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Role of programmed cell death and the inflammasome pathway in Theiler's murine encephalomyelitis

University of Veterinary Medicine Hannover

Department of Pathology

Dandan Li

Hannover 2020

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University of Veterinary Medicine Hannover

Department of Pathology

Center for Systems Neuroscience Hannover

Role of programmed cell death and the inflammasome pathway in Theiler's murine encephalomyelitis

THESIS

Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by

Dandan Li

Suihua, Heilongjiang, China

Hannover, Germany 2020

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Supervisor: Prof. Dr. Wolfgang Baumgärtner, PhD

Supervision Group: Prof. Dr. Wolfgang Baumgärtner, PhD Prof. Dr. Martin Stangel

Prof. Dr. Ulrich Kalinke

1st Evaluation: Prof. Dr. Wolfgang Baumgärtner, PhD Department of Pathology,

University of Veterinary Medicine Hannover, Germany

Prof. Dr. Martin Stangel

Clinical Neuroimmunology and Neurochemistry Department of Neurology

Hannover Medical School, Germany

Prof. Dr. Ulrich Kalinke

Institute for Experimental Infection Research

Centre for Experimental and Clinical Infection Research, Germany

2nd Evaluation: Prof. Andrew J. Steelman Department of Animal Sciences University of Illinois, USA

Date of final exam: 9th October 2020

Dandan Li received a scholarship from the China Scholarship Council (CSC), File No. 201606170128.

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To my family

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Parts of the thesis have been published in peer-reviewed journals previously:

Gerhauser I*, Li L*, Li D*, Klein S, Elmarabet SA, Deschl U, Kalkuhl A, Baumgärtner W, Ulrich R, Beineke A.

Dynamic changes and molecular analysis of cell death in the spinal cord of SJL mice infected with the BeAn strain of Theiler’s murine encephalomyelitis virus.

Apoptosis 2018; 23: 170-186.

*authors contributed equally to this paper

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Contents I

Contents

1 Chapter 1: Aims of this study ... 1

2 Chapter 2: General introduction ... 3

2.1 Theiler’s murine encephalomyelitis virus ... 3

2.2 Multiple Sclerosis ... 4

2.3 Inflammasome ... 5

2.3.1 Activation of the NLRP3 inflammasome ... 5

2.4 Interferons ... 6

2.5 Programmed cell death ... 8

3 Chapter 3: Apoptosis and inflammasome activation in TMEV-IDD ... 11

4 Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice ... 13

5 Chapter 5: General discussion ... 25

6 Chapter 6: Summary ... 31

7 Chapter 7: Zusammenfassung ... 35

8 Chapter 8: References ... 39

9 Chapter 9: Appendix ... 55

9.1 Solution and buffers ... 55

9.2 Protocols ... 56

10 Chapter 10: Acknowledgements ... 75

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List of abbreviations II

List of abbreviations

AIM2 absent-in-melanoma 2

Apaf-1 apoptotic protein activating factor 1

ASC apoptosis-associated speck-like protein containing a caspase activation and recruitment domain

CH25h cholesterol 25-hydroxylase

CIS clinically isolated syndrome

CNS central nervous system

COX-2 cyclooxygenase 2

DAMPs damage-associated molecular patterns

DISC death-inducing signaling complex

DNA deoxyribonucleic acid

dpi days post infection

DCs dendritic cells

EAE experimental autoimmune encephalomyelitis

ERK extracellular-signal regulated kinase

Fas first apoptosis signal

FADD Fas-associated protein with related death domain

FasL first apoptosis signal ligand

FLIP Fas-associated death domain-like interleukin 1β

conversion enzyme inhibiting protein

GSDMD gasdermin D

HIAP-1 human inhibitor of apoptosis-1

IAPs inhibitor of apoptosis proteins

IFN interferon

IFNAR IFN-I receptor

IFN-I type I IFN

IL interleukin

IL-1R IL-1 receptor

IL-10R IL-10 receptor

IL-18BP IL-18 binding protein

IRAK IL-1R-associated kinase

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List of abbreviations III

IRF IFN regulatory factor

ISG IFN-stimulated gene

ISRE IFN-stimulated response element

ISG15 IFN-stimulated protein of 15 kDa

LRR leucine-rich repeat

MAPK mitogen-activated protein kinase

Mpges-1 membrane-bound prostaglandin E synthesis-1

MS multiple sclerosis

MyD88 myeloid-differentiation primary-response gene

NF-ΚB nuclear factor κB

NK nature killer

NLR NOD-like receptor

NO nitric oxide

NOD nucleotide-binding oligomerization domain

OAS1 2’5’-oligoadenylate synthetase 1

ORF open reading frame

PAMP pathogen-associated molecular pattern

PD-L1 programmed death-ligand 1

PGE2 prostaglandin E2

PKR protein kinase R

PPMS primary progressive multiple sclerosis

PRR pattern recognition receptor

SMAC second mitochondria-derived activator of caspase

SPMS secondary progressive multiple sclerosis

SREBP sterol regulatory element-binding protein

STAT signal transducer and activator of transcription

ssRNA single-stranded RNA

RNA ribonucleic acid

ROS reactive oxygen species

RRMS relapsing-remitting disease

Th1 T helper 1

Tim-3 T-cell immunoglobulin mucin-3

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List of abbreviations IV

TIR toll-IL-1 receptor

TLR toll-like receptor

TMEV Theiler’s murine encephalomyelitis virus

TMEV-IDD TMEV-induced demyelinating disease

TNF tumor necrosis factor

TO Theiler’s original

TRADD TNF receptor-associated death domain

TRIF toll/interleukin-1 receptor-domain-containing adapter inducing interferon-β

UTR untranslated region

25HC 25-hydroxycholesterol

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Chapter 1: Aims of the study 1

1 Chapter 1: Aims of this study

Theiler’s murine encephalomyelitis virus (TMEV) was first described in 1934 and is a single- stranded RNA virus belonging to the Picornaviridae family (Theiler, 1934; Theiler, 1937). TMEV strains can be divided into two subgroups based on their virulence. The first subgroup includes the GDVII and FA strains, which are highly neurovirulent and have a high fatality rate (Lipton and Friedmann, 1980). The second subgroup is the Theiler’s original (TO) subgroup and includes the BeAn and DA strains (Fu et al., 1990a; Fu et al., 1990b; Lipton et al., 1991). After intracerebral infection with TMEV strains of the TO subgroup, resistant C57BL/6 mice develop an acute polioencephalomyelitis, but clear the virus from the central nervous system (CNS) within a few weeks. In contrast, these TMEV strains persist in the spinal cord of susceptible SJL/J mice and leads to a chronic demyelinating disease, which is used as an animal model for multiple sclerosis (MS) (Mecha et al., 2013).

