Department of Small Animal Medicine and Surgery Center for Systems Neuroscience Hannover
Investigation of a Th17 skewed immune response and the potential role of the endocannabinoid system in the pathogenesis of canine Steroid-Responsive Meningitis-
Arteritis
Thesis
Submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy
(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Jessica Freundt Revilla Caracas, Venezuela
Hannover, Germany 2016
Supervisor: Prof. Dr. Andrea Tipold Supervision Group: Prof. Dr. Andrea Tipold
Prof. Wolfgang Baumgärtner, PhD Prof. Dr. Martin Stangel
1st Evaluation: Prof. Dr. Andrea Tipold
Department of Small Animal Medicine and Surgery University of Veterinary Medicine, Hannover, Germany Prof. Dr. Wolfgang Baumgärtner, PhD
Department of Pathology
University of Veterinary Medicine, Hannover, Germany Prof. Dr. Martin Stangel
Department of Neurology
Hannover Medical School, Hannover, Germany
2nd Evaluation: Prof. James Anderson School of Veterinary Medicine University of Glasgow, Scotland Date of final exam: 29.11.2016
Sponsorship: PhD studies of Jessica Freundt Revilla were funded by the German Academic Exchange Service (DAAD), and an “Abschluss-Stipendium” from the ZSN der Tierärztlichen Hochschule Hannover.
Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes
Parts of the thesis have been submitted to peer reviewed journals:
Jessica Freundt-Revilla, Arianna Maiolini, Regina Carlson, Martin Beyerbach, Kai Rentmeister, Thomas Flegel, Andrea Fischer, Andrea Tipold (2016). Th17 skewed immune response and cluster of differentiation 40 ligand in canine steroid-responsive meningitis- arteritis, a large animal model for neutrophilic meningitis. Journal of Neuroinflammation, under revision.
Jessica Freundt-Revilla, Franciska Heinrich, Alexander Zörner, Felix Gesell1, Martin Beyerbach, Merav Shamir, Anna Oevermann, Wolfgang Baumgärtner, Andrea Tipold (2016).
The potential role of endocannabinoid system in canine Steroid-Responsive Meningitis- Arteritis. Submitted to PLoS One.
Parts of the thesis have been presented at national and international scientific congresses:
As poster presentations:
J. Freundt-Revilla, A. A. Zörner, F. K. Gesell, M.H. Shamir, A. Tipold: “Endocannabinoids may influence the cell population in canine cerebrospinal fluid”. In: 26th Annual Symposium of ESVN and ECVN “Neuro-Emergency and Critical Care” 26-28.09.2013 in Paris, France.
Abstract published in Proceedings of the Symposium (2013).
J. Freundt-Revilla, R. Carlson, A. Maiolini, A.Tipold: “Interleukin-17 and CD40 ligand in canine Steroid-Responsive Meningitis-Arteritis”. In: 24. Jahrestagung der FG “Innere Medizin und klinische Labordiagnostik” der DVG (InnLab), 29-30.01.2016 in Berlin, Germany. Abstract published in Tierärztliche Praxis Kleintiere/Heimtiere Abstracts (2016) p.
17.
J. Freundt-Revilla, F. Heinrich, M.H. Shamir, A. Oevermann, W. Baumgärtner, A.Tipold:
“Cannabinoid receptor type 2 expression in canine Steroid-Responsive Meningitis-Arteritis and Intraspinal Spirocercosis”. In: 29th Annual Symposium of ESVN and ECVN “Applied Translational Neuroscience”, Edinburgh, Scottland, 16 and 17.09.2016. Abstract published in Proceedings of the Symposium (2016) p.44. Awarded with “Best ECVN Poster Award”.
As oral presentations:
J. Freundt-Revilla, A. A. Zörner, F. K. Gesell, M.H. Shamir, A. Tipold: “Endocannabinoids may influence the cell population in canine cerebrospinal fluid”. In: 22. Jahrestagung der FG
“Innere Medizin und klinische Labordiagnostik” der DVG (InnLab), 31.01-01.02.2014 in Gießen, Germany. Abstract published in Tierärztliche Praxis Kleintiere/Heimtiere Abstracts (2014) V01, A2.
J. Freundt-Revilla, R. Carlson, A. Maiolini, A.Tipold: “Interleukin-17 and CD40 ligand in canine Steroid-Responsive Meningitis-Arteritis”. In: 28th Annual Symposium of ESVN and ECVN “Movement disorders”, Amsterdam, Netherlands, 18 and 19.09.2015. Abstract published in Proceedings of the Symposium (2015) p.48
A. Tipold and J. Freundt-Revilla: “Does a Th17 skewed response in Steroid-Responsive Meningitis-Arteritis exists?” In: 2016 AVIM Forum, Denver, CO, USA, 8-11.06.2016.
Abstract published in Proceedings #23, p.42.
To my family: Mami, Papi, Lore and Tico
i
Table of content
List of abbreviations iii
List of figures and tables vii
1. Summary ix
2. Zusammenfassung xi
3. Aims of the study 1
4. General Introduction and Literature Review 3
4.1. Steroid-Responsive Meningitis-Arteritis 3
4.1.1. Background 3
4.1.2. Classification and clinical features 4
4.1.3. Etiopathogenesis 5
4.1.4. Role of cytokines 6
4.1.5. Diagnosis 9
4.1.6. Treatment and prognosis 9
4.1.7. Pathological findings 11
4.2. T helper cells 12
4.2.1. Th1/Th2 paradigm 12
4.2.2. Th17 subset 13
4.2.3. Interleukin 17 (IL-17) 14
4.2.4. Implications of Th17 and IL-17 in autoimmunity 15
4.3. Cluster of differentiation 40 ligand (CD40L) 15
4.4. Endocannabinoid system 16
4.4.1. Endocannabinoids (ECs) 16
4.4.2. Cannabinoid receptors (CBs) 19
4.4.3. Endocannabinoid system in neurotransmission 21
4.4.4. Endocannabinoid system in inflammation 22
5. Manuscript I: Th17 skewed immune response and cluster of differentiation 40 ligand expression in canine Steroid-Responsive Meningitis-Arteritis, a large
animal model for neutrophilic meningitis 25
5.1. Abstract 26
5.2. Background 27
5.3. Methods 29
5.4. Results 36
5.5. Discussion 43
ii
5.6. Conclusion 51
5.7. Declarations 52
5.8. References 54
5.9. Supplementary information 58
6. Manuscript II: The potential role of the endocannabinoid system in canine
Steroid-Responsive Meningitis-Arteritis 63
6.1. Abstract 64
6.2. Introduction 65
6.3. Materials and Methods 67
6.4. Results 73
6.5. Discussion 83
6.6. Conclusion 89
6.7. Declarations 90
6.8. References 91
6.9. Supplementary information 95
7. General Discussion 97
8. References 105
9. Acknowledgements 115
iii
List of abbreviations
ABC avidin-biotin-peroxidase complex AEA anandamide, arachidonoylethanolamide BBB blood brain barrier
BSA bovine serum albumin CB1 cannabinoid receptor type 1 CB2 cannabinoid receptor type 2 CBD Cannabidiol
CD cluster of differentiation
CD40L cluster of differentiation 40 ligand CDV canine distemper virus
CNS central nervous system CSF cerebrospinal fluid DAG 1.2-diacylglicerol
dL deciliter
DMSO dimethyl sulfoxide DRG dorsal root ganglia
EAE experimental autoimmune encephalomyelitis ECs endocannabinoids
EDTA ethylene diamine tetraacetic acid ELISA enzyme-linked immunosorbent assay ELISpot enzyme-linked immunospot assay et al. et alii
FAAH fatty acid amide hydrolase
Fig. figure
g gram
GABA gamma-aminobutyric acid
h hour
H2O2 hydrogen peroxide
iv HIV-1 Human immunodeficiency virus 1 IBD inflammatory bowel disease IE idiopathic epilepsy
IgA immunoglobulin A
IHC immunohistochemistry
IL interleukin
IVDH intervertebral disc herniation
KD Kawasaki Disease
Kg kilogram
M molar
mg milligram
MHC major histocompatibility complex
min minutes
mL milliliter
MRI magnetic resonance imaging
MUO meningoencephalitis of unknown origin
µg microgram
µl microliter
NAPE-PLD N-acylphasphatidylethanolamine-hydrolyzing phospholipase D
ng nanogram
nM nanomolar
OPC oligodendrocyte progenitor cells PBMCs peripheral blood mononuclear cells PBS phosphate-buffered saline
pM picomolar
