• Keine Ergebnisse gefunden

Virus and host factors involved in herpes simplex virus infection of the human nervous system

N/A
N/A
Protected

Academic year: 2022

Aktie "Virus and host factors involved in herpes simplex virus infection of the human nervous system"

Copied!
59
0
0

Wird geladen.... (Jetzt Volltext ansehen)

Volltext

(1)

Research Center for Emerging Infections and Zoonoses

Virus and Host Factors Involved in Herpes Simplex Virus Infection of the Human Nervous System

INAUGURAL DOCTORAL THESIS

Submitted in partial fulfillment of the requirements for the degree of Doctor of Natural Sciences

- Doctor rerum naturalium - (Dr. rer. nat.)

awarded by the University of Veterinary Medicine Hannover

by

Johanna Gracia Mitterreiter

from

Nuremberg

Hannover, Germany 2017

(2)

Supervision group: Prof. Dr. med. vet. Wolfgang Baumgärtner, PhD Prof. Dr. rer. nat. Peter Claus

Prof. Dr. rer. nat. Beate Sodeik

1st examination: Prof. Dr. Albert Osterhaus, DVM, PhD

Research Center for Emerging Infections and Zoonoses University of Veterinary Medicine Hannover, Germany Prof. Dr. med. vet. Wolfgang Baumgärtner, PhD Institute of Pathology

University of Veterinary Medicine Hannover, Germany Prof. Dr. rer. nat. Peter Claus

Institute of Neuroanatomy

Hannover Medical School, Germany Prof. Dr. rer. nat. Beate Sodeik Institute of Virology

Hannover Medical School, Germany

2nd examination: Prof. Dr. med. habil. Andreas Sauerbrei Institute of Virology and Antiviral Therapy Jena University Clinic, Germany

Day of oral examination: 06 October 2017

(3)

Mitterreiter JG, Titulaer MJ, van Nierop GP, van Kampen JJ, Aron GI, Osterhaus AD, Verjans GM, Ouwendijk WJ. Prevalence of intrathecal acyclovir resistant virus in Herpes simplex encephalitis patients. PLoS One. 2016 May 12;11(5):e0155531. doi:

10.1371/journal.pone.0155531. eCollection 2016.

Mitterreiter JG, Ouwendijk WJ, van Velzen M, van Nierop GP, Osterhaus AD, Verjans GM.

Satellite glial cells in human trigeminal ganglia have a broad expression of functional Toll- like receptors. Eur J Immunol. 2017 Jul;47(7):1181-1187. doi: 10.1002/eji.201746989. Epub 2017 Jun 1.

Poster presentations:

Mitterreiter JG, Titulaer MJ, van Nierop GP, Osterhaus AD, Verjans GM, Ouwendijk WJ.

Prevalence of acyclovir resistance in Herpes simplex encephalitis patients.

• 19th Molecular Medicine Day, Rotterdam 2015

• 2nd N-RENNT Symposium on Neuroinfectiology, Hannover 2015

Mitterreiter JG, Ouwendijk WJ, Osterhaus AD, Verjans GM. Comparative analysis of Toll- like receptor expression and function by glia cells in the central and peripheral nervous system.

• 20th Molecular Medicine Day, Rotterdam 2016

• 3rd N-RENNT Symposium on Neuroinfectiology, Hannover, 2016

Mitterreiter JG, Titulaer MJ, van Nierop GP, van Kampen JJ, Aron GI, Osterhaus AD, Verjans GM, Ouwendijk WJ. Prevalence of intrathecal acyclovir resistant virus in Herpes simplex encephalitis patients.

• 41st International Herpesvirus Workshop, Madison (WI), USA, 2016

Mitterreiter JG, Ouwendijk WJ, van Nierop GP, Osterhaus AD, Verjans GM. Broad expression of functional Toll-like receptors on satellite glial cells in human trigeminal ganglia.

• 9th Graduate School Days, Hannover, 2016

Oral presentation:

Mitterreiter JG. Toll-like receptor expression by satellite glial cells in human trigeminal ganglia. 8th Graduate School Days, Bad Salzdetfurth, 2015.

Sponsorship:

This research was supported by the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) from the Ministry of Science and Culture of Lower Saxony, Germany.

(4)
(5)
(6)
(7)

Contents

List of Abbreviations III

1 General Introduction 1

Structure of the human nervous system ... 1 1.1.1 Anatomy

1.1.2 Cellular composition

Herpes simplex virus infection of the nervous system... 5 1.2.1 Herpes simplex virus

1.2.2 Herpes simplex encephalitis

Immune responses in the nervous system ... 11 1.3.1 TLR overview

1.3.2 TLR ligands

1.3.3 TLR signaling pathways 1.3.4 TLR functions

1.3.5 TLR expression 1.3.6 TLR in diseases

Aims and outline of this thesis ... 15 2 Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex

Encephalitis Patients 17

3 Satellite glial cells in trigeminal ganglia have a broad expression of functional Toll-

like receptors 19

4 General Discussion 21

Pathogenesis of herpetic encephalitis ... 21 Viral factors in HSV infections of the human nervous system ... 22 Host factors in HSV disease of the human nervous system ... 23 4.3.1 HSV sensing by TLRs

4.3.2 Genetic predisposition for herpetic encephalitis 4.3.3 Cell types involved in herpetic encephalitis

Concluding remarks ... 30

5 Summary 31

6 Zusammenfassung 33

7 References 35

8 Acknowledgements 49

(8)
(9)

List of Abbreviations

ACV Acyclovir

ACVS Acyclovir-sensitive ACVR Acyclovir-resistant APC Antigen presenting cells ATP Adenosine triphosphate CD Cluster of differentiation CNS Central nervous system

CpG Deoxycytidylate-phosphate-deoxyguanylate CSF Cerebrospinal fluid

DAMP Damage/danger-associated molecular pattern (ds)DNA (double-stranded) Deoxyribonucleic acid DRG Dorsal root ganglion/ganglia

e.g. Exempli gratia et al. Et alii

HSE Herpes simplex encephalitis HSV Herpes simplex virus

IFN Interferon

IL Interleukin

IRAK Interleukin-1 receptor-associated kinases IRF Interferon regulatory factor

KO Knockout

LPS Lipopolysaccharide

MHC-II Major histocompatibility complex class II

MyD88 Myeloid differentiation primary response gene 88 NAWM Normal-appearing white matter

NF-κB Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells PAMP Pathogen-associated molecular pattern

PBMC Peripheral blood mononuclear cell PCR Polymerase chain reaction

pDC Plasmacytoid dendritic cell PNS Peripheral nervous system poly(I:C) Polyinosinic-polycytidylic acid PRR Pattern-recognition receptor

(ss/ds)RNA (single-stranded/double-stranded) Ribonucleic acid SGC Satellite glial cell

TBK1 TANK-binding kinase 1 TG Trigeminal ganglion/ganglia

(10)

TK Thymidine kinase TLR Toll-like receptor

TNF-α Tumor necrosis factor alpha TRAF TNF receptor associated factor

TRIF TIR-domain-containing adapter-inducing interferon-β

UV Ultraviolet

VZV Varicella zoster virus

wt Wildtype

(11)

1 General Introduction

Structure of the human nervous system 1.1.1 Anatomy

As the main coordinator of the body and mind, the human nervous system is not only responsible for coordination processes within the body, but also for the interaction of the body with the environment as well as the integration of internal and external stimuli. To master the vast amount of tasks it has to fulfill, the nervous system has a complex structure.