Apoptotic oligodendrocytes can be found in early lesions of MS patients indicating that this form of programmed cell death might be critically involved in the formation of the typical demyelination process of this debilitating disease (Barnett and Prineas, 2004; Henderson et al., 2009). On the other hand, the overexpression of anti-apoptotic factors might cause a failure of myelin-reactive immune cells to undergo apoptosis in progressive forms of MS (Hebb et al., 2008a). Pyroptosis is another form of programmed cell death, which can be induced by caspase-1 and contributes to inflammatory demyelination (Hebb et al., 2008b). The activation of pyroptosis leads to membrane breakdown and processing and release of proinflammatory cytokines such as interleukin (IL)-1β and IL-18 (Fink et al., 2005; Fernandes-Alnemri et al., 2007). IL-1β can cause inflammation and fever reactions (Garlanda et al., 2013). The blocking of its receptor binding can be used to treat a variety of inflammatory diseases (Dinarello et al., 2012). IL-18 induces T cells and natural killer (NK) cells to produce IFN-γ, which in turn leads to T helper type 1 (Th1)-mediated inflammation. Receptor binding of IL-1β and IL-18 induces an intracellular signaling cascade via Toll-IL-1 receptor (TIR), myeloid differentiation primary response 88 (MyD88), and IL-1 receptor (IL-1R)-associated kinase (IRAK) activating the transcription factor nuclear factor kappa B (NF-κB), which induces proinflammatory signals (Dinarello et al., 2013). The inflammasome is a multi-protein processing platform necessary for the processing and release of IL-1β and IL-18 (Labzin et al., 2016). Interestingly, it has been reported that TMEV persists in C57BL/6 mice with IL-1R deficiency and even induces demyelination of the spinal cord. These immunodeficient C57BL/6 mice showed higher initial

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Chapter 1: Aims of the study 2

type I interferon (IFN) responses, increased expression of the immunosuppressive molecules PD-L1 and Tim-3 and insufficient T cell activation resulting in the persistence of the virus (Kim et al., 2012). However, administration of IL-1β in C57BL/6 mice induced a pathogenic Th17 response that made them susceptible to TMEV-induced demyelinating disease (TMEV-IDD) (Kim et al., 2012).

The first part of this study aimed to perform a detailed temporal analysis of apoptotic processes and inflammasome activation in the spinal cord of TMEV-infected SJL/J mice using histology, immunohistochemistry, immunofluorescence, transmission electron microscopy, and publicly available microarray data. The second part of this study was based on the hypothesis that blocking the activation of IL-1β and IL-18 allows virus persistence in C57BL/6 mice causing TMEV-IDD. For this purpose, the viral load, inflammation, expression of important cytokines, and clinical outcome was compared between C57BL/6 mice deficient in essential inflammasome components and wild type littermates after TMEV infection.

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Chapter 2: General introduction 3

2 Chapter 2: General introduction 2.1 Theiler’s murine encephalomyelitis virus

TMEV was discovered and isolated by Max Theiler in 1934 from the CNS of mice with flaccid paralysis (Theiler, 1934; Theiler, 1937). TMEV is a positive-sense single-stranded RNA (ssRNA) virus belonging to the Picornaviridae family (Cusick et al., 2014). The virus genome comprises 8098 nucleotides with a 5‘ untranslated region (UTR), an open reading frame (ORF), a 3‘ UTR and a poly A tail (Tsunoda and Fujinami, 2010; Mecha et al., 2013; Cusick et al., 2014). The structural proteins of TMEV are composed of four capsid proteins VP1, VP2, VP3 and VP4. The non-structural proteins including 2A, 2B, 2C, 3A, 3B, 3C and 3D are needed for virus replication.

According to their neurovirulence, TMEV strains can be divided into GDVII and TO subgroups, which are identical in more than 90% of their nucleotides and amino acids (Tsunoda and Fujinami, 2010; Mecha et al., 2013).

The GDVII subgroup includes GDVII and FA strains, which are highly neurotoxic to mice and cause severe encephalitis and eventually death within one to two weeks (Yamada et al., 1991;

Mecha et al., 2013). After GDVII infection, severe axonal damage and neuronal apoptosis can be observed in specific white matter locations (Tsunoda et al., 2003). However, GDVII subgroup strains cannot persist (Martinat et al., 1999). The TO subgroup includes DA and BeAn strains, which have a 93% amino acid homology and are currently used in experimental research (Daniels et al., 1952; Lorch et al., 1981; Zoecklein et al., 2003). TO subgroup strains induce a transient meningoencephalomyelitis after intracerebral infection. Resistant C57BL/6 mice eliminate the virus from the CNS and do not develop a demyelinating disease. However, these mice can develop transient early seizures with hippocampal neurodegeneration and after several weeks epilepsy with spontaneous recurrent seizures (Lipton and immunity, 1975;

Mecha et al., 2013). In contrast, the virus persists and induces a chronic demyelinating disease in susceptible SJL/J mice. In addition, large doses of TMEV induce cardiac lesions in CH3 mice after intraperitoneal inoculation (Mecha et al., 2013; Gerhauser et al., 2019).

In the acute phase of the infection, TO and GDVII strains induce both a severe polioencephalitis. However, there are fewer apoptotic neurons in mice infected with TO strains such as DA (Tsunoda et al., 1997). In the chronic phase of DA infection, gray matter inflammation subsides, but perivascular mononuclear cell infiltrates can still be observed in the ventrolateral white matter of the spinal cord (Tsunoda and Fujinami, 2010; Mecha et al.,

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Chapter 2: General introduction 4

2013). BeAn-infected mice also suffer from a polioencephalitis within the first two weeks after intracerebral inoculation, but then inflammatory cells begin to infiltrate into the white matter (Navarrete-Talloni et al., 2010). Approximately one month after infection, demyelination begins, which also marks the chronic phase. Compared to the BeAn strain, the DA strain is more virulent. However, the degree of inflammation and demyelination caused by the two virus strains is similar (Zoecklein et al., 2003). A higher viral replication can be found in DA- infected mice, whereas BeAn-infected mice develop higher anti-TMEV antibody titers (Zoecklein et al., 2003).

2.2 Multiple Sclerosis

MS is an autoimmune CNS disease affecting an estimated 130 million people worldwide. MS typically occurs between the ages of 20 and 50, and is twice as common in women as in men (Milo and Kahana, 2010; Heine et al., 2015). The disease was first described in 1868 as a plaques lesion in the white matter of the spinal cord and brain (Compston, 1988). The disease causes the destruction of nerve cells in the brain and spinal cord, which impairs the ability of the nervous system to transmit signals. Clinical signs include an impaired vision or even blindness, muscle weakness, and mental problems (Oleszak et al., 2004). Demyelination can be associated with T cell-mediated and/or antibody-dependent inflammatory responses directed against myelin components. MS has been classified into relapsing and progressive forms. As the disease progresses, some symptoms may disappear, but neurological problems persist. In addition, the clinical course of progressive diseases is not uniform, and may develop rapidly or remain stable for a period of time (Lublin et al., 2014). Due to the development of novel diagnostic methods, MS phenotypes have been re-examined in 2011 and new disease courses of MS including clinically isolated syndrome (CIS), relapsing-remitting disease (RRMS), primary progressive disease (PPMS) and a secondary progressive disease (SPMS) have been defined (Lublin et al., 2014). MS is not a hereditary disease but some studies have shown that specific genetic variations can increase the risk of disease. Likewise, people who are related to a MS patient have a higher risk for developing this disease and the closer the relationship is the greater is the risk of disease. If both parents are affected, children have a ten times higher incidence than the general population (Dyment et al., 2004; Milo and Kahana, 2010).

