blood vessels of the brain. Commonly a thrombus is formed at an atherosclerotic site in larger extracranial arteries, which then passages to distal sites to occlude the vessel. LAAS causes cortical, subcortical, cerebellar or brain stem infarcts which are greater than 1.5 cm in diameter. When ischemic damage to the tissue is relatively small, less than 1.5 cm in diameter, then it is called lacunar stroke (LAC). It is usually associated with diabetes and hypertension and primarily emerges from occlusion of a small intracerebral artery to produce subcortical or brain stem lesions. Strokes with other determined etiology (ODE) are relatively rare and are classified by supporting clinical diagnosis of coexisting vascular abnormalities or hematologic disorders. Finally, strokes without defined etiology (UDE) are cases that do not fit into any of these groups.

Independent risk factors contribute to about 90% of ischemic stroke cases[16]. These factors include gender, ageing, recent transient ischemic attack (TIA), defined as brief disruption in the blood supply to part of the brain, coronary heart disease, hypertension, hyperlipidemia, hyperhomocysteinemia, tobacco use, excess body fat, lack of physical activity, diabetes and alcohol misuse. Also, a family history of ischemic stroke occurrence elevates the probability of having a stroke by 30% for close relatives[17]. TIA substantially increases the risk of acute ischemic stroke with a projection for 90 day stroke risk of 10.5%[18], but appropriate management of TIA can effectively prevent cerebral stroke occurrence. Men have higher propensity for ischemic stroke than women in individuals younger than age 55, however after that both sexes face equal risk for having a stroke[19]. While stroke generally affects older people (aged 55-75), the incidence of ischemia in young adults (<55 years of age) is steadily increasing due to a rising prevalence of associated risk factors caused by poor life style choices of the general population[20]. Many of these factors are modifiable, hence adequate control of stroke risk factors is certainly one the essential means to reduce the socio-economic impact of cerebral ischemia. For instance, high blood pressure control can limit the risk of stroke by 30%[21]. Increased blood LDL levels (>130 mg/dL) are highly predisposing to cerebral attack, therefore controlling the lipid levels by appropriate diet and overall healthy lifestyle is critical in the

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prevention of brain ischemia[22]. As a convincing example, tobacco users reach baseline risk levels for ischemic stroke after 5 years of non-smoking lifestyle[19].

2.1.2 Epidemiology and socio-economic burden

Stroke is a leading cause of disability and mortality worldwide. On average, every 40 seconds, someone in the United States has a stroke[1]. In the US, the support costs for ischemic stroke patients are exceeding $73 billion annually, and this is projected to increase to $143 billion by 2035[23]. In Finland such expenditures amount to €1.6 billion, accounting for around 7% of national healthcare costs[24]. Particularly, recurrent ischemic strokes representing around 20% of total number of strokes in the US are characterized by higher mortality rates, greater degree of disability, and increased costs in comparison to first stroke incidents[1]. Later in life, stroke survivors face other health risks, including disability and handicap, cardiac events, poor quality of life, dementia, cognitive decline, and depression, requiring further costly treatments. Statistics from the American Heart Association show that 15–30%

of ischemic stroke patients are left permanently disabled, and 20% require institutional care for at least 3 months after stroke occurrence[1]. Also contributing to the increased financial burden of post-stroke care is the limited awareness in the general public of cerebral ischemia symptoms, including FAST warning signs: Facial drooping, Arm weakness, Speech difficulties and Time to call emergency services.

This eventually delays patient admission to the emergency room within the therapeutic time window, thus preventing acute treatment.

The Global Burden of Disease 2017 statistics show that over the last two decades mortality rates dropped, while the absolute number of stroke incidents survivors and stroke-related deaths are gradually increasing[25]. In 2010 there were 11.6 million ischemic strokes worldwide, with majority of them occurring in low- and middle- income countries. There has been a 25% increase in the worldwide incidence of stroke among adults aged 20 to 64 years over the past 30 years[26]. Thus, implementing strategies to improve the stroke awareness and focus on prevention of risk factors are critical to reduce the current estimates for ischemic stroke.

