Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s

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murine encephalomyelitis1

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Vanessa Herder1,2*, Cut Dahlia Iskandar1,2*, Kristel Kegler1,2, Florian Hansmann1,2,

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Suliman Ahmed Elmarabet1, Muhammad Akram Khan1,2, Arno Kalkuhl3, Ulrich

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Deschl3, Wolfgang Baumgärtner1,2, Reiner Ulrich1,2,, Andreas Beineke1,2,4,

Accepted Article

1

6 7

1

8

Germany

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2

10

3

11

KG, Biberach (Riss), Germany

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*authors have contributed equally; authors have contributed equally

Department of Pathology, University of Veterinary Medicine Hannover, Hannover,

Center for Systems Neuroscience, Hannover, Germany Department of Non-Clinical Drug Safety, Boehringer Ingelheim Pharma GmbH & Co.

13 14

4

15

Prof. Dr. Andreas Beineke, Dipl. ECVP

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Department of Pathology

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University of Veterinary Medicine Hannover

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Bünteweg 17

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D-30559 Hannover, Germany

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Mail: [email protected]

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Phone: 0049-511-953-8640

22

Fax: 0049-511-953-8675

corresponding author

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between thiversion and the Version of Record. Please cite this article as doi: 10.1111/bpa.12238

1 This article is protected by copyright. All rights reserved.

Accepted Article

24 25

Abstract

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Microglia and macrophages play a central role for demyelination in Theiler’s murine

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encephalomyelitis (TME) virus-infection, a commonly used infectious model for

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chronic-progressive multiple sclerosis. In order to determine dynamic changes of

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microglia/macrophage polarization in TME, the spinal cord of SJL mice was

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investigated by gene expression profiling and immunofluorescence. Virus persistence

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and demyelinating leukomyelitis was confirmed by immunohistochemistry and

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histology. Electron microscopy revealed continuous myelin loss together with abortive

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myelin repair during the late chronic infection phase, indicative of incomplete

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remyelination. A total of 59 genes out of 151 M1- and M2-related genes were

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differentially expressed in TMEV-infected mice over the study period. The onset of

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virus-induced demyelination was associated with a dominating M1-polarization, while

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mounting M2-polarization of macrophages/microglia together with sustained

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prominent M1-related gene expression were present during the chronic progressive

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phase. Molecular results were confirmed by immunofluorescence, showing an

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increased spinal cord accumulation of CD16/32+ M1-, arginase-1+ M2- and Ym1+ M2-

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type cells associated with progressive demyelination. The present study provides a

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comprehensive database of M1/M2-related gene expression involved in the initiation

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and progression of demyelination supporting the hypothesis that perpetuating

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interaction between virus and macrophages/microglia induces a vicious circle with

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persistent inflammation and impaired myelin repair in TME.

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2 This article is protected by copyright. All rights reserved.

Accepted Article

47 48

Introduction

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Multiple sclerosis (MS), one of the most frequent central nervous system (CNS)

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diseases in young adults, is a chronic demyelinating disease of unknown etiology

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and possibly multifactorial causes (13). Based on the generation of myelin-specific

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immune responses, MS is regarded as an autoimmune disease (4, 68), presumably

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triggered by virus infections (33, 63). Due to clinical and pathological similarities,

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Theiler’s murine encephalomyelitis (TME) represents a commonly used infectious

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animal model for the chronic-progressive form of human MS (14, 59, 62, 74).

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Following intracerebral infection with a low virulent BeAn-strain of Theiler’s murine

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encephalomyelitis virus (TMEV) susceptible mouse strains develop persistent CNS

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infection with immune mediated spinal cord demyelination and remyelination failure

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(26, 29, 41, 46, 55, 60, 86, 87, 90).

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Microglia and CNS-infiltrating macrophages play a central role in the pathogenesis of

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TMEV-induced demyelination. They represent target cells for viral persistence during

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the chronic disease phase (40, 76) and contribute to myelin damage by the release

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of myelinotoxic factors (bystander demyelination), delayed-type hypersensitivity

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reaction and induction of myelin-specific autoimmunity (48, 56). Similarly, microglia

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induce myelin damage also in autoimmune and toxic rodent models for MS, such as

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experimental

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demyelination,

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microglia/macrophages plasticity describes different cell populations with distinct and

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even opposing functions. For instance, M1-type microglia/macrophages promote

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inflammation, which leads to protective immunity against pathogens but if

autoimmune respectively

encephalomyelitis (47,

80,

93).

(EAE) The

and

cuprizone-induced

current

concept

of

3 This article is protected by copyright. All rights reserved.

uncontrolled also to immune mediated tissue damage by the release of pro-

72

inflammatory cytokines, reactive oxygen species and nitric oxide (69, 71). In contrast,

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M2-type cells exhibit neuroprotective properties usually during advanced disease

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stages due to phagocytosis of debris, promoting tissue repair and termination of

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neuroinflammation by down-regulating M1- and Th1-immune responses (43).

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So far, only few reports mention the polarizing effects of TMEV upon microglia in vitro

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(24). Moreover, M1- and M2-type cells represent merely two extremes of the

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polarization continuum and macrophages/microglia with an intermediate activation

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status can be observed inter alia in demyelinating MS lesions (92), demonstrating the

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need for quantitative analyses of M1/M2-related factors in myelin disorders. Thus, the

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aims of the present study were to (i) select candidate genes involved in

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macrophage/microglia polarization by DNA microarray analyses and (ii) to compare

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their transcription levels to get insights in M1/M2 balances during the initiation and

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progression of virus-induced demyelination. In addition, (iii) dynamic changes of M1-

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and M2-type cells in TME were determined with the aid of immunofluorescence.