RPMI Roswell Park Memorial Institute rSpear Spearman´s rank correlation coefficient SCI spinal cord injury
SFCs spot forming cells
v SRMA steroid-responsive meningitis-arteritis Tab. Table
TGF-ß1 transforming growth factor beta 1
Th T helper
THC ∆9 –tetrahydrocannabinol TLR Toll-like receptor
Treg regulatory T cells
TMB 3.3´.5.5´ Tetramethylbenzidine TLRs toll-like receptors
VEGF vascular endothelial growth factor 1AG 1-arachinodoyl glycerol
2AG 2-arachinodoyl glycerol
% percent
°C degree Celsius
vi
vii
List of figures and tables
(not including the ones presented in manuscripts)Figure 1: “Hunched posture” in a Boxer dog with SRMA in the acute phase 5 Figure 2: The role of cytokines in the immunopathology of SRMA 8
Table 1: Investigated cytokine expression in SRMA 8
Figure 3: T helper cells subsets 13
Figure 4: Chemical structure of anandamide (AEA), 1-arachidonoyl glycerol
(1AG) and 2-arachidonoyl glycerol (2AG) 19
Table 2: Main characteristic of CB1 and CB2 receptors 21
Figure 5: Endocannabinoid system dysregulation in neuroinflammation 103
List of supplementary figures and tables
Table S1: CSF and serum concentrations of IL-17 and CD40L 58 Table S2: Means and medians of wells triplicates of spot forming cells (SFCs)
counted for each patient at 2.5x105 cells/ml dilution 61 Table S3: Values of Anandamide (AEA) and total AG in CSF and serum 95
viii
ix
1. Summary
Investigation of a Th17 skewed immune response and the potential role of the endocannabinoid system in the pathogenesis of canine Steroid-Responsive Meningitis- Arteritis
Jessica Freundt Revilla
Steroid-Responsive Meningitis-Arteritis (SRMA) is an immune-mediated disorder, characterized by inflammatory stenotic lesions of the vessels particularly in the cervical meninges and a neutrophilic pleocytosis. Glucocorticosteroids play a major role in its treatment and in most cases a complete remission is achieved. However, relapses during or following therapy are frequent. In these patients a lasting improvement is not achieved and/or the required dosage of glucocorticosteroids leads to unacceptable severe side effects.
The aim of the current study was to increase the knowledge of the pathogenesis of SRMA that could lead to the application of new therapeutic approaches targeting specific aspects of the immune response. For this purpose, we evaluated a possible Th17 skewed immune response and the potential role of the endocannabinoid system in the pathogenesis of SRMA. Both responses are described to interact with each other.
In the first part of the study, we focused on evaluating the role of IL-17 producing Th17 lymphocytes in the pathogenesis of SRMA, since in former studies increased levels of IL-6 and TGF-ß1 in the cerebrospinal fluid (CSF) of dogs with SRMA were found. Both proteins are essential for the differentiation of naïve CD4+ cells to become Th17 cells. We proved the hypothesis that SRMA is associated with a Th17 skewed immune response using an Enzyme-Linked Immunosorbent Assay (ELISA) for measurement of IL-17 in CSF and serum and an Enzyme-Linked ImmunoSpot (ELISpot) assay to confirm IL-17 production on a
x
single cell level. Markedly increased intrathecal levels of IL-17 in patients with SRMA in the acute stage and during relapses were detected. Moreover, IL-17 CSF levels showed a strong positive correlation with the degree of pleocytosis suggesting that IL-17 might be involved in the massive migration of neutrophils in the CSF and the induction of vascular damage. These findings add to the knowledge of the characterization of the cytokine profile of SRMA and open possible novel therapeutic strategies targeting specific cytokine modulation.
Additionally, increased levels of soluble CD40L were measured in CSF of patients with SRMA in the acute phase and during relapses. Such elevated concentrations may be one of the causes for the vasculitis and the inflammation of the meninges and may open new treatment possibilities to be considered in the management of vasculitis.
The second part of this study focused on investigating the influence of the endocannabinoid system in SRMA. Anandamide was shown to interact with Th17 cells in mice and a similar reaction might occur in dogs. Therefore, the described two mechanisms should be evaluated in the current study. Many autoimmune diseases, like SRMA, show spontaneous exacerbations and remissions, suggesting an unstable relationship between positive and negative regulatory mechanisms. The endocannabinoid system has been proven to have an endogenous protective response attempting to control inflammation. However, a later dysregulation results in exacerbation of inflammation. The two main endocannabinoids, Anandamide (AEA) and 2-arachidonoyl glycerol (2AG), were quantified by mass spectrometry in CSF and serum samples. Our findings show increased levels of endocannabinoids (AEA and 2AG) in patients with SRMA in the acute phase compared with healthy controls and SRMA patients under treatment. Moreover, cannabinoid receptor type 2 (CB2) expression was demonstrated in inflammatory lesions of SRMA. CB2 was strongly expressed on the cellular surface of infiltrating leukocytes, highlighting the endocannabinoid system as an additional potential target for treatment of inflammatory CNS diseases.
xi
2. Zusammenfassung
Th17 dominierte Immunantwort und Einfluß von Endocannabinoiden in der Pathogenese der steril-eitrigen Meningitis-Arteriitis
Jessica Freundt Revilla
Die steril-eitrige Meningitis-Arteriitis (SRMA) ist eine immun bedingte Erkrankung, die sich vornehmlich durch entzündliche Stenosen der Gefäße im Bereich der zervikalen Meningen sowie eine neutrophile Pleozytose manifestiert. Glukokortikoide spielen eine wichtige Rolle in der Behandlung und in den meisten Fällen wird eine komplette Remission erreicht. Allerdings finden häufig Rezidiven während oder nach der Therapie statt. Bei diesen Patienten wird eine nachhaltige Verbesserung nicht erreicht und/oder die erforderliche Dosierung von Glukokortikoiden führt zu inakzeptablen schweren Nebenwirkungen.
Das Ziel der vorliegenden Studie war, den wissenschaftlichen Kenntnisstand der Pathogenese von SRMA zu erweitern, um neue therapeutische Ansätze in bestimmten Aspekten der Immunantwort zu erschließen. Hierzu untersuchten wir eine eventuell Th17 dominierte Immunantwort und die Rolle des Endocannabinoidsystems in der Pathogenese von SRMA.
Der erste Teil der Studie konzentrierte sich auf die Rolle von IL-17 produzierenden Th17-Lymphozyten in der Pathogenese der SRMA. In früheren Studien wurden erhöhte Konzentrationen von IL-6 und TGF-ß1 im Liquor cerebrospinalis von Hunden mit SRMA gefunden. Beide Proteine sind wichtig für die Differenzierung von naiven CD4+ Zellen in Th17 Zellen. Wir konnten die Hypothese beweisen, dass SRMA mit einer Th17 Immunantwort verbunden ist. Mittels enzyme-linked immunosorbent assay (ELISA) wurde IL-17 in Liquor und Serum untersucht und durch einen ELISPOT assay konnte die
xii
Produktion von IL-17 auf Einzelzellebene bestätigt werden. In diesen Untersuchungen fielen deutlich erhöhte intrathekale Konzentrationen von IL-17 bei Patienten mit SRMA in der akuten Phase und während eines Rezidivs auf. IL-17 Konzentrationen im Liquor cerebrospinalis zeigten zudem eine starke positive Korrelation mit dem Grad der Pleozytose.