Anatomically, it can be divided into two parts: (1) the central nervous system (CNS) composed of brain and spinal cord and (2) the peripheral nervous system (PNS) consisting of all remaining nerve structures outside of the CNS. Functionally, however, these two systems are closely intertwined and act in synergy.

The nerve structures of the PNS can be grouped into three major categories: (1) the cranial and (2) spinal nerves with their derivatives that project to all parts of the body and (3) the enteric nervous system that regulates the bowel functions in the gastrointestinal tract. All nerve cell bodies of the sensory nerves are clustered in ganglia. While the dorsal root ganglia (DRG), the ganglia of the spinal nerves, are located bilaterally parallel to the spinal column, ganglia of the cranial nerves lie within the scull.

The trigeminal ganglia (TG) are located at the base of the scull, one on each side of the face (see Fig. 1). They contain the somata of the trigeminal nerve, the fifth cranial nerve that bilaterally delivers sensory input from the face via the TG to the brain. It splits into three major branches of afferent neuron fibers that innervate the eye (ophthalmic nerve), upper (maxillary nerve) and lower jaw (mandibular nerve). The trigeminal nerve also coordinates motor functions such as mastication. From the TG, efferent neuron fibers project to the brainstem [1]. The TG is further of immunological importance as it constitutes, together with the DRG, the location of latency for the human alphaherpesviruses herpes simplex virus (HSV) and varicella zoster virus (VZV) (see 3.2) [2].

(12)

Figure 1: Structure of the human trigeminal ganglion (TG). (A) Schematic representation of the human TG with its three nerve branches. (B) Schematic representation of TG sensory neurons surrounded by satellite glial cells (green). (C) Hematoxilin and eosin staining of human TG tissue. Arrowhead and asterisk indicate the location of the satellite glial cells and neuronal cell body, respectively. Original magnification was 200x with the inset at higher magnification. Scale bar: 100 µm.

1.1.2 Cellular composition

The nervous system consists of a variety of cell types divided into two main groups: neurons and glial cells. Neurons transmit information via electrical and chemical signals to other neurons, hereby creating a large network of interacting cells. All other non-neuronal cell types of the nervous system are collectively referred to as glial cells. Historically, glial cells were mainly regarded as helper cells providing physical support and stabilization. Nowadays, however, they are considered to execute pivotal functions such as nutrient supply and ensuring homeostasis, but also contribution to the immune system and the regulation of synaptic transmission. The CNS and PNS each harbor a distinct pool of different glial cell subsets.

Central nervous system

The CNS contains two major glial cell populations: macro- and microglial cells. Macroglia, namely oligodendrocytes and astrocytes, are derived from the neuronal crest.

Oligodendrocytes are mainly responsible for providing a protective myelin sheath around axon fibers that guarantee a fast signal transduction [3]. Astrocytes, on the other hand, provide metabolic support to neurons, regulate extracellular ion concentration and recycle neurotransmitters such as glutamate. Additionally, astrocyte processes constitute part of the blood brain barrier, which limits access of cells and large molecules from the blood stream to the brain parenchyma [4, 5].

Microglia are the major CNS-resident innate immune cells. Due to their similar phenotype and function they are often referred to as “macrophages of the brain”. In contrast to

(13)

macroglia, microglia originate from myeloid progenitors. They derive from hematopoietic stem cells of the yolk sac that populate the brain during early embryogenesis [6, 7]. In the adult brain, microglia are not replenished by hematopoietic progenitor recruitment from blood circulation but rather sustain via local self-renewal [8].

Even in their ‘resting’ state inactivated microglia are constantly surveilling the microenvironment with extremely motile processes and protrusions [9]. In case of cellular damage due to intrinsic or extrinsic factors, cell injury signs such as high adenosine triphosphate (ATP) levels induce increased microglia motility and migration to affected sites of the brain [10]. Here, activation leads to a conformational change of microglia from an ramified to an amoeboid phenotype with upregulation of numerous proteins [11].

Activated microglia both initiate and orchestrate an innate and adaptive immunity in response to intracerebral pathological processes. They are professional mononuclear phagocytes that clear their microenvironment from apoptotic cell debris via phagocytic receptors such as P2Y6 or TREM2 [12, 13]. After the upregulation of major histocompatibility complex class II (MHC-II) and T-cell co-stimulatory molecules CD80 and CD86, activated microglia present processed antigens to infiltrating CD4+ and CD8+ T-cells leading to the induction of an adaptive immune response in the brain [14, 15]. Furthermore, activated microglia secrete a variety of pro-inflammatory cytokines and chemokines which can have an either protective or detrimental effect on surrounding brain tissue. For example, endotoxin-activated microglia are neurotoxic by secreting tumor necrosis factor alpha (TNF-α) [16], whereas interleukin 4 and interferon gamma activated microglia support neurogenesis via insulin-like growth factor 1 [17]. Finally, microglia can sense invading pathogens via the expression of pattern- recognition receptors (PRRs) such as Toll-like receptors (TLRs) (see chapters 3.3 and 5).

Peripheral nervous system

The PNS contains two major glial cell populations which are spatially separated: Schwann cells and satellite glial cells (SGCs). Schwann cells completely cover the axons of peripheral nerves, hereby providing an isolating myelin sheath that grants protection to the axon, but more importantly facilitates a fast signal transduction along the axon [3].

SGCs are exclusively located in ganglia, where only a minor fraction of the Schwann cell population can be found. They are derived from progenitors of the neuronal crest [18]. Next to pseudo-unipolar sensory neurons and immune cells, SGCs are among the three main cells types in sensory ganglia. They completely wrap around neuronal somata forming a tight sheath that provides physical support and protection [19, 20] (see Fig. 1). Each neuron is surrounded by four to six SGCs, separating them into functional units that are partially divided by connective tissue [19]. The SGC sheath prevents uncontrolled molecule diffusion around neuronal somata but does not constitute an absolute barrier, as it is permeable for micro- and macromolecules [19]. While ‘resting’ SGCs show a slow proliferation cycle with

(14)

an estimated half-life of 600 days [21], peripheral nerve injury as well as inflammation or virus infection lead to SGC activation and increased SGC proliferation [22-24].

SGCs express both markers of the astrocyte lineage (glutamine synthethase and glial fibrillary acidic protein) as well as the oligodendrocyte and Schwann cell marker S100B [19, 25, 26].

Moreover, they carry receptors for numerous neuroactive agents such as ATP [27], bradykinin [28] or acetyl choline [29], and can produce nerve growth factor [30, 31]. Via neurotransmitter transporters for γ-aminobutyric acid and glutamate, SGCs control the composition of the extracellular space surrounding the neurons and coordinate neuron signaling [32]. The coupling of SGCs via gap junctions further facilitates the regulation the neuronal microenvironment as the gap junctions allow the passage of ions and small molecules [20, 33]. This strong connectivity among SGCs does not only enable signaling between SGCs, but also promotes interactions with and between neurons. The detected cross- excitation of neighboring neurons is mediated by diffusible chemical messengers whose concentration in the extracellular space is regulated by SGCs [34]. Bidirectional Ca2+ waves have been detected between both neuron and SGCs and among SGCs [35].

Several studies have demonstrated an immune-regulatory potential of SGCs. Murine SGCs secrete TNF-α and interleukin 6 (IL-6), which is further increased after irradiation with ultraviolet (UV) light or peripheral nerve injury [36, 37]. Recent studies on human TG implicated that SGCs have an additional role as antigen presenting cells (APCs) in the PNS, as they express MHC-II [38], and various markers (e.g. CD14, CD68 and CD11b/c) that are preferentially expressed by professional APCs such as macrophage and dendritic cells [39].