2.1.3 Clinical treatment strategies

Rapid intervention and appropriate treatment are most essential to counteract the detrimental effects of cerebral ischemia. One of the first steps towards acute stroke clinical management was the introduction of CT imaging in the 1970s[27], which facilitated the discrimination between hemorrhagic and ischemic stroke.

Subsequently, early success of reperfusion therapy with IV tPA in patients with acute coronary artery disease[28] sparked further research in this direction, which eventually led to the approval of IV administration of rtPA for ischemic stroke intervention by the US FDA in 1996[29]. Alteplase cleaves the zymogen plasminogen

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to form plasmin, an endogenous enzyme, which breaks the cross-links between fibrin molecules, leading to thrombolysis[30]. Despite some rising controversies over the years[31], thrombolysis is generally considered safe and beneficial for eligible stroke patients. rtPA IV administration has several important benefits in the clinical practice. First, indications for treatment are relatively straightforward as only blood glucose and brain imaging results are required before thrombolysis implementation[32]. Moreover, the treatment is considered fairly cost-effective.

However, there is a fraction of stroke patients that do not improve after pharmacological thrombolysis, especially when a large artery has been occluded and reestablished reperfusion is insufficient. In such cases, rtPA can be combined with other types of interventions, e.g. intra-arterial therapy (IAT) involving mechanical recanalization of occluded vessels[33].

Endovascular interventions are an alternative strategy for restoring the cerebral blood flow, particularly in larger artery occlusion, such as terminal internal carotid artery and proximal middle cerebral artery[34]. Clot removal is achieved by various thrombectomy tools, such as microcatheters, MERCI and stent retrievers, as well as suction devices[35]. These procedures are particularly recommended when the clot is large and greater than 8 mm in length[36]. A considerable number of studies have shown comparable clinical efficacy of therapeutic interventions with rtPA and endovascular procedures[37–39], which are more applicable for patients having blood coagulation problems. An important benefit of endovascular therapy is the extended treatment time window compared to rtPA which is beyond 4.5 h and in some cases can be extended even up to 24 h[40,41]. One of the main limitations of mechanical recanalization procedures are the need of a specialist with good medical training and expensive technology, which are not widely available.

Taken together, the limited number and effectiveness of therapies available in clinics imposes the need to continue the basic, experimental research in the field of ischemic stroke. Even though so far the majority of neuroprotective treatment strategies in animal models have failed to be translated into clinical reality[42], there is still a large room for improvement of preclinical and translational approaches[43].

Neuroinflammation is an important component of the ischemic cascade and elucidating basic neuroinflammatory mechanisms after stroke on a molecular level is expected to not only improve understanding of stroke pathophysiology, but also to contribute to the development of better stroke treatments.

2.1.4 Pathophysiology

The ischemic brain can be divided in two regions, ischemic core and penumbra, also known as peri-infarct zone (Figure 1). During ischemia, cerebral blood flow in the core typically drops below 20% and neurons rapidly die, which is aggravated by Na⁺ and K⁺imbalance, responsible for cell membrane depolarization[44]. The penumbra remains partially perfused with arterial blood, which causes impairment of neuronal

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function, but renders neurons potentially salvageable. After circulation to the ischemic tissue is restored, the peri-infract region is challenged with excitotoxic and inflammatory factors released by dying cells within the infarct core. Glutamate excitotoxicity is one of the main contributors of neuronal death in the ischemic penumbra. It is mediated through NMDA and AMPA receptors and leads to intracellular Ca²⁺influx, which causes calcium homeostasis disruption[45]. This in turn triggers production of ROS and RNS, as well as mitochondrial dysfunction.

