SRMA Treatment
7. General discussion
The precise mechanisms of the failure of self-tolerance are not known for any autoimmune disease (Tizard 2000). These diseases appear to develop spontaneously and the predisposing causes are rarely obvious (Tizard 2000). After years of research the etiopathogenesis of SMRA remains unknown (Tipold and Schatzberg 2010), accordingly, attempts to find infectious or neoplastic triggers have failed (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). Being an immune-mediated disorder (Tipold and Schatzberg 2010), glucocorticoids play a major role in the treatment of SRMA (Cizinauskas et al. 2000). While in most cases a complete remission and a good management of the clinical signs are achieved with long-term glucocorticosteroid treatment (Tipold and Schatzberg 2010), relapses during or following therapy are frequent (Bathen-Noethen et al.
2008; Biedermann et al. 2016; Lowrie et al. 2009a). In these patients a lasting improvement is not accomplished, relapses are persistent (Biedermann et al. 2016) and/or the required dosage of glucocorticosteroids lead to unacceptable severe side effects (Whitley and Day 2011).
Therefore, novel therapeutic strategies targeting specific aspects of the immune response that are practiced in human medicine should be considered for dogs. A deep understanding and characterization of the cytokine profile leading to a dysregulation of the immune system in autoimmune inflammatory disorders allows implementation of novel therapeutic approaches based on specific cytokine modulation.
For this reason, in the first part of this study, we focused on evaluating the role of IL-17 producing ThIL-17 lymphocytes in the pathogenesis of SRMA. ThIL-17 cells have been extensively studied in humans and have an important role in the development of several autoimmune disorders. Interestingly, our group recently found increased levels of IL-6 and
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TGF-ß1 of CSF in dogs with SRMA (Maiolini et al. 2013). Both proteins are essential for the differentiation of naïve CD4+ cells to become Th17 cells (Bettelli et al. 2008; Mangan et al.
2006). Therefore, the aim of this part of the study was to evaluate IL-17 concentrations in CSF and serum in three different stages of the disease and analyze IL-17 synthesis by peripheral mononuclear cells comparing it to positive and negative controls.
We proved the hypothesis that SRMA is associated with a Th17 skewed immune response. 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.
IL-17 induces the release of pro-inflammatory cytokines, chemokines and metalloproteinases from various tissues and cell types resulting in neutrophil recruitment to tissues (Bettelli et al. 2008; Hellings et al. 2003) and also promoting granulopoiesis (Chen et al. 2003). Th17 cells appear at sites of inflammation with rapid kinetics (Bettelli et al. 2008).
Through the potent induction of chemokines, Th17 cells could bridge the gap between innate and adaptive immunity at later stages of inflammatory responses (Bettelli et al. 2008). This has been shown for M. tuberculosis infection, for which an early Th17 response is required to bring Th1 cells into the infected lung tissue to control the infection (Khader et al. 2007).
Although the combination of TGF-ß and IL-6 efficiently generates Th17 cells in-vitro, it has been shown that these cells do not induce tissue inflammation unless they are further cultured in the presence of IL-23 (Haines et al. 2013; McGeachy et al. 2007; McGeachy et al.
2009; Stumhofer et al. 2007). Indeed, full acquisition of pathogenic function by effector Th17 cells is mediated by IL-23 (Langrish et al. 2005; McGeachy et al. 2007). IL-23 does not act on naïve T cells but acts on differentiated Th17 cells to expand and stabilize them which allows the production of their effector cytokines (IL-17 and IL-22) (Bettelli et al. 2008). These facts
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lead to the division of Th17 cells in two broad groups: on one hand, host protective cells that express IL-10 and low levels of IL-17 (McGeachy et al. 2007), on the other hand, a highly inflammatory population that expresses IL-22 and pathogenic levels of IL-17 (Langrish et al.
2005; Zheng et al. 2007). IL-22 produced by Th17 cells, similarly to IL-17, can disrupt tight junctions between endothelial cells of the BBB (Kebir et al. 2007).
To further prove these findings, it was demonstrated that IL-23 deficient mice are resistant to collagen-induced arthritis and EAE (Cua et al. 2003; Murphy et al. 2003) and IL-17 deficient mice develop attenuated collagen induced arthritis (Nakae et al. 2003) and EAE (Komiyama et al. 2006).