Additionally, they showed active phagocytosis and expressed matching co-stimulatory (e.g.

CD54 and CD80) and -inhibitory ligands (e.g. CD200R and programmed death-ligand 1) as well as cytokines involved in regulation of TG-resident T-cell responses to control HSV latency [39].

Overall, SGCs share characteristics with astrocytes as they are both involved in controlling the neuronal microenvironment and transmission of chemical signals. However, their potential function as APCs within ganglia also shows similarities to microglia.

(15)

Herpes simplex virus infection of the nervous system

Despite the many anatomical and immunological barriers that protect the nervous system, there are various neurotropic pathogens that can infect the nervous system and induce a plethora of diseases depending on anatomical location and causative agent. Infections of the brain (encephalitis), meninges (meningitis) and spinal cord (myelitis) in the CNS and various neuropathies in the PNS can be caused by pathogens such as viruses, bacteria and fungi. Virus infections are among the main causes of infectious encephalitis in moderate climate zones [40], of which HSV infections are the most common [41].

1.2.1 Herpes simplex virus Epidemiology and structure

HSV is a wide-spread human pathogen that belongs to the family of Herpesviridae. Together with VZV, HSV-1 and HSV-2 form the subfamily of alphaherpesviruses within the group of eight human herpesviruses described [42]. HSV-1 infections are endemic worldwide and commonly acquired in the first two decades of life through contact with infected saliva. A study in the USA showed an increasing prevalence of HSV-1 with rising age: from 44% in young adults (12-19 years) to 90% in people older than 70 years [43]. In some African regions HSV-1 prevalence can even reach 97% [44]. In contrast, transmission of HSV-2 mainly occurs via sexual contact. The prevalence of HSV-2 infected women in European countries mostly stays notably below 20% while it exceeds 50% in certain African countries [45].

Global incidence is estimated to reach around 23.6 million new HSV-2 infections every year [46].

Herpesviruses are double-stranded DNA (dsDNA) viruses that are broadly distributed among different species. They have been discovered in mammals as well as birds and reptiles and even in oysters. Every species has its own set of herpesviruses that principally can only infect that, or closely related species [42]. Viral particles of all herpesviruses share the same defined structure (see Fig. 2). The virus core consists of a single molecule dsDNA which is tightly packed and wrapped as a spool around a nucleoprotein core. It is protected by a viral capsid formed by 162 capsomeres in icosahedral symmetry. The virion is enclosed by a lipid bilayer envelope in which 13 distinct glycoproteins are embedded. The tegument is an unstructured, proteinaceous layer located between capsid and envelope. The average virion size is about 190 nm in diameter [2, 47].

(16)

Figure 2: Schematic representation of a herpes simplex virus (HSV) virion. A HSV particle consists of a viral capsid containing a DNA core and surrounded by tegument proteins. The particle is enclosed by a lipid envelope with embedded glycoproteins.

HSV life cycle

Alphaherpesvirinae share two defining biological characteristics: the ability to infect neuronal cells (neurotropism) and the establishment of a life-long quiescent infection (viral latency).

The combination of both attributes distinguishes them from all other human virus families.

During primary infection of mucous membranes, HSV penetrates sensory nerve endings of the infected tissue and migrates through trans-axonal transport to the innervating sensory ganglia. Here, it establishes a life-long latent infection in neuronal somata without production of viral particles or signs of neuropathology. In case of HSV-1 infections, which are mainly contracted via the facial mucosa, TG are the primary location of HSV-1 latency. HSV-2 latency, however, is commonly detected in neurons of the sacral ganglia, as the virus is usually transmitted by sexual contact [2]. Periodically, the virus escapes its quiescent, latent stage and enters a lytic phase characterized by high viral gene expression, active viral replication and virion formation [48]. Known triggers for reactivation are stress, immune suppression, peripheral nerve trauma, and UV light [49-51]. Reactivation of the virus and retrograde transport along the peripheral nerve axon back to the mucosa can lead to the development of recurrent mucocutaneous lesions. However, HSV reactivation is more frequently asymptomatic resulting in shedding and dissemination of infectious virus by the host [52-54].

HSV-associated diseases

Due to the broad cell tropism of HSV and different routes of transmission, HSV infections lead to a variety of different clinical manifestations. These range from mild mucocutaneous blisters to potentially blinding eye infections and life-threatening infections, such as herpes simplex encephalitis (HSE, see 3.3.2). While HSV-1 is widely known as the etiologic agent of

(17)

cold sores, HSV-2 is the main cause of genital herpes. However, recent epidemiologic studies report an increase of HSV-1 genital herpes among adolescents [55, 56] while HSV-2 has also been identified as causative agent for orofacial and ocular infections [57]. Orofacial, ocular and genital infections can be the result of primary, recurrent but also superinfection with a new HSV strain.

1.2.2 Herpes simplex encephalitis

HSE is a rare and potentially life-threatening complication of HSV infection of the brain.

Even though this disease affects only a minority of HSV-infected individuals, its case fatality rate is about 70% if not treated promptly with antivirals such as acyclovir (ACV). Only 2.5%

of untreated patients recover with normal neurologic function [58, 59]. Mortality is reduced to 11% to 19% when prompt antiviral therapy is provided [40, 60, 61]. Nevertheless, approximately 12% to 60% of treated HSE patients develop neurological sequelae [62, 63].

Relapses after disease recovery are uncommon but can occur especially in children [64]. HSE affects around 1 in 250,000 to 500,000 persons per year in the USA [62]. A study in the United Kingdom identified almost 20 percent of all encephalitis cases were caused by HSV [40], making HSE the most common cause of sporadic, fatal encephalitis [41, 65]. Especially individuals at extremes of age are at higher risk of contracting HSE, as one third of all cases involve individuals less than 20 years old and half of the patients are more than 50 years old [62].

HSE can be caused by both HSV subtypes, namely HSV- 1 and HSV-2. Whereas 90% of HSE cases in immunocompetent adults are caused by infection with HSV-1, HSE in neonates and immunocompromised individuals is more commonly associated with HSV-2 [62, 65].

The pathology of HSE has not definitely been clarified yet, but most likely involves the combination of the direct cytopathic effect of the virus on CNS-resident cells and a local inflammatory immune response. Both reactivation of an endogenous latent HSV strain and primary HSV infection are still considered as possible causes of HSE development [41] (see 6.1).

Symptoms and diagnostics

Acute HSE disease manifests within less than a week after HSV infection of the brain with the typical clinical triad of acute viral encephalitis: fever, headache and altered level of consciousness [41, 62]. It typically affects the frontal and temporal lobes, resulting in additional symptoms such as personality changes, cognitive impairment, aphasia, seizures, or focal weakness [41, 65]. Lymphocytes often infiltrate both hemispheres causing bilateral but asymmetric brain lesions [65-67]. Even after successful treatment many patients experience neurological sequelae such as cognitive impairment, memory loss, behavioral abnormalities, or seizures [60]. In up to 17 % of confirmed HSE cases, patients can present a relatively mild or atypical disease phenotype [68].

(18)

In the majority of neonatal HSE cases, newborns acquire HSV-2 infection during delivery from the maternal genital tract resulting in either an isolated CNS infection or, more commonly, in a disseminated HSV infection. In the latter case, diffuse encephalitis is accompanied by a multi-organ HSV infection with an even worse disease outcome than sole CNS infection [62, 66, 69].