Accepted Article

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86 87

4 This article is protected by copyright. All rights reserved.

Materials and Methods

Accepted Article

88 89 90

Experimental design

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Five-week-old female SJL/J mice (Harlan, Borchen, Germany) were inoculated into

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the right cerebral hemisphere with 1.63x106 plaque-forming units/mouse of the

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BeAn-strain of TMEV in 20µl Dulbecco’s Modified Eagle Medium (PAA

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Laboratories, Cölbe, Germany) with 2% fetal calf serum and 50µg/kg gentamicin.

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Mock-infected animals received 20µl of the vehicle only. Inoculation was carried

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under general anesthesia with medetomidine (0.5 mg/kg, Domitor, Pfizer, Karlsruhe,

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Germany) and ketamine (100 mg/kg, Ketamine 10%, WDT eG, Garbsen, Germany).

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All experiments were performed in groups of six TMEV- and 3-6 mock-infected

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mice, euthanized 14, 42, 98 and 196 days post infection (dpi). For histology,

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immunohistochemistry and special stains, thoracic spinal cord segments were

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removed immediately after death and fixed in 10% formalin for 24 hours, decalcified

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in disodium-ethylenediaminetetraacetate for 48 h and subsequently embedded in

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paraffin wax. For microarray analysis and immunofluorescence, spinal cords were

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immediately removed, snap-frozen in liquid nitrogen and stored at -80°C (28, 65,

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90).

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The animal experiments were approved and authorized by the local authorities

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(Niedersächsisches Landesamt für Verbraucherschutz- und Lebensmittelsicherheit

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[LAVES], Oldenburg, Germany, permission number: 33.9.42502-04/07/1331, 509c-

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42502-02/589 and 33-42502-05/963).

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5 This article is protected by copyright. All rights reserved.

Histology

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Leukomyelitis was evaluated on hematoxylin and eosin (HE)-stained transversal

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sections using a semiquantitative scoring system based upon the degree of

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perivascular infiltrates: 0 = no changes, 1 = scattered perivascular infiltrates, 2 = 2

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to 3 layers of perivascular inflammatory cells, 3 = more than 3 layers of perivascular

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inflammatory cells, as described previously (23). For the evaluation of myelin loss,

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serial sections of spinal cord were stained with Luxol fast blue-cresyl violet

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(LFB-CV) and the degree of demyelination was semi-quantitatively evaluated as

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follows: 0 = no change, 1 = 25%, 2 = 25-50% and 3 = 50-100% of the white matter

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affected (23). The scoring was performed separately on all 4 quarters of spinal cord

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transversal sections. For each animal the arithmetic average of leukomyelitis and

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myelin loss was calculated. Histological data used for the present study were

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generated in our previous studies (89, 90).

Accepted Article

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124 125

Immunohistochemistry

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Immunohistochemistry was performed using a polyclonal rabbit anti-TMEV capsid

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protein VP1-specific antibody, as described before (39). Briefly, for blocking of the

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endogenous peroxidase, formalin-fixed, paraffin-embedded tissue sections were

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treated with 0.5% H2O2 diluted in methanol for 30 minutes at room temperature.

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Subsequently, slides were incubated with the primary antibody at a dilution of 1:2000

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for 16 hours at 4°C. Goat-anti-rabbit IgG diluted 1:200 (BA9200, H+L, Vector

132

Laboratories, Burlingame, CA, USA) was used as a secondary antibody for one hour

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at room temperature. Sections used as negative controls were incubated with rabbit

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normal serum at a dilution of 1:2000 (Sigma-Aldrich Chemie GmbH, Taufkirchen, 6 This article is protected by copyright. All rights reserved.

Germany). Slides were subsequently incubated with the peroxidase-conjugated

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avidin-biotin complex (ABC method, PK-6000, Vector laboratories, Burlingame, CA,

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USA) for 30 minutes at room temperature. After the positive antigen-antibody reaction

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visualization by incubation with 3.3-diaminobenzidine-tetrachloride in 0.1M imidazole,

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sections were counterstained with Mayer’s hematoxylin.

Accepted Article

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140 141

Immunofluorescence

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Methanol-fixed frozen sections of the thoracic spinal cord were rinsed in 0.1% Triton

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X-100 (Sigma-Aldrich, Taufkirchen, Germany) in phosphate buffered saline (PBS) for

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30 minutes. Non-specific binding was blocked with 20% goat or horse serum,

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respectively, diluted in PBS/0.1% Triton X-100/1% bovine serum albumin for 30

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minutes After washing with 0.1 % Triton X-100 in PBS, slides were incubated with

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primary CD68- (monoclonal rat anti-mouse antibody, Ab53444, clone FA-11, Abcam

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Ltd.; dilution 1:200) and CD107b- (monoclonal rat anti-mouse antibody MCA2293,

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clone

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macrophages/microglia. For visualization of M1-type macrophages/microglia a

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CD16/32-specific antibody (monoclonal rat anti-mouse, 553141, clone 2.4G2, BD

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Pharmingen; dilution 1:25) and for M2-type cells an arginase-1-specific antibody

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(polyclonal goat anti-human, SC-18351, Santa Cruz Biotechnology; dilution 1:50) and

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a Ym1-specific antibody (polyclonal rabbit anti-mouse antibody, ab93034, Abcam

155

Ltd.; dilution 1:100) were used. Slides were incubated for one hour, followed by

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washing in PBS/0.1% Triton X-100. As negative control, slides were incubated with

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goat, rat or rabbit serum in the same concentration as the primary antibodies.

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Subsequently slides were incubated with secondary DyLight 488-conjugated donkey

M3/84,

AbD

Serotec;

dilution

1:200)

for

the

detection

of

7 This article is protected by copyright. All rights reserved.

anti-goat (Jackson ImmunoResearch Laboratories, Dianova; dilution 1:200), Cy2-

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conjugated goat anti-rabbit IgG antibodies (Jackson ImmunoResearch Laboratories,

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Dianova; 1:200) and Cy3-conjugated goat anti-rat (Jackson ImmunoResearch

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Laboratories, Dianova; dilution 1:200), respectively, for one hour at room temperature

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and afterwards washed in PBS.