Decreasing pH (hypercapnia) in the injured brain microenvironment is caused by reduced pO² and concomitant increase of pCO². In case of severe ischemia anaerobic glycolysis causes lactic acid buildup, known as brain acidosis[46], a condition inducing morphological changes and irreversible neuronal damage. Selective gene expression in the penumbra include induction of caspases, aspartate-specific cysteine proteases central for mediating apoptotic processes[47]. Inflammatory responses after ischemic stroke are particularly prevalent in the peri-infarct zone[48]. Microglia rapidly sense the brain homeostasis disturbance and change their morphology, as well as start to secrete NLRP3 inflammasome-mediated interleukin-1β complex and TNF-α[49]. Under neuroinflammatory conditions, cytokines IL-1β, IL-6, IL-18, TNF- α, as well as enzymes iNOS and MMPs are released by many different cell types, including microglia, macrophages and endothelial cells. These inflammatory mediators attract peripheral leukocytes to the damaged parenchyma, in turn facilitating the recruitment of other circulating immune cells into the damaged site[50]. IL-10 and TGF-β are important immunosuppressive cytokines, inhibiting the expression of production of proinflammatory factors and adhesion molecules[51].

Secretion of growth factors, such as IGF-1, facilitates the neuro- and angiogenesis and extracellular matrix reorganization[52]. For the functional recovery of injured tissue reactive astrocytes play also an important role, as their inhibition may impair the process of neurovascular remodeling, as well as result in worsened behavioral outcome[53,54]. This chapter will focus on selected cell types and inflammatory factors important in the pathogenesis of ischemic stroke. Although indisputably important in ischemic stroke pathophysiology the role of oligodendrocytes and endothelial cells will not be discussed here in detail, as they were not a main focus of this dissertation.

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Figure 1. Morphological and biochemical features of the ischemic penumbra and infarct core.

Reprinted from Dirnagl et al., Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391–397, 1999, with permission from Elsevier.

2.1.4.1 The concept of neuroinflammation

Neuroinflammation is considered one of the main hallmarks of ischemic stroke pathophysiology. It has been shown that inflammatory reactions within the ischemic lesion worsen functional disturbances and lead to further structural damage[55].

Cellular injury results in a rapid activation of resident microglia and astrocytes, which mediate first neuroinflammatory responses. In humans four stages of neuroinflammation have been defined[56]. They are illustrated in Figure 2, including selected cellular responses described more in detail in the subsequent chapters of this thesis. Phase 1 occurs 1–2 days after stroke onset and is characterized by acute neuronal injury, leukocyte infiltration and microglia activation. Phase 2 is defined by acute inflammation, ranging from 3 days up to one month after disease onset, reflected by granulocyte, lymphocyte, and macrophage infiltration. The phase of chronic or subacute inflammation (phase 3; 10 days up to 2 months) is characterized by the presence of lymphocyte and macrophage infiltrates, absence of neutrophils, perivascular cuffing, persistent gliosis and brain tissue cavitation. Finally, phase 4, called resorption or recovery phase, can last from 1 month up to several years after stroke onset. During this phase only low number of macrophages is visible and restorative processes prevail within the infarct area. Importantly, neuroinflammation has both detrimental and beneficial effects, depending on the nature of the primary insult, the type of immune response, the microenvironment within the infarcts and also stage of the disease.

Acute neuronal injury

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Figure 2. Chart illustrating the stages of post-ischemic neuroinflammation. Adapted from Shichita et al., 2014.

2.1.4.2 Cellular responses in the context of ischemic brain injury 2.1.4.2.1 Neurons

It has been estimated that every minute following the ischemic attack due to oxygen and glucose deprivation approximately 2 million neurons die and 14 billion synapses are lost[57]. There are several mechanisms of ischemic cell death: apoptosis, necrosis and autophagy. In the ischemic core, where blood perfusion is severely reduced, metabolic stress is caused by ATP depletion due to compromised oxidative phosphorylation, which subsequently impacts cellular energy dynamics. Low ATP levels trigger neuronal death owing to ion pump failure, membrane depolarization, glutamate release, cell swelling and successive plasma membrane rupture[58].