Prompt diagnosis of HSE and direct antiviral treatment are crucial to reduce HSE-associated morbidity and mortality and promote a positive long-term disease outcome. While most viral encephalitis cases show increased protein levels and pleocytosis in the cerebrospinal fluid (CSF), a definitive HSE diagnosis is currently based on the detection of HSV DNA by polymerase chain reaction (PCR) [62]. Additional visualization of anatomical brain lesions via magnetic resonance imaging further consolidates the diagnosis [41, 62, 65]. A second lumbar puncture and assay repetition is recommended as PCR analysis of CSF can be false negative in the first few days of disease [65].

HSE treatment

As soon as HSE is clinically suspected, preemptive high-dose antiviral treatment should be started with ACV as first-choice medication. ACV is administered intravenously for 14 days, in immunocompromised patients and children for up to 21 days [65, 66, 69, 70]. Delay of treatment for only 2 days is already associated with significantly poorer prognosis [60, 71].

Occasionally, intravenous ACV treatment is followed by its oral formulation valaciclovir, but its beneficial effect remains to be validated [63, 65]. A retrospective study on the effect of additional steroid medication to potentially dampen neuroinflammation has shown a favorable outcome for the combination treatment [72]. Animal studies further showed beneficial effects of the use of steroids in HSE models [73, 74]. However, steroid treatment still remains controversial due to the danger of virus reactivation by steroid-induced immunosuppression [75]. In case of clinically suspected resistance to ACV treatment, viral DNA inhibitors foscarnet and cidofovir are used as second-line antivirals [41, 76].

Acyclovir

Due to its high selectivity and safety profile ACV [9-(2-hydroxyethoxymethyl) guanine]

remains the gold standard in the treatment of different HSV diseases. Discovered by Elion and colleagues in the 1970s [77] and first approved in 1981, it constituted a milestone in treatment of herpesvirus infections worldwide. This was acknowledged with the 1988 Nobel Prize in Medicine to Prof. Elion, partly for the development of ACV. Next to HSE therapy, clinical indication for the application of ACV includes treatment of primary as well as recurrent HSV infections, prophylactic therapy of immunocompromised patients to prevent recrudescent disease and herpes zoster caused by VZV. Especially in cases of recurrent genital herpes infection, oral nucleoside analogues are subscribed both as therapeutic as well as prophylactic treatment. Topical formulations for the lip and exterior parts of the eye (e.g. cornea and

(19)

conjunctiva) exist for herpes labialis and ocular HSV infections, respectively [78-80]. Long- term treatment of ACV shows no severe side effects. Rare side effects after intravenous application include renal toxicity and neurological impairment such as hallucination or disorientation [58].

ACV is a nucleoside analogue that indirectly inhibits the function of the viral DNA polymerases thereby blocking HSV replication (see Fig. 3). Its initial activation is catalyzed by the HSV thymidine kinase (TK) protein followed by two sequential phosphorylations by cellular kinases. The resulting ACV-triphosphate acts as a competitive inhibitor of the viral DNA polymerase as it competes with its substrate desoxyguanosid-triphosphate (dGTP) to be incorporated into the replicating viral genome. Incorporation of ACV at the 3’ end of the elongating DNA leads to termination of DNA elongation and subsequently inhibition of HSV replication. Due to its strong affinity to HSV TK, but not to its cellular counterpart, ACV is a highly selective drug in targeting specifically HSV-infected cells. These characteristics also explain the high safety profile of ACV leading to only minor adverse effects [77, 78, 81]. The prodrug valaciclovir was designed as oral formulation since ACV has a poor oral bioavailability, [82, 83]. After its absorption by the gastrointestinal tract the prodrug is activated into ACV [77-79, 81].

Figure 3: Mechanism of acyclovir-mediated inhibition of HSV replication. HSV thymidine kinase (TK) activates acyclovir (ACV) which is subsequently phosphorylated by two different cellular kinases (cKs).

Triphosphorylated ACV now inhibits DNA replication thereby preventing the formation of new HSV particles. Alternative antiviral drugs foscarnet and cidofovir directly inhibit HSV DNA replication by blocking HSV DNA polymerase (pol).

(20)

Acyclovir resistance development

Since ACV is the first-line treatment of HSV and VZV infections that has been used extensively starting in the 1980s, it harbors the danger of drug resistance development. So far, there is no evidence of increased resistance to ACV in immunocompetent patients despite its general and long-term use [78, 84]. In immunocompetent individuals, the overall prevalence of ACV resistance is around 0.5% or less [85-88]. However, the risk of ACV resistance is notably higher in immunocompromised patients with a general prevalence of 3% to 10% [86, 87, 89]. Depending on the type of immune suppression, its prevalence can even reach 14% to 34% in the subgroup of bone marrow and allogenic stem cell transplant recipients [86, 87, 89, 90]. Also in immune-privileged organs such as the eye, an elevated ACV resistance rate has been observed [91, 92]. Due to the lack of a broad scale study, the prevalence of ACV- resistant (ACVR) HSV in HSE patients is still unknown. An accompanied risk of an elevated resistance rate is envisaged, as HSE patients receive an extremely high-dose ACV treatment.

Case reports on individual HSE cases have already demonstrated ACV refractory disease due to ACVR HSV in previously healthy immunocompetent individuals [93, 94]. In one neonate HSE case, the emergence of intrathecal ACVR HSV during ACV therapy has been reported [95]. In case of clinical signs of ACV refractory disease, direct DNA polymerase inhibitors foscarnet or cidofovir are recommended, since ACVR HSV strains often show cross-resistance to nucleoside analogues penciclovir and ganciclovir [78, 96, 97].

In about 95% of cases, ACV resistance is accredited to mutations in the viral TK, which is responsible for the first activation step of ACV. The remaining 5% of ACVR cases are caused by mutations in the HSV DNA polymerase gene [98]. ACV resistance-associated TK phenotypes can be classified into 3 different groups: (1) TK-negative mutants that lack enzymatic TK activity, (2) TK partial mutants that show a reduced level of TK activity, and (3) TK mutants with altered substrate specificity [78, 84, 99]. The majority of ACVR HSV strains belong to the first two groups with altered viral TK activity.

HSV TK is a 376 amino acid protein encoded by the viral UL23 gene [100]. Its active center consists of three conserved regions: (1) an ATP-binding site, (2) a nucleotide binding site, and (3) a cysteine residue at position 336 or 337 for HSV-1 and HSV-2, respectively, which maintains the three-dimensional structure of the active site [101]. The HSV-1 TK gene is highly polymorphic by nature. Many natural single nucleotide polymorphisms can be found in the non-conserved regions [79, 102, 103]. ACV resistance-associated TK mutations, however, are often detected in the highly conserved regions, which are distributed throughout the protein [80, 104]. Around 50% of these ACVR polymorphisms are insertions or deletions in mutational hotspot, which lead to a frameshift and subsequently a truncated non-functional TK protein [105]. The other half comprises amino acid changes in the TK protein resulting in its altered enzymatic activity [99, 106].