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For double staining, slides were simultaneously incubated for 90 minutes with the

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CD107b-specific monoclonal antibody (see above) and primary antibodies directed

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against CCL5 (polyclonal rabbit anti-human antibody; ABIN674949, antibodies-online

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GmbH dilution 1:10), CXCL10 (polyclonal rabbit anti-mouse antibody; ABIN687442,

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antibodies-online GmbH dilution 1:10), interferon-γ (polyclonal rabbit anti-human

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antibody, ABIN669141, antibodies-online GmbH; dilution 1:20), and TMEV

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(polyclonal rabbit anti-TMEV BeAn VP1 antibody; dilution 1:2000 (39)), respectively.

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Cy2-conjugated goat anti-rat (Jackson ImmunoResearch Laboratories, Dianova;

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dilution 1:200) and Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch

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Laboratories, Dianova; dilution 1:200) secondary antibodies were simultaneously

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used to visualize the respective antigens (see above). Nuclear counterstaining was

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performed with 0.01% bisbenzimide (H33258, Sigma Aldrich) for 10 minutes and

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sections were mounted with Dako Fluorescent Mounting medium (Dako Diagnostika).

Accepted Article

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177 178

Statistical analyses

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For

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immunofluorescence, a Mann-Whitney-U-test was performed. A p-value of less than

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0.05 was considered as statistically significant.

non-category

data

obtained

by

histology,

immunohistochemistry

and

8 This article is protected by copyright. All rights reserved.

Accepted Article

182 183

Electron microscopy

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Electron microscopy was performed as described previously (38, 91). Spinal cord

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samples were fixated with 2.5% glutaraldehyde and incubated overnight at 4°C. Post-

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fixation was performed in 1% aqueous osmium tetroxide and after five washes in

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cacodylate buffer (five minutes each) samples were dehydrated through series of

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graded alcohols and embedded in Epon 812 medium. Semi-thin sections were cut on

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a microtome (Ultracut Reichert-Jung, Leica Microsystems, Germany) and stained

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with uranyl citrate for 15 minutes. After eight washing steps samples were incubated

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with lead citrate for seven minutes. Ultra-thin sections were cut with a diamond knife

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(Diatome, USA) and transferred to copper grids. For descriptive ultrastructural

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analyses of white matter changes one hundred axons and their myelin sheaths per

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animal were examined for the presence of degenerative changes (myelin sheath

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vacuolization, myelin loss) and regeneration (oligodendrocyte-type remyelination,

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Schwann cell-type remyelination) by a transmission electron microscope (EM 10C,

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Zeiss, Germany). For quantification of phagocytic activity (gitter cell morphology,

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presence of myelin fragments and/or apoptotic bodies within the cytoplasm) a total of

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one hundred macrophages/microglia were investigated.

200 201 202

Microarray analyses

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RNA was isolated from frozen spinal cord samples using the RNeasy Mini Kit

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(Qiagen, Hilden, Germany), amplified and labeled using the Message Amp II-Biotin 9 This article is protected by copyright. All rights reserved.

Enhanced Kit (Ambion, Austin, USA) and hybridized to GeneChip mouse genome

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430 2.0 arrays (Affymetrix, Santa Clara, USA) as described (90). Six biological

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replicates were used per group and time point, except for five TMEV-infected mice at

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98 dpi. Background adjustment and quantile normalization was performed using

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RMAExpress (6). MIAME compliant data set are deposited in the ArrayExpress

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database (E-MEXP-1717; http://www.ebi.ac.uk/arrayexpress).

Accepted Article

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Selection of M1- and M2-associated genes

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For molecular characterization of macrophage/microglia polarization a data set of

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genes differentially expressed in the spinal cord of TMEV-infected SJL mice obtained

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in our previous global gene expression analysis was used (90). The present analyses

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focused

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microglia/macrophages (Supplemental Table S1) according to peer-reviewed

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publications (15, 17, 35, 54). The fold change was calculated as the ratio of the

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inverse-transformed arithmetic means of the log2-transformed expression values of

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TMEV-infected versus mock-infected mice. Down-regulations are shown as negative

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reciprocal values.

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Statistics, version 20, IBM Corporation, Armonk, USA) comparing TMEV- and mock-

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infected mice were calculated followed by adaption of the p-values according to the

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method described by Storey and Tibshirani using QVALUE 1.0 (84). Significantly

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differentially expressed genes between TMEV- and mock-infected mice were

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selected employing a q-value 0.05 cutoff combined with a 2.0 or -2.0 fold-change

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filter. The relative percentage of differentially expressed M1- versus M2-marker

on a

list

of

genes

Independent

associated

pair-wise

with

M1- or

M2-polarization of

Mann-Whitney-U-tests (IBM SPSS

10 This article is protected by copyright. All rights reserved.

genes was compared for each time point employing Fisher’s exact tests (p-value

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0.05).

Accepted Article

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230 231

Results

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Histological scoring of spinal cord inflammation and demyelination

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Histological data used for the present study were generated in our previous studies

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(89, 90). Examination of the HE-stained spinal cord sections revealed a

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mononuclear inflammation (leukomyelitis) within the white matter of TMEV-infected

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mice beginning at 14 dpi. The inflammatory changes increased towards 98 dpi and

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were significantly increased compared to mock-infected control animals at all

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investigated time points: 14 dpi (p=0.011), 42 dpi (p=0.002), 98 dpi (p=0.013) and

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196 dpi (p=0.002; Figure 1 and 2). The amount of demyelination increased until 196

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dpi (Figure 1 and 2). At 3 investigated time points (42, 98 and 196 dpi),

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demyelination in the spinal cord of TMEV-infected SJL-mice was significantly

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increased compared to mock-infected control mice (p=0.002, p=0.007, p=0.002) as

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determined by the myelin stain LFB-CV (Figure 1 and 2).