Th17 did not evolve to cause autoimmunity but to provide effective host defence against pathogens (Gaffen et al. 2014). Deficiencies in Th17 cells and IL17 are strongly linked to defects in fungal immunity in mice and humans (Gaffen et al. 2014; Hernandez-Santos and Gaffen 2012). The development of IL-17 and IL-10 producing cells are important for mucosal defence, however, subsequent exposure to IL-23 promotes the development of pathogenic Th17 cells (Gaffen et al. 2014).
Novel therapeutics based on multipotent stromal/stem cells (MSCs) have been proposed since the recent characterization of Th17 cells in canines (Kol et al. 2016). These therapies inhibit Th17 polarization and propose dogs as valuable models for novel MSC based translational research in naturally occurring chronic inflammatory diseases in dogs (Kol et al.
2016). However, inhibition of Th17 polarization may result in detrimental effects.
Our results showed low IL-17 expression in healthy dogs in CSF and serum, which may be necessary for an effective host defence against pathogens. Indeed, IL-17 basal levels have been shown to be protective in mice. Knockout IL-17 mice have increased gut mucosal permeability, predisposing them to IBD (Ogawa et al. 2004). IL-17 levels in CSF in SRMA patients, however, exceeded 30 times the amount detected in healthy dogs. IL-17 was
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produced in PBMCs at a single cell level. Elevated IL-17 levels are therefore not considered to result from the severe neutrophilic pleocytosis in SRMA patients. Although IL-23 has not been evaluated in canines, to our knowledge, the IL-23 receptor has been identified in Th17 polarization of canine Th cells and its expression was markedly upregulated post stimulation (Kol et al. 2016). These findings indicate a pathogenic response of Th17 cells, which might occur in SRMA. Further confirmation of the described findings could be the evaluation of IL-23 and IL-22 in dogs with SRMA.
The recent classification of Th17 cells has important value since IL-17 and IL-23 are emerging essential factors in the pathogenesis of many autoimmune diseases (Gaffen et al.
2014). Currently, a number of pharmaceutical companies are at phase II and III clinical trials testing antibodies targeting IL-17 and IL-23 (Deiss et al. 2013; Gaffen et al. 2014; Leonardi et al. 2012; Sandborn et al. 2012). These antibodies have shown remarkable efficacy and safety profiles for the treatment of psoriasis and are showing promising results in ankylosing spondylitis, multiple sclerosis and Crohn´s disease (Chiricozzi and Krueger 2013; Deiss et al.
2013; Gaffen et al. 2014; Leonardi et al. 2012; Patel et al. 2013; Sandborn et al. 2012). Such therapies should be considered for the treatment of autoimmune diseases in canines, such as SRMA.
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 pathogenesis of vasculitis and inflammation of the meninges (Freundt-Revilla et al.
2016). Kawasaki disease (KD) has striking similarities with SRMA (Burns et al. 1991;
Felsburg et al. 1992). Recently, increased expression of CD40L in CD4+ T cells as well as soluble CD40L was found in patients with KD and the levels correlated with coronary artery lesions (C. L. Wang et al. 2003). Overexpression of CD40L may activate CD40-positive target cells (B-cells, macrophages, endothelial cells and vascular smooth cells)(van Kooten et
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al. 2000), with the potential to activate further the immune system and elicit inflammatory reactions, resulting in vascular endothelial damage (C. L. Wang et al. 2003). Importantly, intravenous immunoglobulin therapy seemed to downregulate CD40L expression and vascular damage in patients suffering from KD (C. L. Wang et al. 2003).
Many autoimmune diseases, like SRMA, show spontaneous exacerbations and remissions, suggesting an unstable relationship between positive and negative regulatory mechanisms (Tizard 2000). The endocannabinoid system has been proven to have an endogenous protective response and attempts to control inflammation. However, depending on the type and stage of the disease a dysregulation of the endocannabinoid system may occur, leading to an exacerbation of inflammation.
In the second part of the study, we therefore concentrated on the endocannabinoid system and found increased levels of endocannabinoids (AEA and 2AG) in CSF and serum of dogs with SRMA. Moreover, high expression of CB2 receptors in inflammatory lesions was shown.