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Chapter 2: General introduction 5

2.3 Inflammasome

The inflammasome is a key component of the innate and adoptive immune response. This multimeric protein complex mediates caspase-1 activation and the maturation of the proinflammatory cytokines IL-1β and IL-18 in response to a virus or microbial infection. Pattern recognition receptors (PRRs) can recognize the presence of harmful stimuli such as invading pathogens, dead cells or environmental stimuli, which are known as pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) (Takeuchi and Akira, 2010). These stimuli induce the oligomerization of inflammasome components to create a platform that cleaves the precursor form of caspase-1 into biological active caspase-1 (Franchi et al., 2009). Several PRRs inducing inflammasome activation have been identified so far including the nucleotide-binding oligomerization domain (NOD) leucine-rich repeat (LRR)- containing proteins (NLR) family members NLRP1, NLRP2, NLRP3, and NLRC4, absent-in- melanoma 2 (AIM2) and pyrin (Lamkanfi and Dixit, 2014; Ozaki et al., 2015; Sharma and Kanneganti, 2016). The adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) bridges the interaction between these PRRs and caspase-1. The best characterized inflammasome is the NLRP3 inflammasome, which seems to be implicated in various diseases including metabolic disorders, inflammatory bowel disease, cryopyrin-associated periodic fever syndrome as well as MS (Gris et al., 2010;

Shao et al., 2015).

2.3.1 Activation of the NLRP3 inflammasome

The activation of inflammasomes is a complex process, which is dependent on two signals (Shao et al., 2015). The priming signal is essential for the production of NLRP3 inflammasome components. The stimulation of PRRs such as Toll-like receptors (TLRs) or NLRs results in increased expression of NF-κB, which triggers the production of inactive NLRP3, pro-IL-1β and pro-IL-18 (Bauernfeind et al., 2009). Consequently, under resting conditions, the NLRP3 inflammasome cannot be activated and pro-IL-1β is not produced. In contrast, the priming signal does not seem to influence the expression levels of ASC, caspase-1 and pro-IL-18 (Bauernfeind et al., 2009; Franchi et al., 2009).

The second step of inflammasome activation is induced again by PAMPs or DAMPS. This signal triggers the oligomerization the inflammasome components NLRP3, ASC, and procaspase-1 into a complex. Finally, the activation of caspase-1 leads to the processing and secretion of

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Chapter 2: General introduction 6

IL-1β and IL-18. The inflammasome can be activated by a wide range of irritants including ATP (Mariathasan et al., 2006), heme (Erdei et al., 2018), uric acid crystals (Martinon et al., 2006), asbestos and silica (Dostert et al., 2008), pathogen-associated RNA or DNA, and bacterial and fungal toxins (Greaney et al., 2015; Mathur et al., 2019). Moreover, ion flux, mitochondrial functional impairment and the release of reactive oxygen species (ROS) and lysosomal damage can induce NLRP3 inflammasome activation (Kelley et al., 2019).

2.4 Interferons

In 1957, Isaacs and Lindenmann studied influenza viruses and discovered a protein that protects cells from viral infections (Isaacs and Lindenmann, 1957). This protein has been termed interferon due to its strong antiviral activity and can be secreted in an autocrine and paracrine manner. According to different attributes, it can be divided into three subsets, type I IFN (IFN-I), type II IFN (IFN-γ) and type III IFN (IFN-λ). IFN-I includes IFN-α, IFN-β, IFN-κ, IFN-ω and IFN-ε. In addition, there are 13 IFN-α subtypes in humans (Kraus et al., 2004; Kay et al., 2013; Annibali et al., 2015). Most nucleated cells can produce IFNs. IFN-I is mainly produced by monocytes and fibroblasts, IFN-γ is only produced by T cells and NK cells and IFN-λ is mainly produced by epithelial cells (Boehm et al., 1997; Parkin and Cohen, 2001; Hermant and Michiels, 2014). The IFN-I receptor IFNAR1/IFNAR2 can be expressed by all nucleated cells including neurons (Sommereyns et al., 2008). In contrast, the IFN-γ receptor α chain is only expressed at moderate levels by nearly all cells and the IFN-γ receptor β chain is constitutively expressed at extremely low levels and upregulated only in certain cell types (Bach et al., 1997).

The IFN-λ receptor IFNLR1/IL-10R2 can mainly be found on plasmocytoid dendritic cells and epithelial cells especially of the stomach, intestine, and lungs (Ank et al., 2008; Sommereyns et al., 2008).

IFNs have been extensively studied in recent decades. A large number of studies have proved that especially IFN-I play an important role in the antiviral immune response. IFN-β can ameliorate TMEV-IDD and reduce CD4+ T cell responses. IFN-β has been used as a first-line drug for the treatment of MS and attenuates the development of experimental autoimmune encephalomyelitis (EAE) (Inoue and Shinohara, 2013). IFN-β increases IL-10 expression, inhibits IL-1β production, maintains the stability of the blood-brain barrier and blocks T cell activation (Kraus et al., 2004; Kay et al., 2013; Annibali et al., 2015). The expression of IFN-I can be induced by different viral and bacterial PAMPs, which are recognized by specific PRRs

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Chapter 2: General introduction 7

such as TLRs (Andrejeva et al., 2004; Kraus et al., 2004; Yoneyama et al., 2004; Yoneyama et al., 2005; Takaoka et al., 2007; Kato et al., 2008). The detection of PAMPs triggers different intracellular signaling pathways, such as Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and MyD88-dependent pathways, which activate IFN-I transcription via interferon regulatory factors (IRF) family members (IRF1, IRF3, IRF5, IRF7) and NF-κB (Bonjardim et al., 2009; Boo and Yang, 2010; Hall and Rosen, 2010). IFN-I receptors (IFNAR1 and IFNAR2) are located on the cell surface and linked to Janus kinase 1 and tyrosine kinase 2. These two kinases phosphorylate signal transducer and activator of transcription (STAT) 1 and STAT2, which then combine with interferon regulatory factor 9 (IRF9) to form IFN-stimulated gene factor 3 (ISGF3). This ternary complex enters the nucleus and binds to IFN-stimulated response elements (ISRE) to induce the expression of IFN-stimulated genes (ISGs) (Feng et al., 2002). So far more than 300 ISGs have been discovered (Der et al., 1998; Sadler and Williams, 2008).

Many of them represent antiviral effector proteins, which act at different stages of the virus replication cycle. Nevertheless, the exact antiviral mechanisms have been investigated in detail only in a few cases such as IFN-stimulated gene 15 (ISG15), protein kinase R (PKR) and 2’5’-oligoadenylate synthetase 1 (OAS1) (Schoggins, 2014). In TMEV-infected SJL/J mice, the translation of the ISGs gene appears to be partially blocked limiting their antiviral function. In contrast, ISG15 and PKR proteins were highly expressed in C57BL/6 mice most likely preventing the spread of TMEV in C57BL/6 mice, virus persistence and consequently TMEV- IDD (Li et al., 2015).