Dies deutet darauf hin, dass IL-17 an der massiven Migration von neutrophilen Granulozyten in den Liquor und der Induktion von Gefäßschäden beteiligt sein könnte. Diese Ergebnisse ergänzen den Kenntnisstand über die Charakterisierung des Zytokinprofils von SRMA und erschließen neue therapeutische Strategien für spezifische Zytokin-Modulationen. Zusätzlich wurden erhöhte Konzentrationen von löslichem CD40L in Liquorproben von Patienten mit SRMA in der akuten Phase und während eines Rezidivs gemessen. Solche erhöhten Konzentrationen können ursächlich an der Pathogenese der Vaskulitis und Meningitis beteiligt sein, was ebenfalls neue Behandlungsmöglichkeiten eröffnen kann.
Der zweite Teil dieser Studie beschäftigte sich mit dem Einfluss des Endocannabinoid-Systems in SRMA. Endocannabinoide können vermutlich mit Th17-Zellen interagieren. Daher sollten beide Systeme in dieser Arbeit untersucht werden. Viele Autoimmunerkrankungen, wie SRMA, zeigen spontane Exazerbationen und Remissionen, welche auf eine instabile Beziehung zwischen positiven und negativen Regulationsmechanismen hindeuten. Das Endocannabinoid-System entwickelt eine gewisse endogene Schutzreaktion, um Entzündungen zu kontrollieren. Doch eine spätere Dysregulation dieses Systems resultiert in einer Verschlimmerung der Entzündung. Die beiden wichtigsten Endocannabinoide, Anandamid (AEA) und 2-arachidonoyl glycerol (2AG) wurden durch Massenspektrometrie in Liquor und Serum-Proben quantifiziert. Die Werte dieser Endocannabinoide (AEA und 2AG) waren bei Patienten mit SRMA in der akuten Phase im Vergleich zur gesunden Kontrollgruppe und SRMA Patienten unter Glukokortikosteroidbehandlung deutlich erhöht. Darüber hinaus wurden CB2-Rezeptor-
xiii
Expressionen in entzündlichen Läsionen von SRMA mittels Immunhistochemie demonstriert.
CB2 wurde vor allem an der Zelloberfläche von infiltrierenden Leukozyten exprimiert. Beide Ergebnisse heben die Bedeutung des Endocannabinoidsystems als potentielles Ziel für die Behandlung von entzündlichen ZNS-Erkrankungen hervor.
xiv
1 3.
Aims of the study
The main goal of this study was to further analyze the pathogenesis of Steroid-Responsive Meningitis-Arteritis (SRMA) and evaluate a possible Th17 skewed immune response.
Moreover, we aimed to evaluate the potential role of the endocannabinoid system in the pathogenesis of SRMA.
In the first part of the current study, the possible role of Th17 lymphocytes in the pathogenesis of SRMA should be evaluated. In former studies increased intrathecal production of IL-6 and TGF-ß1 let suggest that Th17 lymphocytes occur in SRMA (Maiolini et al. 2013). To prove this hypothesis, we measured IL-17 levels in cerebrospinal fluid (CSF) and serum in dogs in three different stages of the disease and analyzed IL-17 synthesis by peripheral mononuclear cells and compared it to positive and negative controls.
In the second part of the study, we evaluated the involvement of the endocannabinoid system in the pathogenesis of SRMA. For this purpose, we measured two frequently occurring and bioactive endocannabinoids (Anandamide and 2-arachidonoyl glycerol) in CSF and serum of patients in various stages of the disease. Since Anandamide and 2-arachidonoyl glycerol seem to be involved in the inflammatory response in SRMA the receptors should be evaluated in the central nervous system (CNS) using immunohistochemical analyses.
Cannabinoid receptor type 2 (CB2) was stained in the canine spinal cord of healthy dogs and dogs with SRMA to provide novel insights for further characterization under pathophysiological conditions and the development of new treatment strategies.
2
3
4. General introduction and Literature Review
4.1. Steroid-Responsive Meningitis-Arteritis 4.1.1. Background
Steroid-Responsive Meningitis-Arteritis (SRMA) is the most frequently diagnosed inflammatory central nervous system (CNS) disorder presented in dogs (Fluehmann et al.
2006) and the most common canine meningitis (Meric 1988). It is a systemic immune mediated disorder characterized by inflammatory stenotic lesions of the vessels specially in the cervical meninges (De Lahunta and Glass 2009). It affects mainly juvenile to young adult dogs, and although dogs from 3 months (Harcourt 1978) to 9 years of age may be affected (Cizinauskas et al. 2000), the age of onset is typically between 6 and 18 months (Tipold and Schatzberg 2010). SRMA may affect any breed, however, a predisposition for Beagles, Boxers, Bernese mountain dogs (De Lahunta and Glass 2009; Tipold and Jaggy 1994), Weimaraners, Nova Scotia duck tolling retrievers (Tipold and Schatzberg 2010) and Petit Basset Griffon Vendéen (Vos et al. 2012) was shown; no sex predisposition has been reported so far (Lowrie et al. 2009a).
SRMA was initially reported in young laboratory Beagles (Hayes et al. 1989) and through the years several terms have been used referring to the main clinical and pathological expressions: “Polyarteritis” (Harcourt 1978), “Necrotizing Vasculitis” (Brooks 1984; Scott- Moncrieff et al. 1992), “Beagle Pain Syndrome” (Hayes et al. 1989), “Corticosteroid- Responsive meningomyelitis” (Irving and Christian 1991), “Canine Pain Syndrome” (Burns et al. 1991) and “Canine Juvenile Polyarteritis Syndrome” (Felsburg et al. 1992; Hogenesch et al. 1995) and “Aseptic Suppurative Meningitis” (Behr and Cauzinille 2006). However, Steroid-Responsive Meningitis-Arteritis (SRMA) is the term that describes the pathological
4
and clinical characteristics and treatment of choice for this syndrome. This term is most commonly used in the literature (Tipold and Schatzberg 2010).
4.1.2. Classification and clinical features
Two forms of SRMA have been identified, an acute “classic” form and a chronic
“protracted” form (Tipold and Schatzberg 2010). The most important clinical signs observed in the acute phase of the disease are pyrexia, cervical hyperesthesia, reluctance to move and a stiff gait (De Lahunta and Glass 2009; Tipold and Jaggy 1994). Because of the extreme pain in the neck a “hunched posture” might be even manifested (Figure 1) (Tipold and Schatzberg 2010). In most cases the neurological examination is unremarkable beside the manifestation of pain. However, a decreased menace reaction or slight deficits in postural reactions may be found (Tipold and Jaggy 1994). The signs may show a waxing and waning course (Lowrie et al. 2009a). On the other hand, in the protracted form further neurological deficits may appear such as gait abnormalities, pacing and tetra- or paraparesis, reflecting the extension of the inflammation to the CNS parenchyma (Tipold and Jaggy 1994). In rare cases an involvement of the cranial nerves (Tipold and Jaggy 1994) and of the brain parenchyma leading to seizures may occur (Wrzosek et al. 2009).
5
Figure 1: “Hunched posture” in a Boxer dog with SRMA in the acute phase. Patients in the acute phase of SRMA may present with a “hunched posture” because of extreme pain in the cervical region. Further clinical signs observed in this phase are pyrexia, reluctance to move and stiff gait. The Boxer is a predisposed breed for SRMA.
4.1.3. Etiopathogenesis
Despite years of substantial research, the etiopathogenesis of SRMA remains vague (Tipold and Schatzberg 2010), nontheless, a genetic predisposition has been described in Nova Scotia duck tolling retrievers (Wilbe et al. 2010). Up to now, no infectious or neoplastic triggers have been consistently confirmed (Cizinauskas et al. 2000; Harcourt 1978; Lazzerini et al. 2015; Meric et al. 1985; Rose and Harcourt-Brown 2013; Scott-Moncrieff et al. 1992;
Tipold and Jaggy 1994). However, the pathogenetic factors that contribute to inflammatory cell influx in SRMA have been studied in detail (Spitzbarth et al. 2012). Upregulation of the integrin CD11a has been demonstrated on CSF leukocytes in dogs in the acute phase using flow cytometric analysis. CD11a may be involved in leukocyte recruitment to the CNS and might be a key factor for neutrophil recruitment to the subarachnoidal space (Schwartz et al.