(21)

Immune responses in the nervous system

Historically, the CNS was believed to be an immune-privileged site representing an organ with restricted access of the immune system, especially of infiltrating cells from the adaptive immunity. It is protected from invasion via the blood circulation by a specialized blood-brain barrier. The blood-brain barrier is composed of an endothelial cell layer interconnected by tight junctions and a tight layer of astrocyte end-feet processes. It shields the brain from incoming pathogens and infiltrating cells and strictly regulates the crossing of solutes from the blood into the brain parenchyma [5, 107]. Nowadays, however, it is known that this barrier is not absolute and that both innate and adaptive immune system play an important role in combatting incoming pathogens and other pathologic processes within the brain. In this thesis, the studies focused on the mechanisms of the innate immune system to detect and antagonize invading pathogens such as HSV.

One strategy of the innate immune system is to detect invading pathogens and pathologic cellular changes via specialized PRRs. PRRs can recognize conserved pathogen- and danger- associated molecular patterns (PAMPs or DAMPs, respectively) leading to immune activation by the induction of pro-inflammatory cytokines. They can be located both intracellularly or embedded in the cellular surface membrane and are part of the first-line defense mechanisms that result in a quick and unspecific immune activation. Prominent members of this group are intracellular NOD-like or RIG-I-like receptors, but also C-type lectin surface receptors and the family of TLRs.

1.3.1 TLR overview

In 1989, Charles Janeway already postulated that the ability of the immune system to discriminate self from non-self includes the “recognition […] of certain characteristics or patterns common to infectious agents, but absent from the host” [108]. Less than 10 years later the first human TLRs were discovered. This discovery was later awarded with the 2011 Nobel Prize in Medicine or Physiology to Jules Hoffmann [109] and Bruce Beutler [110].

Currently, 13 TLRs have been described in mammals. Only TLR1-TLR10 are expressed in humans at variable levels, depending on the phenotype, activation and differentiation status of cells [111-115].

TLRs are type I integral membrane receptors with a N-terminal ligand recognition ectodomain, a single transmembrane helix and an C-terminal cytoplasmic signaling TIR (Toll/interleukin-1 receptor homology) domain [116]. The TLR proteins comprise around 800-1,000 residues with a molecular weight of about 84-121 kilodalton [117]. While TLR1, TLR2, TLR4, TLR5 and TLR6 are expressed at the cell surface, TLR 3 and TLR7-TLR9 are located intracellularly embedded in endosomal membranes. TLR1 and TLR6 form heterodimers with TLR2, the remaining TLRs operate in homodimeric complexes [118-120].

(22)

1.3.2 TLR ligands

So far, the targets and biological function of TLR1-TLR9 have been well characterized. Only TLR10 remains an orphan receptor as its stimulating ligand has not been identified yet. The list of TLR activating PAMPs comprises different molecular structures from peptides to nucleic acids present in numerous pathogens. The major PAMPs of different species are listed in table 1.

Table 1: List of human Toll-like receptors (TLRs) and their major ligands1

TLR PAMP2 Species References

TLR1/TLR2 Triacyl(ated) lipopeptides Bacteria [121]

TLR2/TLR6

Diacyl lipopeptide Zymosan

Lipoteichoic acid

Mycoplasma Yeast Bacteria

[118, 121]

[122]

[123]

TLR3 dsRNA Virus [124]

TLR4 LPS

Envelope proteins

Bacteria

Virus (e.g. RSV)

[110]

[125]

TLR5 Flagellin Bacteria [126]

TLR7 ssRNA RNA virus [127, 128]

TLR8 ssRNA RNA virus [127]

TLR9 CpG DNA Bacteria, DNA virus [129, 130]

TLR103 Undetermined Undetermined [115, 131]

1 The overview is not complete. It lists the major TLR agonists of different species

2 PAMP: pathogen-associated molecular pattern. LPS: lipopolysaccharide. RSV: respiratory syncytial virus.

ssRNA: single-stranded RNA. CpG: deoxycytidylate-phosphate-deoxyguanylate.

3 The activating ligand of TLR10 has not yet been identified.

Endosomal TLRs are pivotal for the detection of viruses as they are specialized in recognizing nucleic acids. Whereas TLR3 is activated by double-stranded RNA (dsRNA), TLR7 and TLR8 preferentially interact with single-stranded RNA viruses such as influenza, vesicular stomatitis or human immunodeficiency virus [127, 128, 132]. Through the recognition of CpG (deoxycytidylate-phosphate-deoxyguanylate) DNA motives, TLR9 senses infection with adenoviruses [133] and HSV [134]. Additionally, surface TLRs have also been shown to contribute to the antiviral response. The envelope F-protein of respiratory syncytial virus is capable of inducing an immune response via TLR4 and CD14 [125]. Furthermore, HSV infection of dendritic cells is detected through sequential recognition by TLR2 and TLR9 [134].

1.3.3 TLR signaling pathways

Depending on the stimulus, as well as the type and location of the respective TLR, different signaling pathways (reviewed in [135-137]) can be induced which finally lead to cell activation by transcription factor translocation to the nucleus (see Fig. 4). Hereby, one can distinguish between pathways initiated via the adaptor protein MyD88 (myeloid differentiation primary response gene 88) or TRIF (TIR-domain-containing adapter-inducing interferon-β)-induced signaling. The latter is only initiated after TLR3 or TLR4 activation.

(23)

In the absence of a stimulus, surface TLRs are expressed as monomers. Following ligand recognition, PAMP-induced dimerization leads to formation of m-shaped TLR homo- (TLR4 and TLR5) or heterodimers (TLR1/2 and TLR2/6) [117, 119, 120, 138, 139]. This activates the intracellular adaptor molecule MyD88 which in turn recruits a complex of interleukin-1 receptor-associated kinases (IRAKs) and TNF receptor associated factor 6 (TRAF6). Finally, TLR stimulation leads to the activation of mitogen-activated protein kinases and the transcription factor NF-κB [135, 136]. Analogous to cell surface-exposed TLRs, endosomal TLR7-TLR9 are also expressed as inactive homodimers that are activated by conformational change after ligand binding [140, 141]. However, the recruitment of a different IRAK-TRAF3 complex by MyD88 leads to a direct activation of transcription factor IRF7 (interferon regulatory factor) [142]. Recruitment of TRAF6 can also activate the same downstream signaling cascade as described for surface TLRs. In contrast, TLR3 signals exclusively via the adaptor molecule TRIF [143], leading to an activation cascade of TRAF3, followed by kinase TANK-binding kinase 1 (TBK1) and finally the transcription factor IRF3 [144, 145].

Figure 4: Overview of Toll-like receptor (TLR) signaling pathways (reviewed in [135-137, 146]). AP-1:

activator protein 1. CREB: cAMP response element-binding protein. IKK: inhibitor of kappa B kinase.

IRAK: interleukin-1 receptor-associated kinase. IRF: interferon regulatory factor. MyD88: myeloid differentiation primary response gene 88. TAB: TAK1-binding protein. TAK1: transforming growth factor β- activated protein kinase 1. TBK1: TANK-binding kinase 1. TRAF: TNF receptor associated factor. TRIF:

TIR-domain-containing adapter-inducing interferon-β.

(24)

1.3.4 TLR functions

Even though different TLRs utilize different signaling cascades, they all lead to a similar response: immune system induction by transcription upregulation of various pro- inflammatory cytokines and other regulatory and antimicrobial proteins. Important mediators are type I interferons (e.g. IFN-α), interleukins IL-6 or IL-12 and TNF-α [147, 148]. In addition to innate immune system stimulation, TLRs also function as a linker between the innate and adaptive immune system. Stimulation of TLRs leads to the upregulation of MHC- II and T-cell co-stimulatory molecules (e.g. CD40, CD80 and CD86) on APCs thereby supporting a prompt T-cell response [149]. TLRs can further regulate neutrophil function and promote B-cell proliferation and survival [150, 151]. Additionally, TLRs have various functions that are not primarily related to immune system stimulation. These range from transcription induction of long non-coding RNAs [152], to nuclear reprogramming during generation of induced pluripotent stem cells [153], and modulation of adult hippocampal neurogenesis [154].