Caspase 3 activation leads to cleavage and excessive activation of its downstream targets, such as PARP-1[59], which diminishes intracellular NAD⁺levels. NAD⁺ depletion impacts mitochondrial functionality, manifested by restriction in glycolysis and decreased ATP production, resulting in further aggravated energy failure. Sodium ion (Na⁺) influx into neurons is mediated via NMDARs, AMPARs, and other channels permeable to monovalent ions, while potassium ions (K⁺) flow out of via NMDARs[60]. This in turn promotes edema formation, as water passively starts to enter the cells. In contrast, although neurons in penumbra also display energy reduction, they neither undergo anoxic depolarization nor face profound imbalance of extracellular K⁺[61].

Excitotoxicity following cerebral ischemia is related to the high levels of excitatory amino acids, such as glutamate, which overactivate ionotropic NMDARs, AMPARs and kainate receptors, as well as metabotropic glutamate receptors, leading to intracellular calcium overload[62]. This subsequently triggers synaptic glutamate release and spreading of the excitotoxicity to surrounding neurons. Also, hypoxic depolarization causes the reverse activity of glutamate transporters in astrocytes, further increasing the accumulation of extracellular glutamate. In focal ischemia the extracellular glutamate concentration rises up to 30-50 fold (16–30 μM) in the ischemic core, in comparison to normal physiological levels[63]. Non-excitotoxic mechanisms include the excessive activation of Transient Receptor Potential and Acid sensing ion channels, contributing to membrane depolarization, calcium accumulation, cell swelling, and acidosis-evoked neuronal injury[64,65].

Another mechanism of cell death involves mitochondrial membrane depolarization.

Ischemia-triggered apoptotic and necrotic signals include Ca²⁺ elevation, which breaks down outer mitochondrial membrane integrity, allowing cytochrome c and other apoptotic factors to be released into the cytoplasm[66]. Cytochrome c, together with Apaf-1 and dATP, form the apoptosome, responsible for recruiting procaspase 9 to be cleaved and released as caspase 9 in the active dimer form[62].

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NO and free radical overproduction constitute additional mediators of excitotoxicity and neuronal death. In the brain, NO is synthesized mainly by the neuronal isoform nNOS and cytokine-inducible iNOS[67]. After ischemia primarily caused by NMDAR-mediated Ca²⁺ overload, NO and superoxide anion are responsible for oxidative damage of DNA and lipid peroxidation[68]. By reacting with •O²⁻, NO can produce peroxynitrite (ONOO⁻), which is able to oxidize key mitochondrial enzymes, such as cytochrome c oxidase. Peroxynitrite damages the mitochondrial membrane, which facilitates release of AIF, responsible for PARP-1 activation, and also reacts with membrane unsaturated fatty acids to initiate lipid peroxidation[69,70]. Other free radicals, such as superoxide anion, hydroxyl radical, singlet oxygen (all ROS), and nitric oxide derivatives (RNS), are also crucial mediators of neuronal injury, since they disturb the membrane potential, damage DNA, and prompt lipid peroxidation to induce cellular necrosis or apoptosis. Nucleic acids structural damage occurs by breaking the strands or chemical modification of the bases, as well as protein cross-linking to DNA. ROS activate also TRPM7 channels, known as one of the key mediators of anoxic neuronal death[71]. Moreover, NMDARs excessive activation leads to superoxide overproduction by cytoplasmic enzymes NADPH oxidase and NOX2 and thereby they have a pivotal impact on the outcome of stroke[72]. Other global detrimental effects mediated by free radicals are damage of various proteins by side chain oxidation and disulfide bond destruction[73]. Finally, dying neurons release various molecules that can be categorized as help-me, find-me and eat-me signals. Glutamate, RAGE activator HMGB1, CCL21, heat shock proteins (HSP60), fractalkine and nucleotides trigger microglial and astrocytic activation[74] to initiate neuroinflammatory responses.