244 245

Quantification of virus load in the spinal cord and virus detection in CD107b+

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microglia/macrophages

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Immunohistochemistry for the detection of virus protein in the spinal cord of TMEV-

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infected mice revealed infection at all investigated time points (14, 42, 98, and 196

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dpi). While at 14 dpi infected cells were found scattered in the grey and white matter,

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with the onset of demyelination at 42 dpi positive cells were located predominantly in 11

This article is protected by copyright. All rights reserved.

lesions of the ventral spinal cord white matter (Supplemental figure S1). No positive

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signals were observed in mock-infected control mice.

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Immunofluorescence

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macrophage/microglia infection. Results showed that at 14 dpi 25-50%, at 42 dpi 40-

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57%, at 98 dpi 38-67%, and at 196 dpi 33-58% of TMEV-infected cells represent

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CD107b+ macrophages/microglia (Supplemental figure S2).

Accepted Article

251

double

staining

was

performed

to

demonstrate

257 258

Characterization of myelin alterations and regeneration by electron microscopy

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Descriptive ultrastructural analyses revealed subtle myelin changes before the onset

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of overt demyelination at 14 dpi in an average of 0.3% of investigated axons,

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characterized by vacuolization of myelin sheaths. At 42 dpi 2.2% of axons showed

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myelin sheath vacuolization and 5.8% of axons showed a complete loss of myelin

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(Figure 3). At 98 dpi an average of 2.8% of vacuolated myelin sheaths were observed

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and 8.4% of axons were totally denuded in demyelinated foci. At 196 dpi 5.0% of

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axons within white matter lesions showed a complete loss of myelin sheath, while

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2.5% of axons showed oligodendrocyte-type remyelination and 0.7% Schwann cell-

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type remyelination (Figure 3; Supplemental table S2), indicative of beginning but

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abortive myelin repair (91). Remyelination by Schwann cells was characterized by

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the presence of oval to signet ring-shaped cells in close proximity to axons,

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ensheathing axons with myelin on a one-to-one basis (Figure 3; 20, 96).

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Phagocytosis of myelin fragments associated with denuded axons, representing a

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hallmark of active demyelination, was observed starting 42 dpi. At this time point an

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average of 40.2% of microglia/macrophages displayed gitter cell morphology with 12

This article is protected by copyright. All rights reserved.

phagocytized myelin in the cytoplasm (myelinophages; Figure 3). At 98 and 196 dpi,

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50.1% and 51.5% of investigated macrophages/microglia represent myelinophages.

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In addition, phagocytized apoptotic bodies were present in an average of 9.3% of

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macrophages/microglia at 42 dpi, followed by a decline at 98 (0.7%) and 196 dpi

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(0.5%; Supplemental table S2).

Accepted Article

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279 280

Quantification of M1- and M2-related gene expression by DNA microarray

281

analyses

282

In order to get insights into polarization related to microglia/macrophages, DNA

283

microarray analyses of spinal cord tissue have been performed. A total of 151 genes

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related to macrophages/microglia-polarization were extracted from peer-reviewed

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publications, of which 72 and 66 were unequivocally assigned as M1- and M2-marker

286

genes, respectively. Thirteen genes were assigned to both polarization types

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(Supplemental table S1).

288

A total of 59 genes (39.1%) were differentially expressed in TMEV-infected mice over

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the study period (Figure 4, supplemental table S3). Most strikingly, although the

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number of differentially expressed genes increased over the study period for both

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phenotypes, comparison of the relative proportion of differentially expressed M1-

292

versus M2-marker genes revealed a significantly higher percentage of differentially

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expressed M1-marker genes at 14 (p=0.035) and 42 dpi (p = 0.016). In addition, a

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statistical tendency (p = 0.078) of increased M1-associated genes was observed at

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98 dpi, whereas a comparable proportion of M1- and M2-marker genes was detected

296

at later time points (Figure 4). 13

This article is protected by copyright. All rights reserved.

According to the function, differentially expressed genes were assigned to seven

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pathways, including chemotaxis (group I; 15 genes), phagocytosis, antigen

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processing and presentation (group II; 16 genes), cytokine and growth factor

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signaling (group III; 12 genes), Toll-like receptor signaling (group IV; 2 genes),

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apoptosis (Group V; 4 genes), extracellular matrix interaction and cell adhesion

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(group VI; 5 genes), and miscellaneous genes not related to a specific pathway

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(group VII; 5 genes; supplemental table S3). In group I, 53.3% of genes (8/15 genes)

304

were up-regulated on 14 dpi, while at subsequent time points nearly all genes were

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significantly up-regulated. In group II and III 62.5% of genes (10/16 genes) and

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50.0% of genes (6/12), respectively were up-regulated at 14 dpi, followed by an up-

307

regulation of nearly all genes at 42, 98, and 196 dpi in both groups. Tlr1 (group IV)

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was significantly transcribed at 42, 98, and 196, while expression of Tlr2 was

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observed during the entire observation period. 75% of apoptosis-related genes (3/4

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genes; group V) were significantly up-regulated in infected mice at 14 dpi and 100%

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at subsequent time points. While at 14 dpi 40.0% of genes (2/5 genes), all genes

312

(100%) were up-regulated at 42, 98, and 196 dpi. Miscellaneous genes not assigned

313

to a specific pathway (group VII) included Atf3, Arg1, Cepba, Chi3l3 and Hexb. No

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genes were differentially expressed at 14 dpi. Atf3, Arg1, and Cebpa were

315

significantly increased at 42, 98 and 196 dpi, while the M2-marker Chi3l3 (aka Ym1)

316

was only transcribed during the late chronic phase at 196 dpi (Supplemental table

317

S3).