IFN-β is a well-established first line drug for the treatment of MS patients (Wandinger et al., 1998; Zadeh et al., 2019). The expression levels of IRF1, IRF2, OAS and MxA in MS patients are lower than in healthy individuals, which might be linked to subnormal phosphorylation of STAT1 (Feng et al., 2002). In addition, IRF1/IRF2 ratio is reduced in MS patients compared to controls. IRF2 can act as an inhibitor of ISG transcription suggesting that the low IRF1/IRF2 ratio may also diminish IFN signaling in MS patients (Feng et al., 2002). Current research indicates that IFN-β acts via blocking T cell activation, regulating the production of pro- inflammatory and anti-inflammatory cytokines and maintaining the stability of the blood- brain barrier. Nonetheless, the exact mode of action of IFN-β used in the treatment of MS is still unclear and more research is needed to develop more effective treatment options.

An important mechanism mediating the immunosuppressive effect of IFN-β is to suppress the inflammasome. IFN-β is able to induce the expression of an IL-1R antagonist (Sciacca et al.,

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Chapter 2: General introduction 8

2000) attenuating the IL-1 signaling pathway (Guarda et al., 2011). IFN-β can also indirectly inhibit the release of IL-1β from macrophages in two ways. Firstly, IFN-β induces the expression of IL-10 and cholesterol 25-hydroxylase (Ch25h). After binding to the IL-10 receptor (IL-10R), IL-10 activates STAT3 to inhibit the expression of pro-IL-1β mRNA (Moore et al., 2001).

CH-25h can convert cholesterol into the antiviral lipid 25-hydroxycholesterol (25-HC), which can antagonize sterol regulatory element-binding protein (SREBP) processing to inhibit pro-IL- 1β transcription (Reboldi et al., 2014). Secondly, IFN-β and IFN-γ can stimulate the induction of nitric oxide synthase, thereby increasing the production of endogenous nitric oxide (NO).

NO inhibits the oligomerization of the NLRP3 inflammasome (Hernandez-Cuellar et al., 2012;

Mishra et al., 2013). Finally, IFN-β can prevent NLRP3 activation by inhibiting reactive oxygen species (ROS) production (Inoue et al., 2012). Consequently, IFN-I can impair the assembly and activation of inflammasomes as well as the transcription and maturation of pro-IL-1β.

2.5 Programmed cell death

Apoptosis is a type of programmed cell death that can occur in healthy and damaged cells (Pistritto et al., 2016). Apoptosis signals lead to characteristic cellular morphological changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. Apoptosis plays a very important role in the growth and development of living organisms. Cells produce apoptotic bodies (cell debris) during the process of apoptosis, which can be recognized and cleared by phagocytic cells. Apoptosis is not just an important biological phenomenon but dysregulation of apoptotic processes is also involved in various diseases. Excessive apoptosis causes organ shrinkage, while insufficient apoptosis can result in uncontrolled cell proliferation such as cancer (Pistritto et al., 2016). The restoration of normal apoptosis is the main objective in the treatment of diseases with deficient apoptotic cell death. Moreover, alterations in the apoptotic machinery affect the efficiency of chemotherapy and radiation treatment of cancer, because they mainly kill target cells by inducing apoptosis (Fulda and Debatin, 2006).

Apoptosis is a precisely regulated multi-step cell death program, which can occur in every cell of the body and cannot be stopped once it has started. Apoptosis can be initiated in two ways, which are dependent on internal signals and external signals (Wong and Research, 2011). In the intrinsic pathway, apoptosis is triggered by signals inside the cell such as viruses and stress.

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Chapter 2: General introduction 9

The extrinsic pathway is activated by ligand binding to specific cell surface receptors (Raychaudhuri, 2010). Radiation, malnutrition, viral infections, glucocorticoids or excessive intracellular ion concentration can cause apoptosis (Hardy et al., 2003; Mattson and Chan, 2003). Mitochondria are necessary for cell survival and the basis for activating the intrinsic pathway of apoptosis. The activation of this pathway requires the formation of pores in the mitochondrial outer membrane by Bcl-2 protein family members such a Bak and Bax leading to increased permeability and mitochondrial swelling (Uren et al., 2017). Subsequently, mitochondria release cytochrome C, which combines with apoptotic protein activating factor 1 (Apaf-1) and ATP and then with pro-caspase-9 to form an apoptosome. The apoptosome cleaves pro-caspase-9 to caspase-9, which processes pro-caspase-3 into active caspase-3. The permeabilisation of the outer mitochondrial membrane also results in the release of the second mitochondria-derived activator of caspase (SMAC) into the cytoplasm, which blocks inhibitor of apoptosis proteins (IAPs) (Fesik and Shi, 2001).

The extrinsic pathway can be induced by the binding of TNF-α or first apoptosis signal ligand (FasL) to TNF receptors or the Fas receptor, respectively (Wajant, 2002). TNF-α is an important proinflammatory cytokine and an important exogenous mediator of apoptosis. Most cells have TNF receptors, which can activate caspases via the recruitment of the adapter protein TNF receptor-associated death domain (TRADD). Moreover, TNF signaling can induce apoptosis in a caspase-independent manner through the lysosomal-mitochondrial pathway (Chen et al., 2005). Due to the pro-apoptotic effects of TNF-α, abnormalities of TNF signaling can cause various diseases, especially autoimmune diseases (Bradley and Ireland, 2008). The Fas receptor that binds to FasL is a transmembrane protein of the TNF receptor superfamily (Wajant et al., 2003). Fas and FasL combine to form a death-inducing signaling complex (DISC), which contains Fas-associated protein with related death domain (FADD), caspase-8 and caspase-10. These initiator caspases directly activate the executioner caspase-3 to trigger apoptosis. Furthermore, caspase-8 can promote cytochrome C release from mitochondria to induce apoptosis indirectly via the intrinsic pathway (Kuwana et al., 1998).

Before an apoptotic cell is disposed of by phagocytes, there is a complex process of disassembly, which proceeds in three steps. Initially, the cell membrane forms irregular buds, which continuously grow and cause drastic changes in cell shape. An important regulator of apoptotic cell membrane blebbing is rho associated coiled-coil-containing protein kinase 1 (ROCK1). Subsequently, some cell types may develop thin extensions of the cell membrane

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Chapter 2: General introduction 10

called membrane protrusions. Three different types of these membrane protusions have been described: microtubule spikes, apoptopodia (feet of death), and beaded apoptopodia (the latter having a beads-on-a-string appearance). Pannexin 1 is an important component of membrane channels involved in the formation of apoptopodia and beaded apoptopodia.

Finally, the cell breaks apart into multiple vesicles called apoptotic bodies, which undergo phagocytosis. The plasma membrane protrusions can assist in this process by bringing apoptotic bodies closer to phagocytes (Atkin-Smith and Poon, 2017).

In addition to apoptosis, other pathways have been described that mediate programmed cell death including autophagy, pyroptosis, necroptosis, and eryptosis (Fink et al., 2005; Lang et al., 2006; Fayaz et al., 2014). Autophagy is involved in maintenance of cellular homeostasis, because it degrades excessive organelles and proteins for energy and amino acid recycling (Liang and Le, 2015). Pyroptosis is a gasdermin D (GSDMD)-mediated form of programmed cell death, which is induced by caspase-1 and caspase-11/4/5 and results in the massive release of proinflammatory mediators (Bergsbaken et al., 2009; Shi et al., 2017). Necroptosis is a necrotic cell death pathway, which is triggered by TNF-α under caspase-8-deficient conditions (Ofengeim et al., 2015). The suicidal death of erythrocytes has been termed eryptosis, which is morphologically characterized by cell shrinkage and membrane blebbing and can be induced by oxidative stress (Lang et al., 2006).