2.1.4.2.2 Astrocytes

Astrocytes are brain-resident glial cells that essentially contribute to CNS homeostasis by providing structural support for neurons, maintaining water and ion balance, regulating levels of neurotransmitters and regulatory amino acids, providing antioxidant protection and supplying energy, mainly by glycolysis. They are also an integral part of the neurovascular unit and active component of the information network in the brain. In addition, they are important regulators of BBB function[75]. Astrocytes constitute approximately 30% of the cells in the mammalian CNS. Human astrocytes have especially unique characteristics, as they are by volume 20 times larger and contact up to ten times more synapses than their rodent counterparts[76].

Immediately after ischemic stroke, cytokines and ROS released by neurons and glial cells alter astrocytic phenotype, so they become hypertrophic and start to proliferate.

Reactive astrocytes enhance the intermediate filament marker GFAP and S100β expression, and under certain conditions upregulate their progenitor markers, such as vimentin and nestin[77]. The process of astrocyte activation following brain injury is initiated approximately 4–6 h post-stroke, peaks at 2–3 days, and develops up to

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around one week after ischemic attack[78], whilst microglial activation may last up to several months after the stroke onset[79]. Upon ischemic damage, injured neurons and other brain cells release various molecules, which act as DAMPs or alarmins by activating specific PRRs on different cells, including astrocytes[80]. ATP released from damaged cells activates P2Y receptors on astrocytes[81], resulting in the secretion of inflammatory cytokines, such as IL-1β, or enzymes, such as MMPs, that are involved in BBB disintegration and edema formation[82]. Other immune functions of astrocytes under ischemia include antigens presentation, phagocytosis and pro-MMP2 production[83].

Astrocytes also participate in protective responses by taking up extracellular glutamate[83], contributing to BBB reconstitution[84] and releasing neurotrophic factors[85]. Certain findings indicate that reactive astrogliosis may have a protective role in the brain response to acute injuries, as specific GFAP ablation has been associated with increased ischemic brain damage after pMCAo[86]. A new classification of ischemia-induced, reactive astrocytes has been established quite recently, describing harmful A1-type, which strongly upregulate classical complement cascade genes previously shown to destroy synapses, and protective A2-type, expressing neurotrophic factors[87]. Taken together, the exact function of reactive astrocytes in the progression of ischemic cerebral infarction highly depends on the astrocyte polarization towards distinct phenotypes[87,88] and on their crosstalk with the surrounding microenvironment created by neighboring damaged neurons and activated microglia/macrophages.

2.1.4.2.3 Microglia and other brain resident macrophages

Microglia, resident hematopoietic cells in the CNS parenchyma, maintain homeostasis and surveil the environment in the healthy brain[89]. These cells originate from yolk sac-derived erythromyeloid precursors, which in a process called microgliogenesis, populate the brain during early stages of fetal development[90–

92]. According to these findings, yolk sac macrophages enter the murine brain rudiment via the bloodstream and migrate to the neuroepithelium around E9.5.

These cells resemble macrophages in terms of their morphology and F4/80 and CD11b expression. It has been shown that microgliogenesis is dependent on Irf8/PU.1[93]. Under healthy conditions, microglial cells are highly ramified, express low levels of CD45, MHCII, and Fc surface markers and their replenishment rate is relatively low[94]. Ischemic stroke leads to microglial cell activation, resulting in morphological changes to an ameboid shape, which facilitates an effective response to the environmental changes. Subsequently, microglia start to proliferate and migrate toward lesions, release various inflammatory factors and phagocyte cell debris.

There are several types of non-parenchymal macrophages, e.g. perivascular, subdural meningeal and choroid plexus macrophages, which have been shown to

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participate in brain immune responses[95]. These cells originate from hematopoietic precursors, and with the exception of choroid plexus macrophages, represent a stable population with a low turnover with blood-derived monocytes[95]. As these cells are residing in strategical locations within CNS and interact with the vasculature, one of their main responsibilities during neuroinflammation includes antigen presentation to circulating lymphocytes[96]. It has been recently shown in a rat model of tMCAo, that CD163⁺cells defined as perivascular and meningeal macrophages are key players in control of leukocyte chemotactic infiltration, as well as prominently impact BBB integrity in the acute phase of ischemic stroke[97].