Accepted Article

297

318 319

Temporal changes of macrophages/microglia subsets and verification of DNA

320

microarray results by immunofluorescence 14

This article is protected by copyright. All rights reserved.

Immunofluorescence was used to confirm the results obtained by gene expression

322

profiling. The number of microglia/macrophages increased over time in the spinal

323

cord

324

microglia/macrophages in the late stages of the disease. CD16/32 + M1- and also

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arginase-1+ M2-type cells were significantly increased compared to non-infected

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animals at 42, 98 and 196 dpi (Figure 5 and 6). Interestingly, a significant increase of

327

Ym1+ M2-type cells was found only at 98 and 196 dpi (Figure 5 and 6), suggestive of

328

late M2-polarization during the chronic demyelinating phase. To further substantiate

329

this, statistical analyses between the early (42 dpi) and late demyelinating phase

330

(196 dpi) have been performed. Results revealed a significant time-dependent

331

increase of CD107+ macrophages/microglia (p = 0.006), arginase-1+ M2-type cells (p

332

= 0.042), and Ym1+ M2-type cells (p = 0.009), while no significant temporal

333

differences were observed for CD68+ macrophages/microglia and CD16/32+ M1-type

334

cells (data not shown). Accordingly, the ratio of arginase-1+ M2-type cells to

335

CD16/32+ M1-type cells (p = 0.044) and the ratio of Ym1+ M2-type cells to CD16/32+

336

M1-type cells (p = 0.008) significantly increased over time (Figure 5), characteristic of

337

mounting M2-responses during disease progression.

338

Employing the Spearman’s rank correlation coefficient, the amounts of all

339

investigated macrophage/microglia proteins (CD68, CD107b, CD16/32, arginase-1,

340

Ym1) were significantly, positively correlated with the expression level of the

341

respective genes (Table 1).

342

In order to demonstrate expression of M1-related chemokines (CCL5 and CXCL10)

343

and interferon (IFN)-γ in macrophages/microglia during the early (42 dpi) and late

344

demyelinating phase (196 dpi) immunofluorescence double staining has been

Accepted Article

321

of

infected

mice

with

highest

numbers

of

CD107b+

and

CD68+

15 This article is protected by copyright. All rights reserved.

performed. Results revealed that both chemokines are preferentially expressed by

346

macrophages/microglia in spinal cord white matter lesions, since at 42 dpi 91-100%

347

and at 196 dpi 80-92% of CCL5-positive cells represent CD107+ cells (Supplemental

348

figure S3 and S4). Similarly, co-localization with CD107b was observed in 70-100%

349

at 42 dpi and in 80-94% of cells expressing CXCL10 at 196 dpi (Supplemental figure

350

S3 and S4). At 42 dpi 52-80% and at 196 dpi 72-93% of IFN-γ-positive cells are co-

351

labeled with CD107b (Supplemental figure S3 and S4), showing that in addition to

352

CNS-infiltrating lymphocytes (64) also macrophages/microglia contribute to IFN-γ

353

production in demyelinating lesions of TMEV-infected mice.

Accepted Article

345

354

355

16 This article is protected by copyright. All rights reserved.

Discussion

357

The present study provides a comprehensive database of M1/M2-related genes

358

expressed during the initiation and progression of TME. Although most molecules are

359

produced also by other resident CNS cells and recruited lymphocytes (11, 27), all

360

selected genes can be transcribed by macrophages/microglia and are involved in

361

their polarization, respectively (15, 17, 35, 54). Results revealed an imbalance of

362

M1/M2-responses during the onset of virus-induced demyelination, characterized by

363

the dominance of CD16/32+ M1-type cells and disproportionally elevated M1-related

364

gene expression in the spinal cord of infected mice. With disease progression an

365

accumulation of arginase-1+ and Ym1+, potentially neuroprotective M2-type cells (12,

366

61, 79) together with mounting transcription of M2-related genes was found.

367

However, sustained prominent M1-responses emphasize the importance of innate

368

immunity for immunopathology and progressive myelin loss in demyelinating

369

disorders, as discussed for MS (52, 92).

370

Differentially expressed M1-related genes at 14 dpi in the spinal cord of TMEV-

371

infected mice predominately consist of factors, such as chemokines, involved in the

372

CNS recruitment of macrophages, T cells and B cells (Table S3, group I).

373

Simultaneously, migration of CD68+ antigen presenting cells and activation of genes

374

related to innate and adaptive immunity within the CNS-draining cervical lymph node

375

has been observed in TMEV-infected mice during the acute phase of the disease in

376

our previous study (66). Under neuroinflammatory conditions, chemokines and their

377

receptors are produced by different cell types, such as microglia, astrocytes, neurons

378

and infiltrating leukocytes (85). In the present study, double labeling revealed that

379

macrophages/microglia are a major source of CCL5 (aka RANTES) and CXCL10

Accepted Article

356

17 This article is protected by copyright. All rights reserved.

(aka IP-10) within demyelinating lesions, which are preferentially expressed by M1-

381

type cells (51). In TME, both chemokines have been shown to critically control

382

leukocyte CNS influx and antiviral immune responses, respectively (57, 75, 77). In

383

addition, CCL5 and CXCL10 are up-regulated in the EAE model and the

384

cerebrospinal fluid of MS patients during demyelinating events, demonstrating their

385

functional role in immune mediated damage (18, 50, 81). Interestingly Ym1, detected

386

by microarray analysis and immunofluorescence, also displays chemotactic activity

387

and has been demonstrated to promote Th2 cytokine expression (9, 94), which might

388

reduce Th1-mediated immunopathology but probably also protective antiviral

389

immunity in TMEV-infected susceptible mice strains.