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11 Chapter 3: Apoptosis and inflammasome activation in TMEV-IDD

3 Chapter 3: Apoptosis and inflammasome activation in TMEV-IDD

Abstract

Theiler’s murine encephalomyelitis (TME) is caused by the TME virus (TMEV) and represents an important animal model for multiple sclerosis (MS). Oligodendroglial apoptosis and reduced apoptotic elimination of encephalitogenic leukocytes seem to participate in autoimmune demyelination in MS. The present study quantified apoptotic cells in BeAn- TMEV-induced spinal cord white matter lesions at 14, 42, 98, and 196 days post infection (dpi) using immunostaining. Apoptotic cells were identified by transmission electron microscopy and double-immunofluorescence. The mRNA expression of apoptosis-related genes was investigated using microarray analysis. Oligodendroglial apoptosis was already detected in the predemyelinating phase at 14 dpi. Apoptotic cell numbers peaked at 42 dpi and decreased until 196 dpi partly due to reduced T cell apoptosis. In addition to genes involved in the classical pathways of apoptosis induction, microarray analysis detected the expression of genes related to alternative mechanisms of cell death such as pyroptosis, necroptosis, and endoplasmic reticulum stress. Consequently, oligodendroglial apoptosis is involved in the initiation of the TME demyelination process, whereas the development of apoptosis resistance of T cells potentially favors the maintenance of inflammation and myelin loss.

Keywords: Apoptosis; Endoplasmic reticulum stress; Multiple sclerosis; Necroptosis;

Pyroptosis; Theiler’s murine encephalomyelitis virus

Published in Apoptosis 2018; 23: 170-186

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Chapter 3: Apoptosis and inflammasome activation in TMEV-IDD 12

The extent of Dandan Li’s contribution to the article is evaluated according to the following scales:

A. has contributed to collaboration (0-33%) B. has contributed significantly (34-66%)

C. has essentially performed the study independently (67-100%)

1. Design of the project including the design of the individual experiments: A 2. Performing of the experimental part of the study: A

3. Analysis of the experiments: A

4. Presentation and discussion of the study in article form: A

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 13

4 Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice

ASC- and caspase-1-deficient C57BL/6 mice do not develop demyelinating disease after infection with Theiler’s murine encephalomyelitis virus

Dandan Li1, Melanie Bühler1, Sandra Runft1, Georg Beythien1, Wolfgang Baumgärtner1, Till Strowig2, Ingo Gerhauser1*

1 Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany; Melanie.Buehler@tiho- hannover.de (M.B.); Sandra.Runft@tiho-hannover.de (S.R.); Georg.Beythien@tiho-hannover.de (G.B.);

Wolfgang.Baumgaertner@tiho-hannover.de (W.B.)

2 Department for Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany;

Till.Strowig@helmholtz-hzi.de (T.S.)

* Correspondence: Ingo.Gerhauser@tiho-hannover.de (I.G.); Tel.: +49-(0)511-953-8660; Fax: +49-(0)511-953-8675

Abstract: Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus of the Picornaviridae family. After intracerebral infection of SJL mice, TMEV induces an acute polioencephalomyelitis and a chronic demyelinating leukomyelitis due to its persistence in the spinal. This TMEV-induced demyelinating disease (TMEV-IDD) has widely been used as an animal model of human multiple sclerosis (MS). C57BL/6 (B6) mice eliminate the virus from the central nervous system (CNS) within few weeks and generally do not develop TMEV-IDD.

However, TMEV persists in interleukin-1 receptor (IL-1R)-deficient B6 mice making them susceptible for the demyelinating process. The proinflammatory cytokine IL-1β is activated by the inflammasome pathway, which consists of a pattern recognition receptor (PRR) molecule sensing microbial pathogens, the adaptor molecule Apoptosis-associated speck-like protein containing a CARD (ASC), and the executioner caspase-1. To analyze the contribution of the inflammasome pathway to the resistance of B6 mice to TMEV-IDD, the course of the disease was compared between TMEV (BeAn)-infected ASC- and caspase-1-deficient mice and wild type littermates until 98 days post infection (dpi). CNS inflammation and TMEV protein expression were investigated using histology and immunohistochemistry. Real-time PCR was used to quantify TMEV RNA and IFN-β, ISG15, PKR, TNFα, IL-1β, IL-6, IL-10, IL-12 (p40) and IFNγ mRNA in the brain of TMEV-infected mice at 4 dpi. Unexpectedly, ASC- and caspase-1-deficient mice eliminated the virus and did not develop TMEV-IDD. Moreover, similar mRNA levels of IFN-β, ISG15, PKR and all cytokines were found in immunodeficient mice and their wild type littermates. Consequently, ASC- and caspase-1-deficient mice can compensate lack of inflammasome-dependent IL-1β activation preventing TMEV-IDD. Future studies are needed to determine the cellular origin of alternative proteases able to activate IL-1β, the exact role of role of additional caspase-1 substrates such as IL-18 and IL-33 and inflammasome- independent functions of ASC, which can all contribute to neuroinflammatory diseases including MS.

Keywords: ASC; caspase-1; demyelination; IL-1β; immunohistochemistry; inflammasome;

knockout mice; RT-qPCR; TMEV

Sent to the International Journal of Molecular Sciences

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 14

The extent of Dandan Li’s contribution to the article is evaluated according to the following scales:

A. has contributed to collaboration (0-33%) B. has contributed significantly (34-66%)

C. has essentially performed the study independently (67-100%)

1. Design of the project including the design of the individual experiments: A

2. Performing of the experimental part of the study: C

3. Analysis of the experiments: B

4. Presentation and discussion of the study in article form: B

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 15

ASC- and caspase-1-deficient C57BL/6 mice do not develop demyelinating disease after infection with Theiler’s murine encephalomyelitis virus

Dandan Li1, Melanie Bühler1, Sandra Runft1, Georg Beythien1, Wolfgang Baumgärtner1, Till Strowig2, Ingo Gerhauser1*

1 Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany;

Melanie.Buehler@tiho-hannover.de (M.B.); Sandra.Runft@tiho-hannover.de (S.R.); Georg.Beythien@tiho- hannover.de (G.B.); Wolfgang.Baumgaertner@tiho-hannover.de (W.B.)

2 Department for Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany; Till.Strowig@helmholtz-hzi.de (T.S.)

* Correspondence: Ingo.Gerhauser@tiho-hannover.de (I.G.); Tel.: +49-(0)511-953-8660; Fax: +49-(0)511-953-8675 Abstract: Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus of the Picornaviridae family. After intracerebral infection of SJL mice, TMEV induces an acute polioencephalomyelitis and a chronic demyelinating leukomyelitis due to its persistence in the spinal.

This TMEV-induced demyelinating disease (TMEV-IDD) has widely been used as an animal model of human multiple sclerosis (MS). C57BL/6 (B6) mice eliminate the virus from the central nervous system (CNS) within few weeks and generally do not develop TMEV-IDD. However, TMEV persists in interleukin-1 receptor (IL-1R)-deficient B6 mice making them susceptible for the demyelinating process.