1.3.5 TLR expression

TLRs are expressed ubiquitously in the human body by numerous cell types in various tissues, as they mark the first-line immune defense to invading pathogens, but also sense tissue damage by the recognition of DAMPs [155]. Especially professional APCs, such a dendritic cells [156] and monocytes/macrophages [155, 157], show a broad expression of TLRs which play a central role in the induction of the primary cytokine storm after pathogen infection [158].

In the human CNS, TLRs are expressed by both neurons and glial cells. Restricted TLR expression was observed in human neurons (TLR3) and oligodendrocytes (TLR2 and TLR3) [159, 160]. More abundant and diverse TLR expression was shown in human astrocytes and microglia. Human astrocytes express moderate levels of TLR1–TLR5 and TLR9, whereas microglia express all TLRs except TLR10 [160, 161]. However, most TLR expression profiles on human CNS-resident cells are largely based on detection of their transcripts, not the encoded protein. Compared to the CNS, the expression pattern and function of TLRs in sensory ganglia of the human PNS is ill-defined. TLR3, TLR7 and TLR9 expression was identified on sensory neurons in human DRG [162], as well as TLR4 on neurons in human TG [163]. The role of TLRs or other innate immune sensors on human SGCs, however, has not been studied yet. So far, only studies on mice and rats discovered TLR2 and TLR4 expression in SGCs, respectively [164, 165].

1.3.6 TLR in diseases

Due to their broad expression pattern and the plethora of genes induced upon TLR activation, TLRs are linked to various diseases ranging from classical autoimmune diseases (e.g.

(25)

systemic lupus erythematosus) to multifactorial diseases (e.g. multiple sclerosis) and cardiovascular diseases [166, 167]. The TLR7 agonist imiquimod has even been approved as a drug for cancer treatment of superficial basal cell carcinoma [168]. Additionally, mutations in the TLR3 pathway were identified as a predisposing factor for HSE development (see chapter 6).

Aims and outline of this thesis

The severity of HSV infections in the nervous system depends on (1) viral factors that drive the dissemination of the virus and (2) the host’s immune system aimed to limit the detrimental impact of the invading virus. However, the contributors of this delicate interplay between virus and host immune system are still ill-defined. Therefore, the studies presented in this thesis were aimed to further characterize the involvement of both the viral and host determinants that influence the outcome of HSV infection in the human nervous system.

Chapter 4 covers the genetic variability of HSV isolates and its implications for the treatment of HSE. In HSE a quick response to antiviral treatment is pivotal for the survival of the patient and the quality of life after recovery from disease. Studies on ocular HSV infections showed that in immune-privileged organs, the prevalence of ACV resistance is higher compared to HSV infections in organs with unrestricted immune system access [91, 92]. The presence of intrathecal ACVR HSV in HSE patients is limited to case reports or incidental small cohorts [93, 95, 169, 170]. Consequently, our study aimed to determine the prevalence of ACVR HSV in a cohort of 4 HSV-1 and 8 HSV-2 HSE cases in order to evaluate the likelihood of ACV treatment failure and the necessity to quickly change the antiviral treatment regimen for HSE patients.

In Chapter 5, we focused on the host’s innate immunity to detect foreign pathogens and induce an opposing immune response in the nervous system. Both microglia and SGCs have been shown to act as local APCs in the CNS and PNS, respectively [39]. While microglia are known to express a variety of TLRs [160, 161], there is no data on pathogen recognition by human SGCs that may potentially contribute to the control of HSV infections in sensory ganglia. Consequently, our study aimed to characterize and compare the TLR expression profile and function on human TG-derived SGCs and microglia in order to improve our knowledge on the role of TLR-mediated innate immunity by glial cells in the human nervous system.

(26)
(27)

2 Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex Encephalitis Patients

Johanna G. Mitterreiter1,2, Maarten J. Titulaer3, Gijsbert P. van Nierop3,2, Jeroen J.A. van Kampen2, Georgina I. Aron2, Albert D.M.E. Osterhaus1,2, Georges M.G.M. Verjans2,1 and Werner J.D. Ouwendijk2

1 Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Germany

2 Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands

3 Department of Neurology, Erasmus Medical Center, Rotterdam, the Netherlands

Published in: PLoSOne. 2016 May 12;11(5):e0155531. doi: 10.1371/journal.pone.0155531.

eCollection 2016.

Personal contribution:

JGM performed the experiments, analyzed the data and wrote the manuscript. MJT analyzed clinical data. JJAvK performed and analyzed one experiment. GPvN and GIA assisted with experiments. ADMEO wrote the manuscript. GMGM designed the study and wrote the manuscript. WJDO analyzed data and wrote the manuscript.

(28)

Abstract

Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real- time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients.

Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients.

Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV- sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients.

(29)

3 Satellite glial cells in trigeminal ganglia have a broad expression of functional Toll-like receptors

Johanna G. Mitterreiter1,2, Werner J.D. Ouwendijk2, Monique van Velzen3, Gijsbert P. van Nierop2,4, Albert D.M.E. Osterhaus1 and Georges M.G.M. Verjans2,1

1 Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Germany

2 Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands

3 Department of Anesthesiology, Leiden University Medical Center, Leiden, the Netherlands

4 Department of Neurology, Erasmus Medical Center, Rotterdam, the Netherlands

Published in: Eur J Immunol. 2017 Jul;47(7):1181-1187. doi: 10.1002/eji.201746989. Epub 2017 Jun 1.

Personal contribution:

JGM performed the experiments, analyzed the data and wrote the manuscript. MvV and GPvN performed experiments. GMGM and WJDO designed the study and wrote the manuscript. ADMEO wrote the manuscript.

(30)

Abstract

Toll-like receptors (TLRs) orchestrate immune responses to a wide variety of danger- and pathogen-associated molecular patterns. Compared to the central nervous system (CNS), expression profile and function of TLRs in the human peripheral nervous system (PNS) are ill-defined. We analyzed TLR expression of satellite glial cells (SGCs) and microglia, glial cells predominantly involved in local immune responses in ganglia of the human PNS and normal-appearing white matter (NAWM) of the CNS, respectively. Ex vivo flow cytometry analysis of cell suspensions obtained from human cadaveric trigeminal ganglia (TG) and NAWM showed that both SGCs and microglia expressed TLR1–TLR5, TLR7 and TLR9, although expression levels varied between these cell types. Immunohistochemistry confirmed expression of TLR1–TLR4 and TLR9 by SGCs in situ. Stimulation of TG- and NAWM- derived cell suspensions with ligands of TLR1–TLR6, but not TLR7 and TLR9, induced interleukin 6 (IL-6) secretion. We identified CD45LOWCD14POS SGCs and microglia, but not CD45HIGH leukocytes and CD45NEG cells as the main source of IL-6 and tumor necrosis factor α upon stimulation with TLR3 and TLR5 ligands. In conclusion, human TG-resident SGCs express a broad panel of functional TLRs, suggesting their role in initiating and orchestrating inflammation to pathogens in human sensory ganglia.