2008a). Moreover, upregulation of metalloproteinases 2 and 9 has been demonstrated using PCR in patients with SRMA and is thought to be involved in the disruption of the blood brain barrier (BBB) and to contribute to the neutrophilic pleocytosis (Schwartz et al. 2010).
6
High expression of Toll-like receptors (TLRs) 4 and 9 were found in peripheral blood monocytes of dogs with SRMA in the acute stage (Maiolini et al. 2012a), being involved in the inflammatory process and enhancing the autoimmune reaction (Maiolini et al. 2012a).
Concentration of vascular endothelial growth factor (VEGF) was elevated in CSF of SRMA patients, having a potential role in the development of the systemic arteritis (Maiolini et al.
2013).
Strongly increased IgA levels have been found in serum and CSF of dogs with SRMA in the acute phase and under treatment with glucocorticoids (Felsburg et al. 1992; Maiolini et al. 2012b; Tipold et al. 1994; Tipold and Jaggy 1994), and recently a sensitivity of 91% for IgA concentrations in serum and CSF as a diagnostic marker for SRMA was reported (Maiolini et al. 2012b).
4.1.4. Role of cytokines
Since neutrophilic pleocytosis and elevated IgA levels represent the key features of SRMA, several studies aimed to analyse the molecular factors mediating such responses (Spitzbarth et al. 2012). Enhanced chemotactic activity of the CSF for peripheral mononuclear cells and neutrophilic granulocytes was shown in patients with SRMA compared to healthy controls (Burgener et al. 1998). Moreover, chemotactic factors including IL-8 correlate with IgA values (Burgener et al. 1998) and both remain elevated in dogs with relapses (Tipold and Schatzberg 2010).
Interestingly, SRMA affected dogs show higher number of helper CD4+ T cells than cytotoxic CD8+ T cells in the peripheral blood (Schwartz et al. 2008b). This predominance of T helper (Th) lymphocytes indicates that in SRMA patients a humoral immune response is present, occurring typically to eliminate extracellular pathogens or in autoimmune diseases (Schwartz et al. 2008b). Expression of several cytokines in peripheral blood mononuclear
7
cells (PBMCs) and CSF leukocytes were investigated in patients in the acute phase of SRMA (Schwartz et al. 2011). Increased levels of IL-4 were found and compared to control dogs, dogs with other CNS diseases and dogs with different inflammatory disorders (Schwartz et al.
2011). Moreover, IL-5 and IL-10 were elevated compared to controls; however, no difference was found when compared to other CNS inflammatory diseases (Schwartz et al. 2011). On the other hand, IL-2 and IFN-γ were downregulated in PBMCs of dogs with SRMA (Schwartz et al. 2011). Down regulation of Th1-related cytokines (IL-2 and IFN-γ) and increased levels of IL-4 lead to the assumption that SRMA is caused by a Th2 polarized immune response (Schwartz et al. 2011), leading to an upregulation of the humoral immune response and excessive IgA production (Schwartz et al. 2011).
Maiolini and others found increased levels of IL-6 and TGF-ß1 in CSF of canines suffering from SRMA. A combined intrathecal increase of these proteins could induce CD4+
progenitors to differentiate into Th17 subset and enhance the autoimmune response (Maiolini et al. 2013). Indeed, in the current study we could find increased levels of IL-17 in CSF samples of patients suffering SRMA in the acute phase and during relapses (Freundt-Revilla et al. 2016). Such intrathecal IL-17 synthesis may induce disruption of the BBB and the massive infiltration of neutrophils to the subarachnoidal space (Freundt-Revilla et al. 2016).
The cytokine expression investigated so far in SRMA is summarized in Table 1.
The role of cytokines in the immunopathology of SRMA is summarized in Figure 2.
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Figure 2: The role of cytokines in the immunopathology of SRMA. Upregulation of IL-4 leads to a Th2 polarization resulting in massive secretion of IgA by plasma cells. While up- regulation of IL-8 expression and integrin CD11a are involved in the massive infiltration of neutrophils to the subarachnoid space. High levels of IL-6 and TGF-ß lead to Th17 differentiation secreting IL-17 which is involved in the recruitment of neutrophils and together with IL-6 in BBB disruption and leakage of the vessel wall (Modified from Spitzbarth et al. 2012).
Table 1: Investigated cytokine expression in SRMA. (Modified from Spitzbarth et al.
2012).
Cytokines Downregulated Upregulated Tissue Reference
TNF, IL-6 IL-6 Serum Hogenesch et al. (1995)
IL-8 IL-8 CSF Burgener et al. (1998)
IL-2, IL-4, IL-5,
IL-10, IFN-γ IL-2, IFN-γ IL-4, IL-5,
IL-10 CSF, PBMCs Schwartz et al. (2011) TGF-ß, IL-6 TGF-ß, IL-6 CSF, Serum Maiolini et al. (2013)
IL-17, IFN-γ IFN-γ IL-17 CSF, Serum,
PBMCs
Freundt-Revilla et al.
(2016)
Th17 CD4
CD4
Th2 Th17
IL-17 IL-6
TGF-ß IL-5 IL-10 IL-4
IL-4 IL-5 IL-10
B
IgA
IgA
IL-6
IL-8
Blood-brain barrier CD11a
CD11a
CD11a Th2
Plasma cell
Neutrophils Neutrophils
Plasma cell Subarachnoidal space
9 4.1.5. Diagnosis
In the absence of a definitive diagnostic test, SRMA is diagnosed based on gathering clinical and laboratory findings: history of acute cervical pain, presentation of fever and stiffness, an inflammatory leucogram (Hayes et al. 1989), high protein levels and pleocytosis in the CSF (Meric et al. 1985), and increased serum and CSF concentrations of acute phase proteins (APPs) may help establishing the diagnosis (Bathen-Noethen et al. 2008; Lowrie et al. 2009b). However, the most precise diagnostic indicator so far is a massive increase in serum and CSF IgA titers (Felsburg et al. 1992; Maiolini et al. 2012b; Tipold et al. 1994;
Tipold and Jaggy 1994). Recently, a sensitivity of 91% for IgA concentrations in paired serum and CSF samples as a diagnostic marker for SRMA was reported (Maiolini et al.
2012b). Finally, the exclusion of other diseases causing fever, cervical pain and stiffness is mandatory (Lowrie et al. 2009a).
Analysis of the CSF in the acute phase is characterized by a neutrophilic pleocytosis and increased protein levels (Tipold 1995), while in the protracted form, CSF analysis may be normal or may show a mild to moderate mononuclear or mixed pleocytosis with normal or slightly increased total protein (Lowrie et al. 2009a; Tipold and Jaggy 1994).
4.1.6. Treatment and prognosis
Glucocorticoids play a major role in the treatment of SRMA (Cizinauskas et al. 2000).
Glucocorticosteroids inhibit vasodilation and increased vascular permeability (Perretti and Ahluwalia 2000), decrease leukocyte migration into inflamed sites (Perretti and Ahluwalia 2000), and repress transcription of many genes encoding pro-inflammatory cytokines, chemokines, cell adhesion molecules and key enzymes involved in the initiation and maintenance of the inflammatory response (Barnes 1998; Coutinho and Chapman 2011). The
10
mechanisms of action of glucocorticoids are, however, unspecific and are associated with potentially serious side effects (Meier 1996; Whitley and Day 2011).
In most cases, management of the clinical signs and a complete remission is achieved in SRMA patients with long-term treatment with prednisolone (Tipold and Schatzberg 2010).
The accepted treatment regime for a minimum of 6 months consists of:
- Prednisolone or Prednisone 4 mg/kg/day for two days.
- Prednisolone or Prednisone reduced to 2 mg/kg/day for one or two weeks.