The proinflammatory cytokine IL-1β is activated by the inflammasome pathway, which consists of a pattern recognition receptor (PRR) molecule sensing microbial pathogens, the adaptor molecule Apoptosis-associated speck-like protein containing a CARD (ASC), and the executioner caspase-1. To analyze the contribution of the inflammasome pathway to the resistance of B6 mice to TMEV-IDD, the course of the disease was compared between TMEV (BeAn)-infected ASC- and caspase-1-deficient mice and wild type littermates until 98 days post infection (dpi). CNS inflammation and TMEV protein expression were investigated using histology and immunohistochemistry. Real-time PCR was used to quantify TMEV RNA and IFN-β, ISG15, PKR, TNFα, IL-1β, IL-6, IL-10, IL-12 (p40) and IFNγ mRNA in the brain of TMEV-infected mice at 4 dpi. Unexpectedly, ASC- and caspase-1-deficient mice eliminated the virus and did not develop TMEV-IDD. Moreover, similar mRNA levels of IFN-β, ISG15, PKR and all cytokines were found in immunodeficient mice and their wild type littermates.

Consequently, ASC- and caspase-1-deficient mice can compensate lack of inflammasome-dependent IL-1β activation preventing TMEV-IDD. Future studies are needed to determine the cellular origin of alternative proteases able to activate IL-1β, the exact role of role of additional caspase-1 substrates such as IL-18 and IL-33 and inflammasome-independent functions of ASC, which can all contribute to neuroinflammatory diseases including MS.

Keywords: ASC; caspase-1; demyelination; IL-1β; immunohistochemistry; inflammasome; knockout mice; RT-qPCR; TMEV

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 16

1. Introduction

Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus of the Picornaviridae family (Theiler 1937). The intracerebral infection of SJL mice with TO subgroup strains of TMEV (BeAn, DA) induces a biphasic disease characterized by acute polioencephalomyelitis and chronic demyelinating leukomyelitis, which represents an important animal model of human multiple sclerosis (MS) (Oleszak, Chang et al. 2004, Mecha, Carrillo-Salinas et al. 2013, Tsunoda, Sato et al. 2016).

TMEV-induced demyelinating disease (TMEV-IDD) is initiated by oligodendrocyte apoptosis and axonal damage and aggravated by a delayed-type hypersensitivity response directed against viral antigens and later myelin components (Miller, Vanderlugt et al. 1997, Tsunoda and Fujinami 2010, Gerhauser, Li et al. 2018). The virus mainly infects hippocampal and cortical neurons in the early phase of the disease but only persists in white matter glial cells and macrophages in advanced stages (Gerhauser, Hansmann et al. 2019). In contrast to SJL mice, C57BL/6 (B6) mice eliminate the virus from the central nervous system (CNS) within few weeks and do not develop TMEV-IDD (Lipton and Dal Canto 1979, Gerhauser, Alldinger et al. 2007). Nevertheless, TMEV can persist in interleukin-1 receptor (IL-1R)-deficient B6 mice and even induces demyelination in their spinal cord (Kim, Jin et al. 2012).

These immunodeficient mice show a higher initial type I interferon (IFN-I) response, which causes an increased expression of the immunosuppressive molecules PD-L1 and Tim-3 and insufficient T cell activation allowing viral persistence. However, the administration of IL-1β to immunocompetent B6 mice induces a pathogenic T helper 17 (Th17) cell response turning them susceptible to TMEV-IDD as well (Kim, Jin et al. 2012). Interestingly, PD-L1 and Tim-3 are down-regulated in peripheral blood mononuclear cells (PBMCs) from MS patients, which leads to an overactivation of immune cells, inflammation and demyelination (Javan, Aslani et al. 2016, Mohammadzadeh, Rad et al. 2018).

The proinflammatory cytokines IL-1β and IL-18 are produced as inactive precursors, which are processed by caspase-1 and caspase-4/5/11 after activation of the inflammasome pathway (van de Veerdonk, Netea et al. 2011, Voet, Srinivasan et al. 2019). Inflammasomes are vital players in innate immunity and involved in the complex host-pathogen-interactions. Typical inflammasomes represent multiprotein complexes composed of a pathogen sensor molecule such as nucleotide binding domain and leucine-rich repeat-containing receptor (NLR) or AIM2-like receptor (ALR), the adaptor molecule apoptosis- associated speck-like protein containing a caspase activation and recruitment domain (ASC) and the executioner caspase-1 (Chakraborty, Kaushik et al. 2010, Ransohoff and Brown 2012, Shrivastava, Leon-Juarez et al.

2016). These molecules are mainly expressed by microglia in the brain but astrocytes and neurons as well as myeloid cells infiltrating from the periphery can produce and activate inflammasomes pathway components (Voet, Srinivasan et al. 2019). Inflammasome activation prevents viral replication by inducing an inflammatory cell death program termed pyroptosis mediated by the pore-forming protein gasdermin D (GSDMD) (Shrivastava, Leon-Juarez et al. 2016, Shi, Gao et al. 2017). The NLRP3 inflammasome pathway and downstream IL-1β signaling are also critical for the control of West Nile virus (WNV) infections (Ramos, Lanteri et al. 2012, Kumar, Roe et al. 2013), whereas wild-type and caspase-1-deficient mice are equally susceptible to encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) infection despite the ability of the NLRP3 inflammasome to detect these viruses (Rajan, Rodriguez et al. 2011). Most importantly, the promotion of T cell pathogenicity and CNS cell infiltration caused by inflammasome signaling contributes to the development of MS lesions (Barclay and Shinohara 2017). Moreover, the inhibition of NLRP3 inflammasome activation by IFN-I plays a major role in the response of MS patients to IFN-β treatment (Inoue and Shinohara 2013, Malhotra, Rio et al. 2015). Similarly, the NLRP3 inflammasome can induce demyelinating lesions in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, but an aggressive disease induction results in NLRP3 inflammasome-independent EAE, which does not respond to IFN-β treatment (Inoue and Shinohara 2013). ASC-/- mice develop milder EAE lesions compared to caspase-1-/- mice due to inflammasome-independent functions of ASC, which support the survival of CD4+ T cells including myelin oligodendrocyte glycoprotein–specific T cells (Shaw, Lukens et al. 2010). Consequently, the different inflammasome pathway components can independently support the EAE demyelination process.

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 17

TMEV infection of IL-1R-deficient B6 mice indicated an indirect antiviral effect of the inflammasome pathway based on the inhibition of immunosuppressive molecules such as IFN-I acting on T cells (McRae, Semnani et al. 1998, Kim, Jin et al. 2012). In contrast, strong activation of the NLRP3 inflammasome and down-stream PGE2 signaling in dendritic cells and CD11b+ leukocytes, spleen cells and bone marrow cells of SJL mice impairs early protective IFN-γ-producing CD4+ and CD8+ T cell responses allowing viral persistence necessary for TMEV-IDD (Kim, Jin et al. 2017). ASC increases serum levels of proinflammatory cytokines, IFN-α and IgM during WNV infection thereby limiting viral replication. Nevertheless, elevated and not reduced levels of IFN-γ, CCL2 and CCL5 were found in the brains from ASC-/- mice correlating with enhanced astrocyte activation, leukocyte infiltration and neuronal cell death (Kumar, Roe et al. 2013). The exact role of specific inflammasome pathway components in the pathogenesis of TMEV-IDD has not been investigated so far. Therefore, the present study aimed to analyze the consequences of ASC or caspase-1 deficiency on virus elimination, CNS inflammation, cytokine and IFN-I expression and clinical outcome in TMEV-infected mice.