390

M1-responses are a hallmark of early innate immunity following viral infection

391

mediated by the interaction between microglial toll-like receptors (Table S3, group IV)

392

and cellular compounds (damage associated molecular pattern) and pathogen

393

associated molecular pattern, respectively (35, 36). However, besides their pivotal

394

role for antiviral immunity, microglia have been demonstrated to induce also myelin-

395

specific adaptive Th1-responses in TMEV-infected mice (67). Similarly, M1-polarized

396

cells foster immunopathology in primary autoimmune CNS disorders (58) and the

397

drug

398

macrophages/microglia from a M1- to a protective M2-phenotype (47). In addition,

399

selective inhibition of M1-type microglia by minocycline treatment reduces

400

neurodegeneration as demonstrated in mouse models for amyotrophic lateral

401

sclerosis (37). Similar to TME, experimental spinal cord injury in mice leads to

402

microglial polarization into a pro-inflammatory and neurotoxic M1-phenotype, which

403

might function as an early trigger of degeneration and immunological events at later

404

disease stages (35). Excessive microglial responses can be observed also in human

Accepted Article

380

Fasudil

ameliorates

the

clinical

severity

of

EAE

by

shifting

18 This article is protected by copyright. All rights reserved.

and canine spinal cord trauma, which leads to potentially destructive effects by the

406

release of pro-inflammatory cytokines, proteolytic molecules and reactive oxygen

407

species (2, 3, 19, 53, 72, 82, 83). Taken together, an imbalance towards M1-

408

dominance represents a potential prerequisite for lesion initiation in TME as currently

409

discussed for MS (22). Similar to findings in the present study, early innate immune

410

responses with activated pro-inflammatory microglia can be detected in pre-

411

demyelinating and early demyelinating MS lesions, which are supposed to induce

412

myelin damage and immunopathology (21, 52).

413

In the present study, the onset of demyelination and phagocytosis of myelin and

414

apoptotic cells is accompanied by an up-regulation of genes involved in antigen

415

processing, presentation and T cell stimulation (Table S3, group II). The functional

416

relevance of phagocytic macrophages/microglia for the pathogenesis of CNS

417

damage is discussed controversially. On the one hand, phagocytosis of myelin debris

418

enhances CNS regeneration following traumatic injury (95). Moreover, ingestion of

419

myelin induces a foamy appearance and anti-inflammatory function of cultured

420

human macrophages and myelinophages within MS lesions acquire a M2-phenotype,

421

which are supposed to contribute to resolution of inflammation and tissue repair (8).

422

In addition, phagocytosis of apoptotic cells by cultured rodent microglia leads to

423

diminished pro-inflammatory cytokine production with a reduced ability to activate T

424

cells (49). On the other hand, incorporation of myelin and cellular debris by microglia

425

is able to enhance their antigen presenting and myelin-specific T cell stimulatory

426

capacity in vitro (5, 10). Furthermore, isolated rat microglia exposed to myelin have

427

been described to develop a neurotoxic phenotype with an increased inducible nitric

428

oxide synthase, tumor necrosis factor-α and glutamate expression (69) .

Accepted Article

405

19 This article is protected by copyright. All rights reserved.

Microarray analysis revealed the transcription of several genes participating in the

430

interferon pathway predominately during the demyelinating phase (Table S3, group

431

III). In TME, microglia/macrophages activated by virus or IFN-γ enhance immune

432

mediated tissue damage by presenting viral antigens and endogenous myelin

433

epitopes to CD4+ T cells, which induces delayed type hypersensitivity and

434

autoimmunity, respectively (7, 16, 34, 70). Moreover, beside its protective antiviral

435

function, IFN-γ increases the migration of macrophages and microglial activation,

436

which induces myelinotoxic substances and free radicals causing progressive myelin

437

loss (bystander demyelination) in TME (46, 59, 88, 89). IFN-γ is the main cytokine

438

associated with M1-activation of microglia and macrophages (35, 71) and has been

439

shown in TMEV-infected mice to be produce by CD4+ and CD8+ T cells (64).

440

Noteworthy, demonstration of the cytokine in CD107b+ cells in the present study is

441

indicative also of an autocrine regulation of M1-polarization, as demonstrated in

442

endotoxin-stimulated macrophages (78).

443

Despite mounting M2-polarization and the expression of regeneration promoting

444

factors, such as insulin like growth factor-1 (igf1) and transforming growth factor-β

445

(Tgfb1) (25, 42, 93), CNS recovery remains abortive and only insufficient

446

remyelination attempts by oligodendrocytes and Schwann cells were found in the

447

spinal cord during the late chronic TME phase. Similar to the present observation,

448

macrophages/microglia with both M1- and M2-properities can be found in active

449

demyelinating MS brain lesions (92). Recent studies have demonstrated that the

450

switch of M1- into M2-type cells is required for efficient oligodendrocyte differentiation

451

and myelin repair following toxin-induced demyelination in rodents and that M2-

452

conditoned media drive oligodendrocyte maturation in vitro (61). In addition, M2-type

453

macrophages/microglia protect from EAE through deactivation of encephalitogenic

Accepted Article

429

20 This article is protected by copyright. All rights reserved.

Th1 and Th17 cells (73). Consequently, continuous M1-polarization observed till the

455

late chronic phase (196 dpi) in TMEV-infected mice has the potential to antagonize

456

neuroprotective effects of M2-microglia/macrophages. In agreement with previous

457

reports (40, 97), CD107+ microglia/macrophages represent a target for virus infection