2.1.4.2.3.1 Microglial activation in ischemic stroke

Activated microglia express typical myeloid markers and acquire round amoeboid shape, which makes them barely distinguishable from blood-borne macrophages[98], a major challenge in neuroinflammation research. Nevertheless, recent studies of distinct molecular and functional signatures in microglia have pinpointed several microglia-specific genes, like surface markers P2ry12, Gpr34, Mertk, secreted C1qa, Pros1 and Gas6 in humans, and Fcrls, Tmem119, Olfml3, Hexb, Tgfbr1, and Sall1 in mice[99]. In addition, CD11b and Iba1 remain typical markers of activated microglia, together with molecules associated with antigen presentation, such as MHC-II[100]. Under brain ischemia microglial activation is regulated by a number of receptors, including purinergic ATP receptors, such as P2RX7 and P2Y12, TLR4, CX3CR1, PPAR-γ, CB2, TREM2 and CD200R[101]. Microglia become activated very rapidly after the ischemic insult, their reactivity peaks a few days after the stroke and may persist for several weeks. This is evident both in animal stroke models[102,103] and in patients[104,105]. Spatiotemporal characteristics of immune cell activation in cerebral ischemia are summarized in Figure 3.

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Figure 3. Spatiotemporal profile of microglia and myeloid immune cell polarization in transient brain ischemia. Adapted from Benakis et al., 2015.

Polarization of activated microglial cells results in acquiring M1 or M2-like phenotype, mediating different cellular responses. Classically activated M1-type microglia/macrophages produce proinflammatory cytokines like IL-1β, IL-6, IL-23, while M2-type, alternatively activated cells are able to induce Th2 cell responses and release anti-inflammatory cytokines like IL-4[106]. Of note, neutrophils are also capable of acquiring N1 pro- or N2 anti-inflammatory phenotype, and their activation profiles resemble those of microglia and macrophages (Fig. 3). The types of microglial and macrophage activation are summarized in Figure 4. M1 phenotype, modulated primarily through TLRs, is generally considered detrimental, since persistent release of proinflammatory cytokines, like TNF-α[107], MCP1, and MIP-1 α[108], triggers increased ROS production, representing a potential source of cytotoxicity[109]. M1-type microglial cells also release other factors, such as reactive oxygen and nitrogen species[110], MMPs and other proteases. MMPs activity, apart from the phagocytosis of endothelial cells by perivascular microglia, have been highlighted as one of the substantial factors responsible for loss of BBB integrity leading to leukocyte influx into the ischemic penumbra[111]. Harmful mediators derived from M1-type microglial cells play a proven role in ischemic injury and inflammation, as pharmacological or miRNA-induced regulation of microglial activity ameliorates ischemic brain injury[112–114]. Rational immunomodulation in the early stages after ischemic stroke has been highlighted as a promising treatment strategy in experimental stroke models.

M2-type microglia, also called alternatively activated microglia, are characterized by release of anti-inflammatory factors, such as IL-4, IL-10, IL-13, and TGF-β, and are considered beneficial for ischemic stroke outcome[115]. M2-type microglia are mostly found in the early stages after ischemic injury, whereas in later stages the M1- like phenotype becomes increasingly predominant[108]. M2-type microglia polarize into different subpopulations (M2a, M2b, M2c and M2d), which have unique features, distinct gene expression profiles and specific biological functions[116,117].

The M2a (alternative) subtype is induced by IL-4 and IL-13, M2b (immunoregulatory) elicited by IL-1 receptor ligands, immune complexes and LPS, while M2c (immunosuppressing) is mediated by IL-10, TGF-β and glucocorticoids[118]. IL-6, TLR agonists and adenosine are responsible for macrophage/microglial polarization into M2d subtype[119]. Interestingly, some studies have shown that in the tMCAo model, selective ablation of proliferating resident microglia resulted in larger brain infarction, associated with an increased amount of apoptotic neurons[120].