2. Results

Clinical and histological investigation

Clinical signs were absent in all investigated mice after TMEV infection. Moreover, mice continuously gained weight during the investigation period and their motor coordination did not deteriorate (Fig. 1). ASC-/- and caspase-1-/- mice developed mild to moderate inflammatory brain and spinal cord lesions in the acute phase of the disease (4 and 14 dpi), which did not differ significantly from their wild type littermates. In the chronic phase of the disease (98 dpi) perivascular mononuclear cell infiltrates were also found in the spinal cord of ASC-/- and caspase-1-/- mice as well as wild type littermates of ASC/- mice but not in the brain of the TMEV-infected mice (Fig. 2).

Fig. 1. Clinical investigation. Weight (A) and Rotarod performance (B) of ASC-/- and caspase-1-/- mice (KO) and wild type littermates (WT) infected with Theiler’s murine encephalomyelitis virus. All mice showed a continuous increase in body weight and no deterioration in motor coordination.

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 18

Fig. 2. Inflammation. Semiquantitative scores of perivascular mononuclear cell infiltrates in the brain (A) and spinal cord (B) of ASC-/- and caspase-1-/- mice (KO) and wild type littermates (WT) infected with Theiler’s murine encephalomyelitis virus at 4, 14 and 98 days post infection (dpi). Mild to moderate inflammation was found at 4 and 14 dpi but not at 98 dpi in the brain (A), whereas mild inflammation was present in the spinal cord of at all investigated time points (B). Significant differences between KO and WT mice were not detected. Note few perivascular mononuclear cells in the hippocampus (A) and spinal cord (B) of ASC-/- (KO) and ASC+/+ (WT) mice. Bars = 300 µm.

Immunohistochemistry

TMEV+ cells were mainly detected at 4 dpi in all investigated mouse groups. Virus antigen was only present in few cells of single animals at 14 dpi and not found at 98 dpi (Fig. 3). The perivascular mononuclear cell infiltrates contained approximately 70% CD3+ T cells, 10% CD45R+ B cells and 20%

Iba-1+ macrophages. No significant differences between ASC-/- and caspase-1-/- mice and their respective wild type littermates were found in the composition of these leukocytes in the perivascular area.

Fig. 3. Viral load. Immunohistochemistry was used to detect virus antigen in the brain of ASC-/- and caspase-1-/- mice (KO) and wild type littermates (WT) infected with Theiler’s murine encephalomyelitis virus (TMEV) at 4, 14 and 98 days post infection (dpi). No significant differences in the number of TMEV+ cells between KO and WT mice demonstrating rapid virus elimination. Note TMEV+ cells in the CA2 area of the hippocampus of ASC-/- (KO) and ASC+/+ (WT) mice. Bars = 300 µm.

RT-qPCR

RT-qPCR did not reveal significant differences in the amount of TMEV RNA as well as IFNβ, ISG15, PKR, TNFα, IL-1β, IL-6, IL-10, IL-12 (p40) and IFNγ transcript numbers in the brain of ASC-/- and caspase-1-/- mice and their respective wild type littermates of TMEV-infected mice at 4 dpi (Fig. 4).

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 19

Fig 4. RT-qPCR. The number of Theiler’s murine encephalomyelitis virus (TMEV) RNA copies/ng RNA as well as IFNβ, ISG15, PKR, TNFα, IL-1β, IL-6, IL-10, IL-12 (p40) and IFNγ mRNA copies /ng RNA was quantified in the brain of ASC-/- and caspase-1-/- mice (KO) and wild type littermates (WT) infected with TMEV at 4 days post infection using RT-qPCR. No significant differences between ASC-/- and caspase- 1-/- mice and their respective wild type littermates were found.

3. Discussion

ASC and caspase-1 are major components of the inflammasome pathway, which plays a critical role in several neuroinflammatory and neurodegenerative diseases including MS (Singhal, Jaehne et al.

2014, Voet, Srinivasan et al. 2019). Inflammasome activation induces the maturation of IL-1β by proteolytic cleavage of its precursor form. This proinflammatory cytokine is involved in the acute phase response and stimulates the expression of proteins adhesion molecules on endothelial cells and chemokines necessary for leukocyte recruitment. IL-1β also orchestrates the differentiation and function of innate and adaptive lymphoid cells (Garlanda, Dinarello et al. 2013). Moreover, the induction of eicosanoids such as PGE2 by IL-1β limits excessive production of immunosuppressive IFN-I, which seems to cause virus persistence and the development of demyelinating disease in TMEV-infected IL- 1R-deficient B6 mice (Kim, Jin et al. 2012, Mayer-Barber, Andrade et al. 2014). Consequently, the authors hypothesized that the elimination of inflammasome-dependent IL-1β maturation might make B6 mice susceptible to TMEV-IDD. However, the present study revealed no consequences of ASC or caspase-1 deficiency on virus elimination, CNS inflammation, cytokine and IFN-I expression and clinical outcome in TMEV-infected B6 mice. These findings can be explained by alternative inflammasome-independent pathways of IL-1β activation, which compensate lack of pro-IL-1β processing by caspase-1. In addition to microorganisms such as Candida albicans, Staphylococcus aureus and Streptococcus pyogenes, monocyte/macrophages, neutrophils, NK cells, mast cells and epithelial cells can produce proteases able to cleave pro-IL-1β including proteinase 3, cathepsin G, elastase, granzyme A, chymase, chymotrypsin, meprin A and meprin β (Fantuzzi, Ku et al. 1997, Netea, van de Veerdonk et al. 2015).

The difference between the previously described IL-1R-deficient mice and the present ASC- and caspase-1-deficient mice might also be related to the functions of other capase-1 substrates such as IL-18 and IL-33. IL-18 has been implicated in several autoimmune diseases including MS, because it stimulates γδ T cells to express IL-17 and promotes the production of IFN-γ from T cells and NK cells (Dinarello, Novick et al. 2013). IL-33 is a potent inducer for a Th2 immune response, but this IL-1-like cytokine is inactivated after maturation by caspase-1 (Cayrol and Girard 2009, Abd Rachman Isnadi, Chin et al.

2018). Therefore, lack of inflammasome activity in ASC- and caspase-1-deficient mice inhibits pathogenic Th17 cell responses, which can be enhanced by IL-18 and IL-33 processing in wild type and IL-1R-deficient B6 mice. This inhibition of pathogenic Th17 cell responses might prevent the initiation of the demyelination process in the present ASC- and caspase-1-deficient mice, which suffer from a reduced IL-1β-dependent antiviral activity (Kim, Jin et al. 2012).

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 20

Finally, inflammasome-independent functions of ASC, which regulate the activity of the mitogen- activated protein kinase (MAPK) extracellular-signal regulated kinase (ERK) in murine and human monocytes/macrophages, antigen-specific IgG responses and chemokine and IFN-I expression, might contribute to TMEV-IDD in IL-1R-deficient mice (Ellebedy, Lupfer et al. 2011, Taxman, Holley-Guthrie et al. 2011, Kumar, Roe et al. 2013). However, these pathogenic functions of ASC, which are not related to its role in the inflammasome pathway, cannot explain lack of demyelinating lesions in caspase-1- deficient mice.