(31)

4 General Discussion

Pathogenesis of herpetic encephalitis

HSE is a life-threatening complication of a HSV infection of the brain. Despite prompt therapy with the antiviral drug ACV, still too many patients die or survive with serious long- lasting neurological sequelae. The histopathology of HSE is characterized by necrosis, hemorrhage and immune cell infiltration primarily in the frontal and temporal lobes [171].

However, the exact mechanisms how and when the virus enters the brain and causes these histopathological changes are not fully understood. The neurotropic nature of HSV makes human studies difficult. Thus far, there is no animal model available that accurately mimics the human disease. Due to the limited availability of human HSE specimens, most studies still focus on HSV infection in the experimental HSE mouse model. However, extrapolation of data from mouse to human has to be regarded cautiously as the mouse is not the natural host of HSV, and there has no equivalent murine alpha herpesvirus been identified yet. The route of virus dissemination to the CNS is yet an unsolved issue, which is crucial in order to understand the initiation and perpetuation of HSE pathogenesis on the cellular level and to identify the cell types and the signal cascades involved.

There are four different possibilities how and when HSV enters the CNS to cause HSE:

1. primary infection of the CNS with entry via olfactory or trigeminal nerves, 2. primary infection of the CNS by hematogenous spread,

3. reactivation of latent virus in the TG with spread to the brain, or 4. reactivation of latent virus within the CNS.

Due to the common infection route of HSV via the orofacial and genial mucosa followed by direct invasion into peripheral nerve endings and transaxonal spread to the innervating sensory ganglia, the second option seems rather unlikely as the principle route. Even though a mouse model showed hematogenous spread of HSV-1 after intraperitoneal virus injection, acute HSE in adult individuals is normally not accompanied by HSV viremia [172]. However, a disseminated, potentially blood-borne HSV infection and subsequently HSE may occur in neonates infected with HSV-2 [62].

Serologic surveys of HSE patients suggested that HSE occurs due to primary (30%) and recurrent (70%) HSV infection [173]. Indeed, comparative restriction fragment length polymorphism analysis of sequential HSV isolates from cold sores and HSE of the same individuals identified the involvement of both genetically identical or distinct virus strains [174]. The observation of mainly bilateral lesions, and the young age in childhood HSE cases, supports the hypothesis of a primary HSV infection. Contrastingly, cases of relapsing HSE and the onset of disease in adults at high age suggest that the disease is initiated by reactivation of an endogenous latent HSV strain that entered the brain. Notably, in Chapter 4

(32)

we have shown that identical TK sequences were recovered from successive CSF samples and samples from distinct anatomical sites of the same HSE patients, suggesting that consecutive HSE and mucosal HSV infections within the same individual can be caused by the same virus strain. However, even though the genetic variability of the viral UL23 TK gene facilitates discrimination between HSV-1 strains [10, 17, 18, 30], sequence analysis of other HSV genes and ideally the whole viral genome should be performed additionally to determine the origin of the virus at different anatomic sites within the same individual.

As there are indications for both primary and recurrent HSV infection that trigger HSE, there is also conflicting data on the virus route to the brain. Traditionally, HSV is known to establish latency only in the TG. Thus, potential reactivating virus would enter the brain via the trigeminal nerve. On the other hand, some studies suggest that HSV also establishes latency in the brain and reactivation occurs within the cerebral parenchyma to initiate HSE [175]. Indeed, HSV DNA was discovered in autopsy brain specimens from patients without known neurological disorder on multiple occasions [176-179].

During primary infection, HSV is presumed to also enter the CNS via the olfactory nerve to the olfactory bulb. The olfactory bulb is in closer anatomical proximity to the primary HSE lesion sites in the frontal and temporal lobes compared to the projection site of the trigeminal nerve in the brainstem. Additionally, immunohistochemical analysis of human post-mortem HSE brain specimens located HSV protein expression in the olfactory tract, but not in tissues of the trigeminal pathways [180]. In contrast, detection of HSV in the medulla and pons of mouse brains after ocular HSV-1 infection indicates virus entry via the trigeminal nerve [181]. Some studies in mice and rats suggest the simultaneous usage of both routes [182, 183]. In a HSE mouse model, the observation of infiltrating leukocytes into both the brainstem and olfactory bulb indicated a parallel virus entry to the CNS via the olfactory and the trigeminal route [183].

Altogether, the initiation and perpetuation of HSE pathogenesis probably does not involve a single route of CNS virus entry or common pathological mechanism. It is likely to be the result of both primary HSV infection and reactivation of latent virus depending on the individual case. The virus entry route possibly varies depending on virus transmission route and viral titer.

Viral factors in HSV infections of the human nervous system

Symptomatic HSV infection of the CNS is a rare event occurring in only a minority of HSV infected individuals. Thus far, it is not yet understood which factors contribute to the development of HSE in some patients, while most other HSV infected individuals stay healthy. Viral factors such as virus strain diversity can advance the progression of HSV infection and its disease outcome. So far, it is still unknown if there are specific HSV strains which are more encephalogenic in humans. The influence of virus strain diversity on the

(33)

outcome of HSV-1 CNS infections has been investigated in the mouse model [184, 185]. In a cornea infection model, all mice infected with the HSV-1 strain KOS survived, while three other virus strains (RE, 294.1, and McKrae) induced death by encephalitis [184]. The mortality of the infected mice correlated with viral growth in the brain stem indicating a distinct neurovirulence for each virus strain. Additionally, diverse CNS demyelination events were also detected by different HSV strains [186, 187]. In Chapter 4 we determined the prevalence of ACVR HSV in HSE patients to assess whether there is an increased selection for ACVR virus strains. This phenomenon was already observed in herpetic eye diseases and immunocompromised patients [86, 91]. However, no resistance-associated HSV-TK mutations were detected in a cohort of 12 HSE patients, consisting of 4 HSV-1 and 8 HSV-2 HSE cases. Phenotypic resistance testing revealed two ACVR HSV-2 isolates from patient swabs which are most likely caused by mutations in the herpesviral DNA polymerase gene.

Thus, in our cohort we did not detect an increased prevalence for ACV resistance-associated HSV-TK mutations in HSE patients or selection of specific ACVR virus strains that would likely contribute to a worse disease outcome.

Altogether, different viral strains probably influence the development of HSV infection in the CNS. However, it remains unknown which features define the differential neurovirulence of HSV strains. Our study indicated that antiviral resistance is not a driving force for HSV strain selection and persistence in the CNS of HSE patients.

Host factors in HSV disease of the human nervous system

In addition to viral factors, the host immune system plays an important role in combatting incoming HSV and influencing the outcome of the HSV-induced CNS disease. There is conflicting data on the beneficial or detrimental contribution of the innate and/or adaptive immune system on HSE. The exact cellular mechanisms of HSE pathology have not been completely elucidated. So far, there is no targeted immunotherapy for HSE patients available that complements the antiviral treatment. Trials with corticosteroids to unspecifically dampen a potentially damaging intracerebral immune response have shown beneficial effects in the animal model [73, 74]. However, corticosteroids are not commonly used as supportive HSE therapy in human medicine. In order to find new therapeutic targets in the future, many studies have focused on the role of PRRs, especially TLRs, in sensing and combatting HSV infection and their contribution to HSV induced CNS disease.

4.3.1 HSV sensing by TLRs

TLRs were shown to be broadly expressed on a variety of different cell types in both mice and humans. They range from dendritic cells, macrophages, T-cells and NK cells to non-immune cells such as epithelial cells and keratinocytes [155, 188-190]. TLR transcript expression was also identified on different neuronal and glial cells of the CNS and PNS. In Chapter 5, we

(34)

described a broad TLR protein expression on human microglia and identified a similar TLR profile on human TG-resident SGCs. Thus, studies investigating the sensing of HSV via TLRs analyzed many different cell types both in vitro and in vivo in the mouse model.