458

in the present study. Since TMEV has been demonstrated to preferentially infect

459

activated myeloid cells with M1-charateristics, such as CD16/32 and IFN-γ

460

expression, in vitro (30, 31), it is also tempting to speculate that prolonged M1-

461

polarization contributes to viral persistence in susceptible mouse strains by providing

462

permissive target cells for TMEV. In addition, genes have been identified by the

463

present microarray analysis that might be involved in disturbed viral elimination by

464

influencing the interferon pathway (Table S3, group III). For instance, OASL1, a

465

recently defined type I interferon negative regulator and translation inhibitor of IRF7 is

466

differentially up-regulated in TMEV-infected mice. OASL1 causes T cell suppression

467

in persistent lymphocytic choriomeningitis virus infection of mice, and is regarded as

468

a new target for preventing chronic infectious diseases (44, 45). In agreement with

469

this idea, subpopulations of CNS-infiltrating macrophages have been demonstrated

470

to reduce protective antiviral immunity by inducing T cell exhaustion which leads to

471

virus persistence in TMEV-infected mice (32). Besides this, M2-polarized cells have

472

the ability to reduce antiviral immunity, as described for human cytomegalovirus

473

infection (1).

474

In conclusion, the perpetuating interaction between virus and macrophages/microglia

475

induces a vicious circle with continuous inflammation and impaired myelin repair in

476

the spinal cord of TMEV-infected mice. The present findings support the hypothesis

477

of a dual function of either polarized cells with promoting effects upon antiviral

478

immunity and immunopathology, respectively, in TME. Hence, in contrast to the

Accepted Article

454

21 This article is protected by copyright. All rights reserved.

therapeutic effect of M2-dominence in primary autoimmune diseases, such as EAE,

480

only a well-orchestrated and timely balanced polarization of macrophages/microglia

481

might have the ability to prevent virus persistence and reduce myelin loss in this

482

infectious MS model.

Accepted Article

479

483

484

485

Acknowledgements

486

The authors would like to thank Caroline Schütz, Kerstin Schöne, Danuta Waschke,

487

Bettina Buck and Petra Grünig for their excellent technical support during the

488

laboratory work and Dr. Karl Rohn for statistical analyses. This study was supported

489

by the German Research Foundation (FOR 1103, BA 815/10-2, BE 4200/1-2 and UL

490

421/1-2).

491

492

493

494

495

496

22 This article is protected by copyright. All rights reserved.

Figure legends

498

Figure 1

499

Histological lesions in the spinal cord of Theiler´s murine encephalomyelitis virus-

500

infected mice. A) Lymphocytic meningitis (arrows) and B) mild vacuolization of the

501

spinal cord white matter in an infected animal at 42 days post infection. C) Prominent

502

infiltration of macrophages/microglia in the spinal cord and lymphocytic meningitis

503

(arrow) at 196 days post infection. D) Demyelination of the spinal cord white matter

504

(asterisks) at 196 days post infection. E) Higher magnification of C) showing

505

activated macrophages/microglia with a foamy cytoplasm (gitter cells). F) Note

506

accumulation of myelin debris within the cytoplasm of macrophages/microglia,

507

indicative of myelinophagia. GM = gray matter; bars = 300µm (A-D) and 30µm (E-F);

508

hematoxylin-eosin stain (A,C,E), luxol fast blue stain (B,D,F).

Accepted Article

497

509 510

Figure 2

511

Scoring of demyelinating leukomyelitis in Theiler´s murine encephalomyelitis virus-

512

infected mice. A) Histology reveals inflammatory responses in the spinal cord

513

(leukomyelitis) at all investigated time points. B) Detection of demyelination in the

514

spinal cord white matter at 42, 98 and 196 days post infection. dpi = days post

515

infection; mock = mock-infected control mice; TMEV = Theiler´s murine

516

encephalomyelitis virus-infected mice;  = significant difference (p≤0.05, Mann-

517

Whitney-U-test). Box and whisker plots display median and quartiles with maximum

518

and minimum values.

519

23 This article is protected by copyright. All rights reserved.

Figure 3

521

Ultrastructural analyses of the spinal cord white matter of Theiler`s murine

522

encephalomyelitis virus-infected mice by transmission electron microscopy. A)

523

Macrophages/microglia containing phagocytized myelin fragments (white asterisks)

524

at 42 days post infection, characteristic of myelinophagia (M = nucleus of a

525

macrophage/microglial cell; magnification 13300x). B) Demyelinated axons (black

526

asterisks) lacking myelin sheaths and focal myelin vacuolization (arrow) in an

527

infected mouse at 96 days post infection. For comparison, myelinated axons with

528

intact myelin sheaths are labelled with triangles (magnification 6600x). C)

529

Oligodendrocyte in proximity to multiple remyelinated axons with thin myelin sheaths

530

(black asterisks) during late chronic infection phase (196 days post infection),

531

indicative of oligodendrocyte-mediated remyelination. Normally myelinated axons are

532

labelled with triangles (O = nucleus of an oligodendrocyte; magnification 5300x). D)

533

Schwann cell remyelination in a demyelinated area at 196 days post infection,

534

characterized by comparatively thick newly formed myelin sheaths (arrows) and a

535

one Schwann cell per axon relationship (S = nucleus of a Schwann cell;

536

magnification 6650x).

Accepted Article

520

537

538

Figure 4

539

Expression profile of M1- and M2-related genes in the spinal cord during the course

540

of Theiler´s murine encephalomyelitis. A) Heat map displays fold changes, indicated

541

by a color scale ranging from –4 (relative low expression) in green to 4 (relative high

542

expression) in red. 59 out of 151 selected genes are differentially expressed in

543

infected mice. B) Comparison of the relative proportion (percentage) of differentially 24

This article is protected by copyright. All rights reserved.

expressed M1- versus M2 marker genes employing the fisher´s exact test revealed a

545

significant dominance ( = p0.05) of M1-related genes at 14 and 42 days post

546

infection (dpi). A statistical tendency (p = 0.078) of an increased M1-associated gene

547

expression is observed at 98 dpi, whereas comparable proportions of M1- and M2-

548

marker genes are detected at 196 dpi.