4. Materials and Methods Animal experiment

ASC-/- (B6.129S5-Pycardtm1Flv) mice (http://www.informatics.jax.org/allele/MGI:3686870) and caspase-1-/- (Casp1tm2.1Flv) were generated as described (Mariathasan, Newton et al. 2004, Sutterwala, Ogura et al. 2006, Case, Kohler et al. 2013, Blazejewski, Thiemann et al. 2017). Heterozygous knockout mice were used to breed 27 ASC-/- and 28 caspase-1-/- mice and 60 wild type littermates. Mice were kept under controlled environmental conditions (22–24 °C; 50–60% humidity; 12/12 h light/dark cycle) with free access to standard rodent diet (R/M-H; sniff Spezialdiäten GmbH, Soest, Germany) and tap water.

At the age of five to six weeks, groups of seven to ten mice were inoculated into the right cerebral hemisphere with 1×105 plaque-forming units (PFU) per mouse of the BeAn strain of TMEV in 20 µl Dulbecco’s modified Eagle medium (DMEM; PAA Laboratories, Cölbe, Germany) with 2 % fetal calf serum and 50 μg/kg gentamicin. Inoculation was performed under general anesthesia using medetomidine (0.5 mg/kg, Domitor; Pfizer, Karlsruhe, Germany) and ketamine (100 mg/kg, Ketamin 10 %; WDT eG, Garbsen, Germany). General appearance, activity, and gait was evaluated weekly as previously described (Ulrich, Baumgärtner et al. 2006). Moreover, a rotarod assay (RotaRod Treadmill, TSE Technical & Scientific Equipment, Bad Homburg, Germany) was performed every week to test motor strength and control of TMEV-infected mice (Ulrich, Kalkuhl et al. 2010). Animals were sacrificed at 4, 14 and 98 days post infection (dpi). Segments of the brain and spinal cord of mice were removed immediately after death and either formalin-fixed and paraffin-embedded or snap-frozen in OCT embedding compound (Sakura Finetek Europe, Zoeterwoude, Netherlands) using liquid nitrogen (Markus, Failing et al. 2002). Animal experiments were conducted in accordance with the German Animal Welfare Law and were authorized by the local government (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany, permission number: 33.12-42502- 04-14/1656).

Histological examination

Two micrometers paraffin sections of brain (cerebrum and cerebellum) and spinal cord (cervical, thoracic and lumbar) were routinely stained with hematoxylin and eosin (HE). The degree of inflammation was evaluated using a semiquantitative scoring system of perivascular mononuclear cell infiltrates (Gerhauser, Alldinger et al. 2007).

Immunohistochemistry

Immunohistochemistry was performed as previously described (Gerhauser, Alldinger et al. 2005, Gerhauser, Alldinger et al. 2007, Kummerfeld, Meens et al. 2009, Li, Ulrich et al. 2015, Gerhauser, Li et al. 2018). Briefly, paraffin sections were blocked with 20% goat serum and stained with rabbit polyclonal antibodies directed against TMEV antigen (VP1; 1:2000), CD3 (T lymphocytes; Agilent Technologies Deutschland GmbH, Waldbronn, Germany; A045201; 1:500) and Iba-1 (microglia/macrophages; Wako Chemicals GmbH, Neuss, Germany; 019-19741; 1:1000) or rat monoclonal antibody directed against CD45R (B lymphocytes; BD Biosciences, Heidelberg, Germany; clone RA3-6B2; 553085; 1:1000) overnight at 4°C. Primary antibodies were replaced by rabbit serum (R4505, Merck KGaA, Darmstadt, Germany) or rat serum (R9759; Merck KGaA) as negative control. Biotinylated goat-anti-rabbit IgG (BA- 1000, Vector Laboratories, Burlingame, CA, USA; 1:200) and rabbit-anti rat IgG (BA-4001; Vector Laboratories; 1:200) were used as secondary antibodies. Immunolabeling was visualized by the avidin-

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Chapter 4: ASC- and caspase-1-deficient C57BL/6 mice 21

biotin-peroxidase complex (ABC) method (PK-6100, Vector Laboratories) with 3,3-diaminobenzidine (DAB, Merck KGaA) as substrate and slight counterstaining was performed using Mayer’s hematoxylin.

Polymerase chain reaction

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for TMEV, IFN-β, ISG15, PKR, TNFα, IL-1β, IL-6, IL-10, IL-12 (p40), IFNγ and three housekeeping genes (β-Actin, GAPDH, HPRT) using standard protocols, the AriaMx Real-Time PCR system (Agilent Technologies Deutschland GmbH), and Brilliant III Ultra-Fast SYBR® QPCR Master Mixes as described (Gerhauser, Alldinger et al.

2005, Gerhauser, Hansmann et al. 2012, Li, Ulrich et al. 2015, Waltl, Käufer et al. 2018). Tenfold serial dilution standards were used to quantify the results. Experimental data were normalized using a normalization factor calculated from the three housekeeping genes (Vandesompele, De Preter et al.

2002). Specificity of each reaction was controlled by melting curve analysis.

Statistical analysis

Statistical analysis was performed using Prism 6 (GraphPad Software, La Jolla, CA, USA). Mann- Whitney tests were used to compare clinical, histological, immunohistochemical and PCR data (knockout vs. wild type mice). P < 0.05 was considered as statistical significant.

5. Conclusions

ASC or caspase-1 deficiency had no effect on virus elimination, CNS inflammation, cytokine and IFN-I expression and clinical outcome in TMEV-infected B6 mice. Lack of inflammasome-dependent IL-1β-activation might be compensated by other proteases able to cleave pro-IL-1β, reduced pathogenic Th17 cell responses induced by IL-18 and IL-33 processing as well as inflammasome-independent functions of ASC, which can support the initiation and progression of TMEV-IDD. Nevertheless, future studies are needed to unravel the complex interactions of the inflammasome and other proinflammatory pathways in neuroinflammatory diseases.

Author contributions: I.G., T.S. and W.B. designed the study. D.L., M.B. and S.R. performed the animal experiment. D.L. performed histology, immunohistochemistry and RT-qPCR. G.B. performed the Western Blot analysis. D.L. and I.G. analysed the data, designed the figures and drafted the manuscript.

I.G. supported the laboratory work and finalized the manuscript. All authors have read and approved the final manuscript.

Funding: This work was supported by the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower Saxony. DL was funded by the China Scholarship Council (File No. 201606170128). This publication was further supported by German Research Foundation and University of Veterinary Medicine Hannover, Foundation within the funding program Open Access Publishing.

Acknowledgements: The authors thank Julia Baskas, Petra Grünig, Angela Karl, Christiane Namneck, Caroline Schütz and Danuta Waschke for excellent technical assistance. ASC-/- mice were developed by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA) and kindly provided by Prof. Richard A.

Flavell (Yale University, New Haven, CT, USA). Caspase-1-/- mice were also kindly provided by Prof.

Richard A. Flavell.

Conflict of interest: The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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