- Prednisolone or Prednisone reduced to 1 mg/kg/day.
(Cizinauskas et al. 2000)
Every four to six weeks dogs are re-examined clinically and with laboratory techniques (CSF, hematology and/or acute phase proteins). When clinical signs and CSF values are normal, prednisolone is reduced to half until a dose of 0.5 mg/kg every 48 to 72 hours is given (Tipold and Schatzberg 2010). Untreated dogs typically show a relapsing and remitting disease course (Tipold and Schatzberg 2010).
In refractory cases immunosuppressive drugs are used in combination with glucocorticoids (Biedermann et al. 2016; Tipold and Schatzberg 2010). The most common being azathioprine at 1.5 mg/kg every 48 hours (Tipold 2000). Unfortunately, relapses during or following therapy seem to be frequent (Biedermann et al. 2016). Relapse rates may vary depending on the treatment protocol used and on the owner compliance (Biedermann et al.
2016). Relapse rates of 16% (Bathen-Noethen et al. 2008), 20% (Lowrie et al. 2009a), and recently 34% (Biedermann et al. 2016) have been reported. In these patients a lasting improvement is not achieved, relapses are persistent (Biedermann et al. 2016) and/or unacceptable side effects appear (Whitley and Day 2011). Such relapses might occur due to glucocorticoid resistance, but the true pathogenesis in canines is unknown (Whitley and Day
11
2011). In humans, up to 30% of patients treated for inflammatory diseases develop glucocorticoid resistance (Whitley and Day 2011). Hence, novel therapeutic strategies targeting specific aspects of the immune response are practiced in human medicine and should be considered for dogs.
4.1.7. Pathological findings
The most consistent pathological findings are inflammatory stenotic lesions of meningeal arteries and infiltration of inflammatory cells into the meninges (Meric et al.
1985). Macroscopically, leptomeningeal hemorrhages, meningeal plaques (Tipold and Schatzberg 2010) and/or enlarged thickened vessels (Tipold et al. 1995) may be found.
Histopathological examination in the acute stage reveals an extensive suppurative leptomeningitis involving the meninges of the CNS, most severely in the cervical region, with invasion of macrophages, plasma cells, lymphocytes and polymorphonuclear cells (Meric 1988; Tipold et al. 1995). The vascular lesions may range from histiocytic-lymphocytic periarterial infiltration to transmural arterial inflammation with fibrinoid necrosis and vascular thrombosis involving mostly small- to medium-sized muscular arteries of the heart, cranial mediastinum, and cervical spinal meninges (Scott-Moncrieff et al. 1992; Snyder et al. 1995).
Lesions in the meningeal arteries consist of swelling of the endothelial cells, marked swelling of the nuclei of the tunica muscularis, and hyaline degeneration (Tipold et al. 1995). The most severely affected vessels show necrosis of the wall components, rupture of the elastic lamina and perivascular accumulation of inflammatory cells (Snyder et al. 1995). Massive periarterial accumulations of inflammatory cells are common and often extended into adjacent tissues (Snyder et al. 1995). Vasculitis is more common in the leptomeninges of the spinal cord than around the brain (Tipold and Schatzberg 2010). Nonetheless, vasculitis occasionally occurs in other organ systems (Snyder et al. 1995).
12
In the chronic form, inflammatory infiltration in the meninges is milder and marked fibrous thickening and focal mineralization of the leptomeninges are present (Tipold et al.
1995; Tipold and Schatzberg 2010). Furthermore, nerve root degeneration (Tipold and Schatzberg 2010) and rarely spinal cord infarction, secondary to vessel rupture and hemorrhage may occur (Hoff and Vandevelde 1981). However, acute and chronic vascular lesions may be found in the same section (Snyder et al. 1995), revealing the relapsing nature of SRMA.
4.2. T helper cells
4.2.1. Th1/Th2 paradigm
Lymphocytes induce and regulate immune responses in vertebrates (Bettelli et al.
2008). Naïve CD4+ T cells can differentiate into various subsets of T helper cells depending on the cytokine environment present at the time of the initial engagement of their T-cell receptor in the peripheral immune compartment (Figure 3) (Bettelli et al. 2008; Vojdani and Lambert 2011a). Moreover, for each T helper differentiation program, specific transcription factors have been identified as master regulators (Bettelli et al. 2008). Initially, Th differentiation was proposed to be divided in two subpopulations based on their cytokine expression profiles: Th1 and Th2 (Mosmann and Coffman 1989). Naïve CD4+ cells in the presence of IL-12 and transcription factors T-bet and STAT4 become Th1 cells (Vojdani and Lambert 2011a), which synthetize IFN-γ and mediate protection against intracellular pathogens. On the other hand, in the presence of IL-4 and transcription factors GATA-3 and STAT6, naïve CD4+ cells become Th2 cells (Vojdani and Lambert 2011a), which synthetize IL-4, IL-13 and IL-25 and moderate the clearance of extracellular pathogens (Bettelli et al.
2008). The Th1/Th2 paradigm was challenged following the discovery of a third subset of Th cells, known as Th17 cells (Langrish et al. 2005). In the presence of TGF-ß, IL-23, IL-6 and transcription factor RORγt, naïve CD4+ cells become Th17, which synthetize IL-17 and are
13
potent inducers of autoimmunity and tissue inflammation (Bettelli et al. 2008). Finally, in the presence of TGF-ß and transcription factor FOXP3, naïve T cells become Treg cells (Bettelli et al. 2008) (Figure 3).
Figure 3: T helper cells subsets. Naïve CD4+ T cells can differentiate into various subsets of T helper cells (Th1, Th2 and Th17) depending on the cytokine environment present at the time of the initial engagement of their T cell receptor and costimulatory receptors in the peripheral immune compartment (Modified from Bettelli et al. 2008; Leung et al. 2010).
4.2.2. Th17 subset
Th17 cells constitute a new linage of CD4+ T cells which seem to have a role in the activation of neutrophils and the immunity to bacteria, particularly in mucosal surfaces, through the synthesis of IL-17 (Stockinger et al. 2007; Vojdani and Lambert 2011a). Th17 cells require both TGF-ß1 and IL-6 for their development (Bettelli et al. 2006; Mangan et al.
2006) and in humans IL-23 is essential for the maturation of inflammatory Th17 cells (Cua et al. 2003). It has been shown that IL-6 deficient mice fail to develop a Th17 response and are
Treg FOXP3
Th2 GATA3
Th1 T-bet
Tnaive
Th17 RORуt Immunity
Tolerance
Clearance of
intracellular pathogens Immunopathology Autoimmunity
Clearance of
extracellular pathogens Allergy
Atopy
Clearance of certain extracellular pathogens Tissue inflammation Immunopathology Autoimmunity TGF-ß
TGF-ß + IL-6 IL-4 IL-12
Immunoregulation
(Peripheral tolerance) IL-17
IL-22 IL-4 IL-25 IL-13 IFN-γ
14
resistant to the development of experimental autoimmune encephalomyelitis (EAE) (Bettelli et al. 2006; Korn et al. 2007) and collagen-induced arthritis (Alonzi et al. 1998).
4.2.3. Interleukin 17 (IL-17)
IL-17 has been designated IL-17A to indicate that it is the founding member of the IL- 17 cytokine family, consisting of IL-17A-F members (Korn et al. 2009; Moseley et al. 2003), all of them playing an active role in inflammatory response and in autoimmune diseases (Kolls and Linden 2004). IL-17 is a pro-inflammatory cytokine secreted primarily by activated T-cells (Yao et al. 1995), which acts on the IL-17 receptor (IL-17R) expressed in cells of all tissues examined to date (Moseley et al. 2003). Although T-cells are the major source for IL-17 (Ferretti et al. 2003; Kebir et al. 2007; Kolls and Linden 2004; Korn et al.
2009; Vojdani and Lambert 2011a), IL-17 is also produced by a variety of cell types like neutrophils (Ferretti et al. 2003), eosinophils (Molet et al. 2001) and monocytes (Zhou et al.