Accepted Article

544

549 550

Figure 5

551

Quantification of different macrophage/microglia subsets in the spinal cord of

552

Theiler´s murine encephalomyelitis virus-infected mice by immunofluorescence.

553

Significant increase of A) CD68+ cells, B) CD107b+ cells, C) CD16/CD32+ cells, and

554

D) arginase-1+ cells at 42, 98 and 196 days post infection (dpi) and of E) Ym1+ cells

555

at 98 and 196 dpi in infected mice compared to mock-infected control mice. TMEV =

556

Theiler´s murine encephalomyelitis virus-infected mice; mock = mock-infected control

557

mice;  = significant difference (p≤0.05, Mann-Whitney-U-test). Box and whisker plots

558

display median and quartiles with maximum and minimum values. F) Significantly

559

elevated ratios of arginase-1+ M2-type cells to CD16/32+ M1-type cells (arginase-

560

1:CD16/32) and Ym1+ M2-type cells to CD16/32+ M1-type cells (Ym1:CD16/32) at

561

196 dpi compared to 42 dpi. Columns display median with maximum and minimum

562

values.  = significant difference (p≤0.05, Mann-Whitney-U-test).

563

564

Figure 6

565

Detection of different macrophage/microglia subsets in the spinal cord of Theiler`s

566

murine encephalomyelitis virus-infected mice by immunofluorescence. Accumulation

567

of A) CD107b+ cells, B) CD68+ cells, C) arginase-1 (Arg-1)+ cells, D) CD16/32+ cells, 25

This article is protected by copyright. All rights reserved.

and E) Ym1+ cells in the spinal cord white matter at 196 days post infection. Inserts

569

show higher magnifications of labelled cells. BIS = bisbenzimide (blue nuclear

570

counterstain).

Accepted Article

568

571

572

26 This article is protected by copyright. All rights reserved.

Supporting material:

Accepted Article

573

574 575

Figure S1

576

Detection of Theiler´s murine encephalomyelitis virus in the murine spinal cord by

577

immunohistochemistry. A) Quantification of infected cells at different time points.

578

TMEV = Theiler´s murine encephalomyelitis virus-infected mice; mock = mock-

579

infected control mice; dpi = days post infection;  = significant difference (p≤0.05,

580

Mann-Whitney-U-test). Box and whisker plots display median and quartiles with

581

maximum and minimum values. B) Note virus-specific labeling (brownish signal) in

582

the spinal cord white matter of an infected mouse at 98 dpi. Scale bar = 200 µm;

583

insert; scale bar = 50 µm.

584 585

Figure S2

586

Phenotyping of Theiler´s murine encephalomyelitis virus-infected cells (TMEV) by

587

immunofluorescence double staining. A) Percentage of infected cells representing

588

CD107b+ macrophages/microglia at different days post infection (dpi). Columns

589

display median with maximum and minimum values. B) Co-localization of TMEV

590

(green) and CD107b (red) in an inflammatory spinal cord lesion at 98 dpi. Double-

591

stained cells exhibit a yellow color. Nuclei are stained with bisbenzimide (blue).

592

Scale bars = 50 µm.

593 594

Figure S3

595

Detection of chemokines and interferon-γ (IFN-γ) in CD107b+ macrophages/microglia

596

by immunofluorescence double staining. Percentage of A) CCL5-, B) CXCL10- and 27

This article is protected by copyright. All rights reserved.

C) IFN-γ-positive cells co-expressing CD107b at 42 and 196 days post infection (dpi).

598

Columns display median with maximum and minimum values.

Accepted Article

597

599 600

Figure S4

601

Detection of chemokines and interferon-γ (IFN-γ) in CD107b+ macrophages/microglia

602

in the spinal cord by immunofluorescence double staining. Top row: co-localization of

603

CCL5 (green) and CD107b (red) in an infected mouse at 196 days post infection

604

(dpi). Middle row: co-localization of CXCL10 (green) and CD107b (red) in an infected

605

mouse at 196 dpi. Bottom row: co-localization of IFN-γ (green) and CD107b (red) in

606

an infected mouse at 42 dpi. Double-stained cells exhibit a yellow color (right

607

column). Nuclei are stained with bisbenzimide (blue). Scale bars = 50 µm.

608 609 610

28 This article is protected by copyright. All rights reserved.

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Accepted Article

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Accepted Article

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Accepted Article

896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931

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Table 1: correlation between data obtained by gene expression analyses and immunofluorescence

Accepted Article

932 933

Gene expression

934 935 936 937 938

Immunofluorescence CD107b

CD68

CD16/32

Arginase-1

Ym1

Arginase-1

0.756*

0.686*

0.650*

0.630*

0.662*

Cd68

0.735*

0.751*

0.757*

0.708*

0.717*

Cd32b

0.722*

0.854*

0.760*

0.624*

0.720*

Cd16

0.723*

0.852*

0.804*

0.589*

0.691*

Cd107b

0.629*

0.629*

0.636*

0.476*

0.595*

Ym1

0.320

0.348

0.321

0.449*

0.563*

Spearman’s rank correlation coefficient was used to correlate absolute numbers (positive cells/spinal cord) of CD107b+, CD68+, CD16/32+, aginase-1+ and Ym1+ cells with the respective mRNA level measured by microarray analysis in the spinal cord of Theiler´s murine encephalomyelitis virus-infected mice. Significant differences of the correlation coefficient from zero are marked as follows: * = p≤0.01.

939

940 941

36 This article is protected by copyright. All rights reserved.

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