The highest levels of AEA and total AG in CSF were found in dogs with SRMA in the acute phase. Furthermore, CB2 was strongly expressed on infiltrating leukocytes (i.e.
neutrophils, lymphocytes, plasma cells and macrophages) in spinal cord lesions of dogs with SRMA and CB2 expression seemed to be upregulated both in glial cells and neurons.
Interestingly, CB2 positive glial cells underwent changes in the morphology in SRMA towards a round shape showing less and shorter cytoplasmic extensions. Although further studies are needed to exactly confirm the type of glial cells expressing CB2, it can be speculated that CB2 expressing cells are astrocytes and/or activated microglia. Upregulation of CB2 receptor expression in microglia, macrophages, neurons and astrocytes upon activation has been demonstrated before (Cabral and Marciano-Cabral 2005; Cabral and
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Griffin-Thomas 2009; Carlisle et al. 2002; Galve-Roperh et al. 2013). The rapid activation of cannabinoid receptors by endocannabinoids after SCI is thought to be an endogenous protective response (Arevalo-Martin et al. 2012). However, in diseased CNS tissue, in which the immune system has been activated, the cell-specific expression profile of cannabinoid receptors changes, resulting in higher expression of CB2 receptors in activated microglia (Atwood and Mackie 2010; Di Marzo et al. 2015). Activated microglia can secondarily activate astrocytes leading to further induction of inflammatory factors (Cabral and Griffin-Thomas 2009). As the disorder progresses, the blood brain barrier (BBB) becomes partially disrupted and blood macrophages, B cells, T cells, natural killer cells and other infiltrating leukocytes start upregulating CB2 (Cabral and Griffin-Thomas 2009; Di Marzo 2008; Stella 2009). The activation of the receptors further stimulates the migration and/or activation of these cells into the nervous tissue and towards the endocannabinoids produced by microglia, neurons (Di Marzo 2008) and leukocytes (Di Marzo et al. 1999; Di Marzo et al. 2015; Varga et al. 1998). Endocannabinoid production by microglia increases in this state (Di Marzo et al.
2015) initiating a neuroinflammatory response and causing gliosis, exaggerated microglial activity and neuronal death (Di Marzo 2008) (Figure 5).
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Figure 5: Endocannabinoid system dysregulation in neuroinflammation. Resting microglia express low levels of CB2 and produce low levels of endocannabinoids. Initially as microglial cells become activated they start counteracting cellular damage. As the disorder progresses, the BBB becomes partially disrupted and blood leukocytes upregulate CB2 receptors. The activation of the receptors stimulates cell migration/activation.
Endocannabinoid production by microglial cells increases initiating a neuroinflammatory response (Modified from Di Marzo 2008; Di Marzo et al. 2015).
A dysregulation of the endocannabinoid system during neuroinflammation leads to increased CB2 receptor expression, excessive endocannabinoid production leading to an exacerbated inflammatory response. Thus, both CB2 agonists and antagonists might be beneficial in counteracting the inflammatory consequences depending on the disease phase (Di Marzo 2008), as shown in rheumatoid arthritis (Gui et al. 2015). Gui and others describe
Blood-brain barrier
B
B B
NK
NK Neuron
T
CB2 receptor Endocannabinoids Macrophage
Eosinophil
Neutrophil Microglia
Activated microglia
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in rheumatoid arthritis an inhibition of pro-inflammatory cytokines and of the immune response mediated by T cells (Gui et al. 2015). Moreover, anandamide might attenuate Th17 cell mediated delayed-type hypersensitivity (Jackson et al. 2014) possibly influencing the above described pathogenetic mechanisms in SRMA.
Recently, Kozela and others proved a decrease of the Th17 inflammatory response by cannabinoids (Kozela et al. 2013). Both THC and CBD suppressed IL-17 secretion independently of CB1 and CB2 receptors (Kozela et al. 2013). Although the mechanisms of action are still not fully understood, these targets should be considered for possible treatments.
An interaction between the two responses,Th17 skewed reaction and upregulation of the endocannabinoid system, during an inflammatory CNS disease in dogs could not be proven in the current study, but the described results are giving hint to such a phenomenon occurring in SRMA.
In conclusion, IL-17 producing cells, CD40L and the endocannabinoid system are involved in the pathogenesis of SRMA and should be considered as targets for future therapies.
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