2005). The activation of IL-17R results generally in the induction of other pro-inflammatory cytokines (Moseley et al. 2003). IL-17 has very strong pro-inflammatory effects on many cellular targets, including fibroblasts, epithelial cells, endothelial cells, monocytes/macrophages, keratinocytes and osteoclasts (Weaver et al. 2007). Moreover, IL-17 induces the release of IL-6 and other cytokines that trigger an inflammatory reaction characterized by neutrophil influx (Hellings et al. 2003) and promotes granulopoiesis (Chen et al. 2003). Experimental studies showed that overexpression of IL-17 in the joint space of mice with collagen-induced arthritis leads to increased neutrophil recruitment (Lubberts et al.
2001). Interestingly, the mechanisms by which IL-17 disrupts the tight junctions between endothelial cells of the blood brain barrier (BBB) were recently demonstrated and the ability of Th17 cell to transmigrate efficiently across the BBB was proven (Kebir et al. 2007).
Expression of IL-17 and IL-22 receptors on endothelial cells of the BBB results in the binding of Th17 cells to BBB tight junctions which causes its disruption and allows Th17 cells to
15
transmigrate across the BBB (Vojdani and Lambert 2011b). Furthermore, IL-17 enhances inflammatory cytokine production by microglia and even its synthesis by microglia and astrocytes has been proven (Kawanokuchi et al. 2008).
4.2.4. Implications of Th17 and IL-17 in autoimmunity
IL-17 producing cells have an important role in the development of several autoimmune diseases in humans such as systemic lupus erythematosus (Shah et al. 2010), rheumatoid arthritis (Chabaud et al. 1999), bronchial allergy (Hellings et al. 2003), inflammatory bowel disease (Zhang et al. 2006), multiple sclerosis (Brucklacher-Waldert et al. 2009) and Kawasaki disease (Guo et al. 2015) and also in experimental models such as collagen-induced arthritis (Lubberts et al. 2004) and experimental autoimmune encephalomyelitis (EAE) (Cua et al. 2003). Recently, IL-17 producing cells were found in inflamed tissues of several chronic idiopathic disorders in dogs including inflammatory bowel disease, gingivitis, chronic idiopathic rhinitis and chronic dermatoses (Kol et al. 2016).
Increased levels of IL-17 were found in dog brain tissues with granulomatous meningoencephalomyelitis (GME) (Park et al. 2013). In a study performed in 2013 by Rancan et al., high plasma concentrations of IL-17 were found in dogs with brachycephalic airway obstruction syndrome (BAOS) and the values appeared to be associated with disease severity (Rancan et al. 2013).
4.3. Cluster of differentiation ligand (CD40L)
The CD40 Ligand or CD154 is a transmembrane glycoprotein from the tumor necrosis factor α (TNF α) family primarily expressed on the surface of activated CD4+ T cells which interacts with the CD40 receptor expressed principally on B cells but also in other cells (Cron 2003). Activated T cells not only express CD40L in their membranes but also secrete a soluble form of CD40L, which is biologically active and interacts also with the CD40
16
receptor (Graf et al. 1995). Triggering of CD40–CD40L interaction initiates multiple signaling cascades that lead to the release of key pro-inflammatory mediators (Ramirez et al.
2010). The CD40 receptor is expressed by vascular endothelial cells and its activation by CD40L leads to leukocyte adhesion (Hollenbaugh et al. 1995). Moreover, high levels of soluble CD40L can regulate CNS inflammation at the BBB (Ramirez et al. 2010). CD40 upregulation in the brain vasculature has been reported in neuroinflammation in patients with Human immunodeficiency virus-1 (HIV-1) encephalitis (Ramirez et al. 2010). It is possible that secreted soluble CD40L in the brain exacerbates and perpetuates inflammatory responses by acting on and upregulating CD40 in the brain endothelium (Ramirez et al. 2010). Recently, increased expression of CD40L in CD4+ T cells as well as soluble CD40L was found in patients with Kawasaki disease (KD) and the levels correlated with coronary artery lesions (C.
L. Wang et al. 2003). Most importantly, intravenous immunoglobulin therapy seemed to downregulate CD40L expression and vascular damage (C. L. Wang et al. 2003). KD has striking similarities with SRMA (Burns et al. 1991; Felsburg et al. 1992). Indeed, in the current study, we found increased levels of soluble CD40L in CSF of patients with SRMA in the acute phase and during relapses, such elevated concentrations may be involved in the inflammation of the meninges (Freundt-Revilla et al. 2016).
4.4. Endocannabinoid system 4.4.1. Endocannabinoids (ECs)
Endocannabinoids (ECs) are endogenous lipid transmitters that mimic the actions of ∆9–
tetrahydrocannabinol (THC) by binding and activating specific G-protein receptors known as
“CB1 and CB2 cannabinoid receptors” (Battista et al. 2012). These endogenous agonists of CB receptors include amides, esters, and ethers of long chain polyunsaturated fatty acids (Battista et al. 2012), specifically arachidonic acid, (Di Marzo et al. 2004) that are generally considered to be synthesized and released from cells immediately after biosynthesis “on
17
demand” (Battista et al. 2012; Muccioli 2010). No evidence exists for their storage in secretory vesicles, and several of their biosynthetic enzymes are found in the plasma membrane (Di Marzo 2008).
Five endogenous mammalian substances acting on cannabinoid receptors (CB1 and CB2) have been identified so far (Pacher et al. 2006). These called “endocannabinoids” are arachidonoyl ethanolamide (Anandamide, AEA) (Devane et al. 1992), 2-arachidonoyl glycerol (2AG) (Mechoulam et al. 1995; Sugiura et al. 1995), arachidonoyl ethanolamine (Virodhamine, O-AEA) (Porter et al. 2002), 2-arachidonyl glycerol ether (Noladin ether, 2AGe) (Hanus et al. 2001) and N-arachidonoyl dopamide (NADA) (Bisogno et al. 2000).
The most bioactive ECs and the best studied ones are AEA and 2AG (Battista et al.
2012; Di Marzo et al. 2004; Muccioli 2010). Anandamide was firstly identified in the porcine brain as the first endogenous ligand of the CB1 receptor (Devane et al. 1992), although it binds to both CB1 and CB2 receptors (Glass and Northup 1999). Anandamide is the amide component of arachidonic acid and ethanolamine (Cabral and Griffin-Thomas 2009). Several competing pathways have been described for the biosynthesis of AEA. The best reported one describes AEA synthesis by postsynaptic neurons following membrane depolarization and Ca2+ influx into the cell (Di Marzo et al. 1994). Calcium activates N- acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) forming AEA, which acts as a retrograde messenger molecule to modulate neurotransmitter release from CB1 expressing presynaptic terminals (Di Marzo et al. 1994). Finally, it is inactivated through re-uptake and fatty acid amide hydrolase (FAAH) degradation mechanisms in astrocytes and neurons (Deutsch and Chin 1993; Di Marzo et al. 1994). 2AG was firstly isolated from canine intestine, being the first endocannabinoid isolated from a peripheral tissue (Mechoulam et al.
1995). However, 2AG was also isolated from rat brain (Sugiura et al. 1995). 2AG is an ester derivative of arachidonic acid and glycerol and is synthesized from the hydrolysis of 1.2-
18
diacylglycerol (DAG) by a DAG lipase (Cabral and Griffin-Thomas 2009). Interestingly, 2AG is more bioactive and abundant than AEA in the brain (Cabral and Griffin-Thomas 2009). In general, levels of 2AG in unstimulated tissues and cells are usually much higher than those of AEA and are principally sufficient to permanently activate both cannabinoid receptors (Stella et al. 1997; Sugiura et al. 1995). However, the main reason for such high concentration is that 2AG is at the crossroad of multiple routes of lipid metabolism, where it can serve as an end-product and precursor for different pathways implying that a significant amount of 2AG is involved in housekeeping functions rather than in signaling (Piomelli 2003). Interestingly, 2AG isomerizes spontaneously 1-arachidonoyl glycerol (1AG) by acyl migration in a physiological manner (Figure 4). However, 1AG is inactive on CBs receptors (Zoerner et al. 2011). This phenomenon is of particular importance for choosing the right techniques of quantification of 2AG (Zoerner et al. 2011).