Phagocytosis of damaged cells is a crucial process exerted mainly by microglia and macrophages, supporting structural and functional reorganization of the injured brain[121]. Complement-mediated phagocytosis shifts the cytokine profile from proinflammatory to anti-inflammatory[122], however transient complement

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inhibition in the acute stroke phase can also alleviate neuroinflammation and improve neuronal survival[123]. CD68, a common marker used to visualize active phagocytic cells, is upregulated in the penumbra early in pMCAo, and then gradually increases in the core to peak at 7 dpi[124]. Scavenger receptors MARCO, CD36, mannose receptor CD206, Ym1, and Trem2 are other markers linked to phagocytic activity or prevention of extracellular matrix degradation[125–129].

Neuronal death and functional impairment can be prevented by blocking specific phagocytic pathways. For example, abolishing MerTK and MFG-E8 action effectively suppresses phagocytosis of viable neurons and in turn ameliorates neurological deficits after focal brain ischemia[129].

Figure 4. Scheme representing the types of microglial and macrophage activation states. The division for M1 and M2 polarization is considered rather simplistic, yet practical approach to discriminate between the microglia and macrophage multiple subtypes. Adapted from Rőszer, 2015.

2.1.4.2.4 Blood-derived monocytes/macrophages in cerebral ischemia Increased permeability of the BBB is a typical hallmark of cerebral ischemia. Once BBB becomes disrupted, blood-borne cells and different molecules extravasate into the brain parenchyma to participate in the ongoing inflammatory processes[130].

Increased expression of adhesion molecules, together with the release of interleukins and chemokines, facilitate the infiltration of circulating immune cells to the injured

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brain[55]. The secretion of MMPs also significantly contributes to BBB breakdown, which further promotes the influx of immune cells from the periphery[131,132].

Monocytes and macrophages, detected in the brain tissue already at 24-72 h post- stroke[55], are the first immune cells infiltrating the ischemic lesion, followed by neutrophils, dendritic cells and lymphocytes. Recent studies show that the meninges covering tissue affected by ischemia are the main point of leukocyte recruitment to the brain in the early stages of ischemic stroke[133].

Blood-derived macrophages begin to provide phagocytic activity at the infarct site around one week post stroke[134]. Similar to microglia under inflammatory conditions, macrophages are also able to polarize into M1 or M2 phenotypes. M2- type macrophages are present in the ischemic core region around 3-5 days post stroke, however later on M1-like macrophages start to dominate in this area[135].

Several mechanisms have been proposed to contribute to the regulation of macrophage response in ischemic stroke. CX3CR1 signaling promotes monocyte/macrophage expansion, as well as microglial proinflammatory activation leading to the increase of brain damage[136–138]. CCR2 and MCP-1 activation is thought to be responsible for attracting peripheral cells to enter the cerebral tissue under ischemia[139]. CCR2 positive cells have been implicated either in detrimental[140,141], as well as beneficial actions, through TGFβ-related maintenance and support of vascular integrity[142]. Moreover, spleen-derived monocytes are considered as an important cell population participating in the post- stroke regulation of inflammatory responses. After cerebral ischemia in rodents their spleen undergoes transient atrophy, which has been linked to release of splenic immune cells passaging through the circulation into the ischemic lesion[143]. These cells have been mainly associated with increase of the inflammatory response and exacerbation of brain injury[144,145].

Once macrophages enter the ischemia-damaged brain parenchyma, they become morphologically, as well as in great extent phenotypically indistinguishable from resident microglia and that is one of the main challenges in the context of studying the immune responses after ischemic stroke. However, to circumvent this problem several approaches have been developed that enable discrimination between activated microglia and infiltrated monocyte-derived macrophages[146], for example labeling of peripheral macrophages with GFP[147,148]. The microglial and peripheral macrophage activity within the ischemic infarct has been also distinguished by labeling macrophages with USPIO and analyzing their spatiotemporal activity by MRI. In these studies, macrophage infiltration was identified at 4 dpi in the ischemic core in rats subjected to pMCAo[149,150]. Finally, based on differential expression of CD45, CD11b⁺ microglia and peripheral macrophages can be distinguished by FACS. Resident microglia are characterized as CD45^lo, while activated macrophages express notably higher levels of this marker and thus are CD45^hi[151,152].