In vitro studies on mouse-derived cell cultures mainly focused on the role of TLR2 and TLR9.

In mouse plasmacytoid dendritic cells (pDCs), HSV-1 infection led to the secretion of the major antiviral cytokine IFN-α which was significantly lower in TLR9-deficient pDCs [191, 192]. Infection of murine pDCs with live and UV-inactivated HSV-2 showed a similar dependency on TLR9 for IFN-α production [193]. Experiments on primary mouse cells identified TLR2 as an important PRR in HSV recognition. Both TLR9- and TLR2-deficient macrophages secreted reduced IL-12 and TNF-α levels after HSV-1 infection [194]. In case of mouse microglia, TLR2 was required for the production of a plethora of different cytokines and chemokines in response to HSV-1 [195]. Finally, a study by Sato et al. showed that HSV- 1 and HSV-2 are simultaneously recognized by TLR2 and TLR9 acting in synergy to induce IL-6 an IL-12 secretion by bone-marrow derived murine dendritic cells. This dual recognition occurred in a sequential fashion within the same cell, first by activating surface TLR2, then followed by stimulation of intracellular TLR9 [134].

In vitro studies with human cell cultures also linked several TLRs to recognition of HSV particles. Immortalized human kidney cells as well as peripheral blood mononuclear cells (PBMCs) secreted IL-6 upon TLR2 activation in a dose-dependent manner with increasing infection rates of HSV [196, 197]. Next to the induction of pro-inflammatory cytokines, HSV- 1 infection of human corneal epithelial cells led to an increased TLR7 expression and down- regulation of TLR3 [198]. Additionally, HSV-2 infection of human cervical epithelial cells evoked an increased TLR4 and TLR9 levels [199]. Altogether, in vitro studies in mouse and human cell cultures connected several TLRs to the sensing of HSV. Especially TLR2 and TLR9 seem to play a prominent role in the initiation of pro-inflammatory cytokines after HSV infection.

In vivo studies in a HSE mouse model further analyzed the impact of TLR-mediated virus sensing in HSV-induced CNS disease. Deletion of the gene encoding the TLR adaptor molecule MyD88 first indicated a crucial role of TLR signaling in HSE. MyD88 knockout (MyD88-KO) rendered mice highly susceptible to both HSV-1 and HSV-2 induced encephalitis [200-202]. However, focus on TLR2 and TLR9 due to their important role in vitro provided contradicting data. Several studies examined the effect of TLR2-KO, TLR9- KO and combined TLR2/9-KO on mouse mortality due to HSV-induced CNS inflammation.

While intracranial and intraperitoneal HSV-1 challenge of TLR2-KO mice indicated a detrimental contribution of TLR2 in HSE [196, 203], intranasal HSV-1 infection led to an unaltered survival rate in TLR2-KO mice compared to wildtype (wt) control [194, 200].

Furthermore, no impact on survival was detected in both TLR9-KO and TLR2/9-KO mice after intracranial HSV infection [203]. Contrastingly, intranasal virus inoculation led to the

(35)

opposite effect with increased mortality rates of TLR9-KO and TLR2/9-KO mice [194].

Finally, a HSV-2 mouse model of genital herpes with a genital infection route suggested a beneficial synergistic effect of TLR2 and TLR9 combined [202]. The different outcomes of these HSV infection studies in TLR-deficient mice are most likely related to the different virus inoculation routes. Moreover, these data illustrate once more that experimental mouse models should always be interpreted with great care. Conclusions drawn from these experiments cannot simply be extrapolated to the pathogenesis of the respective disease in humans. While the intranasal HSV-1 infection and genital HSV-2 administration more closely resemble the human infection routes, strain differences and different viral administration titers also contribute to different CNS manifestation phenotypes in mice and human. Additionally, other HSV mouse models exist that suggest a cell type- and timepoint-restricted TLR dependency and cooperation of different PRRs demonstrating a more complex HSV-induced immune response [181, 204, 205].

Altogether, both in vitro and in vivo studies showed that HSV recognition is a multifactorial process that involves different cell types, multiple TLRs and most likely the cooperation with other virus sensing mechanisms of the immune system.

4.3.2 Genetic predisposition for herpetic encephalitis

The discovery of several childhood HSE cases associated with genetic mutations in genes encoding TLR3 signaling proteins, revealed a potential genetic predisposition to develop HSE. In 2006, a study reported on two children with an autosomal recessive UNC-93B deficiency suffering from HSE [206]. UNC-93B is a transmembrane protein required for the trafficking of intracellular TLRs including TLR3 from the endoplasmic reticulum to the endosomal compartment [207]. Subsequent HSE cases revealed both dominant-negative and autosomal recessive TLR3 mutations that led to either a reduced ligand binding or complete loss of protein function, respectively [208, 209]. In all cases, the children showed no signs of other diseases and stimulation of their fibroblasts with HSV-1 or TLR3 agonist poly(I:C) induced impaired cytokine responses. Also, HSV-1 infection of their fibroblasts resulted in increased viral replication and cell death, while no abnormal reaction was detected in response to other viral infections [206, 208, 209]. Following studies identified more childhood HSE patients with mutations in TLR3 signaling cascade proteins: an autosomal-dominant TRAF3 mutation, autosomal dominant and recessive mutations in TRIF, heterozygous TBK1 mutations, and an autosomal dominant IRF3 deficiency [210-213] (for TLR3 pathway see Fig. 4). Fibroblasts from most of these patients showed a similar deficient phenotype as described above. Interestingly, in many cases family members with the same heterozygous mutations did not suffer from HSE despite being latently HSV-1 infected [208, 210, 211]. An epidemiologic survey of 85 children with HSE further confirmed that HSE is indeed a sporadic disease with no familial cases registered. However, in some cases, HSE might be the result of a Mendelian predisposition with incomplete clinical penetrance [214]. Moreover, a

Referenzen

ÄHNLICHE DOKUMENTE

In mice, activation of peripheral and intestinal CD8αα + and CD8αβ + T cells as well as intestinal CD4 + T cells with anti-CD3 in the presence of retinoic acid (RA) results

In alle Vertiefungen 100 µL Stopplösung in der gleichen Reihenfolge und mit den gleichen Zeitintervallen wie bei Zugabe der TMB-Substratlösung pipettieren, dadurch erfolgt

Therefore, the aims of this study were (i) to investigate and compare morphological features and functional properties of canine and murine SGCs and (ii) to analyze the

Key words: Varicella zoster virus, Human neurons, iPSC, ARPE19 cells, Metabolomics, Stable isotope labelling, Non - targeted, Mass isotopomer dis- tribution,

For the second part of the thesis, I investigated HSV1 induced changes in the cortical actin cytoskeleton during the first hours of infection (Koithan et al., in preparation-a),

Dieser Test kann nicht zwischen HSV-1, HSV-2 und VZV differenzieren, ist jedoch für eine schnelle Orientierung bezüglich einer möglichen antiviralen Therapie und

22 The higher incidence of oral HSV in adult patients undergoing chemotherapy than in the pediatric patients might be explained by the previous assumption

Two of the most revealing experiments in the recent past were done using a mouse model underscoring the importance and usefulness of its derivation. The phenotype of mice