Endocannabinoids are produced by a variety of cell types, including endothelial cells (Gauthier et al. 2005), glial cells (Walter et al. 2003), macrophages (Di Marzo et al. 1999), adipocytes (Gonthier et al. 2007) and Purkinje cells (Maejima et al. 2001). Furthermore, they act on their receptors only locally, possibly because of their high lipophilicity, and are immediately inactivated under physiological conditions (Di Marzo and Petrosino 2007).
19
Figure 4: Chemical structure of anandamide (AEA), 1-arachidonoyl glycerol (1AG) and 2-arachidonoyl glycerol (2AG). Isomerization of 2AG to 1AG through acyl migration.
(Modified from Zoerner et al. 2011).
4.4.2. Cannabinoid receptors (CBs)
In mammalian tissues, two main subtypes of cannabinoid receptors (CBs) have been determined (Pertwee 1997). These G protein-coupled receptors transduce many of the effects of the ECs and their distribution has been extensively studied by quantitative autoradiography (Glass et al. 1997; Herkenham et al. 1990), in situ hybridization (Hohmann and Herkenham 1999; Marsicano and Lutz 1999) and immunocytochemistry (Cristino et al. 2006; Eggan and Lewis 2007; Tsou et al. 1998). In general, the main function of CBs appears to be the modulation of ongoing release of chemical messengers, CB2 from immune cells and CB1 mainly from neurons (Mackie 2005). In addition, ECs seem to act on the CBs locally and are immediately inactivated under physiological conditions (Di Marzo and Petrosino 2007).
The CB1 is a seven-transmembrane, G protein-coupled receptor consisting of 472 amino acids (Human) (Sugiura and Waku 2000) and is the major mediator of the effects of cannabis and its derivatives in the brain (Mackie 2005). CB1 receptors are primarily
OH O
N H
Anandamide,
arachidonoylethanolamide (AEA)
2-arachidonoyl glycerol (2AG) 1-arachidonoyl glycerol (1AG)
OH
OH O O
OH OH O O Isomerization
20
expressed in the Central Nervous System (CNS) and Peripheral Nervous System (PNS), mainly on neurons, where they are mostly located on axons and presynaptic terminals (Mackie 2005). ECs act in a retrograde manner to activate CB1 receptors and inhibit the release of neurotransmitters, emphasizing their important role modulating neurotransmission at specific synapses (Mackie 2005; Ohno-Shosaku et al. 2001). The highest expression of CB1 receptors in the brain are in the globus pallidus, substantia nigra pars reticulate, cerebellar molecular layer, hippocampal dentate gyrus and the neocortex (Devane et al. 1992;
Herkenham et al. 1990; Westlake et al. 1994). This pattern, with few variations was detected through autoradiography in humans, rhesus monkeys, rats, guinea pigs and dogs (Herkenham et al. 1990).
Although CB1 receptors are mainly expressed in neurons mostly on axons and presynaptic terminals (Hajos et al. 2000; Katona et al. 2000; Katona et al. 2001), they have been also found on postsynaptic structures (Eggan and Lewis 2007; Pickel et al. 2004; Salio et al. 2002), glial cells (Meng et al. 2014; Navarrete and Araque 2010; Rodriguez et al. 2001;
Stella 2010) and even peripheral cells (cells of the striated ducts of the parotid and mandibular glands, keratinocytes, fibroblasts, macrophages) (Dall'Aglio et al. 2010; Fede et al. 2016; Han et al. 2009; Mercati et al. 2012; Stander et al. 2005).
On the other hand, CB2 receptors are principally expressed on immune cells (Galiegue et al. 1995), where they modulate cytokine release (Pertwee 2006), decrease antigen presentation (Buckley et al. 2000) and modulate cell migration (Miller and Stella 2008) in order to control proliferation, differentiation and survival of both neural and non- neuronal cells (Fernandez-Ruiz et al. 2007). Immune cells express high levels of CB2, and even a hierarchy of CB2 receptor expression has been described within the immune system (B cells > natural killer cells > monocytes > neutrophils > CD8 lymphocytes > CD4
21
lymphocytes) (Galiegue et al. 1995). The level of expression, however, is dependent on the activation state of the cell and the type of stimuli (Rom and Persidsky 2013).
The main characteristics of CB1 and CB2 receptors are summarized in Table 2.
Table 2: Main characteristics of CB1 and CB2 receptors.
Characteristics CB1 receptor CB2 receptor
Gene/Chromosome CNR1/6q14 CNR2/1p36
Main endogenous ligands AEA and 2AG 2AG (AEA partial agonist)
Major tissue locations CNS and PNS Immune system
Other tissue locations Pituitary gland, thyroid gland, adrenal gland, reproductive system,
liver, adipocytes, lungs, kidney
Spleen, tonsils, thymus gland, gastrointestinal tract, osteocytes
Main cellular location Presynaptic glutamate and GABA neurons
Monocytes, macrophages, microglia, B-cells, T-cells
General action Inhibition of release of glutamate and GABA
Modulates cytokine release and immune response
4.4.3. Endocannabinoid system in neurotransmission
CB1 are considered the most abundant G-protein coupled receptors in the mammalian brain (Herkenham et al. 1990). Moreover, they are mostly expressed in axon terminals of GABAergic interneurons (Piomelli 2003). However, they are also expressed in glutamatergic neurons (Di Marzo 2008; Piomelli 2003). A sustained increase in the intracellular calcium leads to endocannabinoid production (Stella 2004). ECs are released on demand from the membrane of phospholipid precursors at postsynaptic neurons and bind to CB1 receptors in the presynaptic terminals after retrograde diffusion (Battista et al. 2012). CB1 receptor activation by endocannabinoids induces activation of MAPK and voltage activated Ca2+
22
channels, stimulates inwardly rectifying K+ channels and inhibits adenylate cyclase and cyclic AMP-protein kinase (PKA) resulting in reduction of neurotransmitter release (Di Marzo et al. 2015).
4.4.4. Endocannabinoid system in inflammation
Increasing evidence supports the immunomodulatory roles of 2AG and AEA (Turcotte et al. 2015). Exogenous application of 2AG and AEA has shown to exert anti-inflammatory effects by decreasing the production of inflammatory mediators (Gui et al. 2015). The anti- inflammatory and neuroprotective effects of endocannabinoids and cannabinoids have been shown in several experimental models (Comelli et al. 2007; Hegde et al. 2008; Karsak et al.
2007; Mestre et al. 2005; Rom and Persidsky 2013; Storr et al. 2009; Yu et al. 2010) where activation of the cannabinoid system has been linked to decreased inflammatory cell recruitment and enhanced anti-inflammatory cytokine production (Turcotte et al. 2015), establishing the endocannabinoid system as a promising target for the treatment of inflammatory disorders.
The expression of CB1 and CB2 receptors in mammalian tissues has been proven (Pertwee 1997), and the presence of cannabinoid receptors in the dogs brain has been evidenced using synthetic cannabinoids by the means of autoradiography (Herkenham et al.
1990). Moreover, CB1 and/or CB2 receptors have been detected in the dogs salivary glands (Dall'Aglio et al. 2010), hair follicles (Mercati et al. 2012), skin and hippocampus (Campora et al. 2012). 2AG has been isolated from the canine intestine (Mechoulam et al. 1995), and both AEA and 2AG have been measured in serum and cerebrospinal fluid in healthy dogs and dogs with idiopathic epilepsy (Gesell et al. 2013). Aside from that, investigations of the role of the endocannabinoid systems in canines are very limited. Despite the lack of information in canines, nowadays several companies sell medical marihuana to be used in pets to treat
23
chronic pain, seizures, inflammation, cancer, diabetes, nausea, anxiety and obesity. There is, however, a lack of reliable research to back those claims regarding the specific distribution of cannabinoid receptors and the role of the endocannabinoids within the healthy nervous system and under pathological conditions.