Monocytes are classified as proinflammatory Ly6C^hiand regulatory Ly6C^lo[153].

CCR2 expression on Ly6C^himonocytes contributes to M2-type polarization, and its

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inhibition results in increased RNS production in macrophages, leading to worsened stroke outcome[154,155]. Ly6C^hiCD43^lo monocyte subset has been linked to tissue repair processes, as these initially proinflammatory cells have the aptitude to polarize into anti-inflammatory phenotype in the ischemic brain environment[156]. Ly6C^lo monocytes are essentially redundant in the progression and recovery of ischemic stroke[157]. Ly6C^loCX3CR1^hi monocytes are a terminally differentiated subset of monocytes not able to polarize further into anti-inflammatory cells. These patrolling monocytes are mainly responsible for endothelial integrity maintenance[158].

An important factor influencing the temporal profile of immune cell activation and infiltration into the brain is the type of cerebral ischemia. Compared to transient ischemia with reperfusion, in permanent ischemia the microglial activation peaks at 1 and 5 dpi and the peripheral immune cells infiltrate into the brain earlier[159,160].

It is thought that mechanical disruption of meninges integrity by craniectomy in pMCAo facilitates peripheral immune cell and molecule entry into the brain parenchyma[133].

2.1.4.3 Inflammatory mediators

Soluble immune factors such as cytokines, growth factors, chemokines and adhesion molecules profoundly contribute to post-ischemic immune responses. Cytokines and chemokines can be categorized according to structural homology of their receptors, or based on their physiological function as pro- (Th1) and anti-inflammatory (Th2) cytokines[161].

The Type I cytokine receptor family, also known as hemopoietic growth factor family, has a 3D motif consisting of an extracellular region containing four α helices[161]. Members of this family include receptors to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, G-CSF and GM-CSF. Certain receptor subfamilies also can be recognized: IL-2R (with IL-2, IL-7, IL-9, IL-15, and IL-21 receptors), IL-4R (with IL-3, IL-4, IL-5, IL-13, and GM-CSF receptors) and IL-12R subfamily (with IL-12, IL-23, and IL-27 receptors). One of the most prominent members of this family, IL-6, is usually classified as proinflammatory cytokine, since its high levels in plasma are correlated with bigger infarcts and worse neurological deficits[162]. However, IL-6 actions also involve STAT3 activation, known to confer neuroprotection by transcriptional regulation of antiapoptotic regulatory factors, such as the Bcl-2 family[163,164].

The Type II cytokine receptor family, also called interferon family, comprises of receptors to IL-10, IL-19, IL-20, IL-22, and 8 types of interferons. Within this family there are two groups: IL-10R and IFNR subfamily[161]. Functions of this family of cytokines include modulation of inflammatory mechanisms, antiviral states induction, inhibition or stimulation of cell growth and regulation of apoptosis[165].

Similar to the Type I cytokine family, Th1 and Th2-type cytokines are included in this family, with IFN-γ being a typical proinflammatory cytokine and IL-10 exerting anti- inflammatory and immunosuppressive actions.

Modulation of neuroimmune responses as potential treatment strategies in ischemic stroke

uef.fi

Dissertations in Health Sciences

NATALIA KOŁOSOWSKA

MODULATION OF NEUROIMMUNE

NATALIA KOŁOSOWSKA

MODULATION OF NEUROIMMUNE RESPONSES AS POTENTIAL TREATMENT

STRATEGIES IN ISCHEMIC STROKE

Natalia Kołosowska

MODULATION OF NEUROIMMUNE RESPONSES AS POTENTIAL TREATMENT

STRATEGIES IN ISCHEMIC STROKE

ACKNOWLEDGEMENTS

LIST OF ORIGINAL PUBLICATIONS

CONTENTS

ABBREVIATIONS

1 INTRODUCTION

2 LITERATURE REVIEW