Therefore, in the current study we evaluated the involvement of the endocannabinoid system in the pathogenesis of SRMA as an example for a painful inflammatory CNS disease, by measuring AEA and 2AG in serum and CSF in various stages of the disease and in healthy controls. Moreover, we evaluated CB2 receptor expression in spinal cord of healthy dogs and in lesions of dogs with SRMA.
24
25
5. Manuscript I: Th17 skewed immune response and cluster of differentiation 40 ligand expression in canine steroid-responsive meningitis-arteritis, a large animal model for neutrophilic meningitis
Jessica Freundt-Revilla1,6,*, Arianna Maiolini1, Regina Carlson1, Martin Beyerbach2, Kai Rentmeister3, Thomas Flegel4, Andrea Fischer5, Andrea Tipold1,6
1 Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany
2 Institute for Biometry, Epidemiology and Information Processing, University of Veterinary Medicine, Hannover, Germany
3 Tierärztliche Praxis für Neurologie, Dettelbach, Germany
4 Department of Small Animal Medicine, University of Leipzig, Leipzig, Germany
5 Clinic of Small Animal Medicine, Centre for Clinical Veterinary Medicine, LMU Munich, Munich, Germany
6 Center for Systems Neuroscience, Hannover, Germany
*Corresponding author: Jessica Freundt Revilla; Bünteweg 9, 30559 Hannover, Germany;
Telephone: +49 511/ 953 - 6282; E-mail address: jessica.freundt.revilla@tiho-hannover.de
Contribution of Jessica Freundt Revilla to this publication: 80%
J. Freundt-Revilla performed the experiments, analyzed the data and wrote the manuscript. A.
Maiolini was involved in the concept and design of the study and critically revised the manuscript. R. Carlson was involved in the concept and design of the study and helped performing the experiments. M. Beyerbach was involved in the statistical analysis.
Rentmeister, T. Flegel and Andrea Fischer were involved in the clinical evaluation of patients and collection of samples and critically revised the manuscript. A. Tipold was involved in the concept and design of the study, the coordination of experiments, analysis of the data, and critically revised the manuscript.
Journal of Neuroinflammation, under revision.
26 5.1. Abstract
Background
Steroid Responsive Meningitis-Arteritis (SRMA) is an immune mediated disorder characterized by neutrophilic pleocytosis and an arteritis particularly in the cervical leptomeninges. Previous studies of the disease have shown increased levels of IL-6 and TGF- ß1 in CSF. In the presence of these cytokines, naïve CD4+ cells differentiate into Th17 lymphocytes which synthetize IL-17. It has been shown that IL-17 plays an active role in autoimmune diseases, it induces and mediates inflammatory responses and has an important role in recruitment of neutrophils. The hypothesis of a Th17 skewed immune response in SRMA should be confirmed by evaluating IL-17 and CD40L, inducing the vasculitis.
Methods
An Enzyme-Linked Immunosorbent Assay (ELISA) was performed to measure IL-17 and CD40L in serum and CSF from a total of 79 dogs. Measurements of patients suffering from SRMA in the acute state (SRMA A) were compared with levels of patients under treatment with steroids (SRMA T), recurrence of the disease (SRMA R), other neurological disorders and healthy dogs, using the two-part-test. Additionally, secretion of IL-17 and IFN- γ from peripheral blood mononuclear cells (PBMCs) was confirmed by an Enzyme-Linked
ImmunoSpot (ELISpot) assay.
Results
Significant higher levels of IL-17 were found in CSF of dogs with SRMA A compared with SRMA T, other neurological disorders and healthy dogs (p<0.0001). In addition, levels of CD40L in CSF in dogs with SRMA A and SRMA R were significantly higher than in SRMA T (p=0.0004) and healthy controls (p=0.014). Furthermore, CSF concentrations of IL- 17 and CD40L showed a strong positive correlation among each other (rSpear= 0.6601;
p<0.0001) and with the degree of pleocytosis (rSpear= 0.8842; p<0.0001 and rSpear= 0.6649;
27
p<0.0001 respectively). IL-17 synthesis from PBMCs in SRMA patients was confirmed, however, IL-17 is mainly intrathecally produced.
Conclusions
These results imply that Th17 cells are inducing the autoimmune response in SRMA and are involved in the severe neutrophilic pleocytosis and disruption of the blood brain barrier (BBB). CD-40L intrathecal synthesis might be involved in the striking vasculitis. The investigation of the role of IL-17 in SRMA might elucidate important pathomechanism and open new therapeutic strategies.
Keywords
Steroid Responsive Meningitis-Arteritis (SRMA), Interleukin-17 (IL-17), Cluster of differentiation 40 ligand (CD40L), Interferon gamma (IFN- γ), Cerebrospinal fluid (CSF), Serum, Canine.
5.2. Background
Steroid-responsive meningitis-arteritis (SRMA) is the most frequently diagnosed meningitis in canines (Meric 1988). It is a systemic immune-mediated disorder (Tipold and Schatzberg 2010) characterized by systemic inflammatory lesions of the vessels, but particularly in the cervical leptomeninges (De Lahunta and Glass 2009; Harcourt 1978) and a recognized large animal model for neutrophilic meningitis (Maiolini et al. 2012a). This disorder affects typically young adult dogs (Cizinauskas et al. 2000), can occur in any breed, although Beagles, Boxers, Bernese mountain dogs (De Lahunta and Glass 2009; Tipold and Jaggy 1994), Weimaraners, Nova Scotia duck tolling retrievers (Tipold and Schatzberg 2010) and Petit Basset Griffon Vendéen (Vos et al. 2012) are over-represented.
Two different forms of SRMA are recognized, a typical acute form and a protracted atypical form (Tipold and Jaggy 1994). In the acute one, common clinical signs include fever,
28
reluctance to move, stiff gait, cervical rigidity and pain (De Lahunta and Glass 2009); the analysis of cerebrospinal fluid (CSF) reveals a marked polymorphonuclear pleocytosis and elevated protein (Tipold and Jaggy 1994). The protracted form may be observed following relapses and neurological deficits like reduced menace response, anisocoria, strabismus and variable degrees of paresis and ataxia might occur; CSF analysis at this stage reveals mononuclear or mixed cell populations (Tipold and Jaggy 1994).
The etiology of SRMA remains unknown, even a genetic predisposition was described in Nova Scotia duck tolling retrievers (Wilbe et al. 2010). Up to now, no infectious agents eliciting the disease have been consistently detected (Cizinauskas et al. 2000; Harcourt 1978;
Lazzerini et al. 2015; Meric et al. 1985; Rose and Harcourt-Brown 2013; Tipold and Jaggy 1994). Several studies (Maiolini et al. 2012b; Maiolini et al. 2013; Schwartz et al. 2008b;
Schwartz et al. 2008a; Schwartz et al. 2011) confirmed an immune-mediated disease.
Furthermore, high concentrations of immunoglobulin A (IgA) have been found both intrathecally and systemically in dogs affected with SRMA (Maiolini et al. 2012b; Tipold et al. 1994).
A predominance of T helper lymphocytes (CD4+) in peripheral blood (Schwartz et al.
2008b) with a prominent Th2-mediated immune response in dogs suffering from SRMA was shown (Schwartz et al. 2011). Furthermore, increased levels of interleukin 6 (IL-6) and transforming growth factor beta 1 (TGF-ß1) have been found in CSF. A combined intrathecal increase of these proteins could induce CD4+ progenitors to differentiate to the recently discovered third T helper subset (Th17) and enhance the autoimmune response in SRMA (Maiolini et al. 2013).
Interleukin 17 (IL-17) synthetizing cells are known to be involved in the pathogenesis of several autoimmune diseases (Cua et al. 2003; Hellings et al. 2003; Lubberts et al. 2004;
Zhang et al. 2006). IL-17 is a pro-inflammatory cytokine secreted primarily by activated T-