Studies by others, our published and ongoing studies in human and MS tissue and mouse models have characterized the importance of growth arrest-specific protein 6 (Gas6) signaling and members of the Tyro3, Axl, and Mertk (TAM) family of receptor tyrosine kinases (RTKs) during inflammatory demyelinating disease and recovery (1-21,41). The maintenance of a Gas6 reservoir in multiple cell types and organs is dependent on the simultaneous expression on the cell surface of receptor tyrosine kinase Axl demonstrated to aid in the resolution of inflammation in tissue (12,18). Immunohistochemical detection of Gas6 in multiple tissues sections including spleen, small intestine, liver and lung from wild-type (WT), Mertk?/? and Tyro3?/? mice was positive. By contrast, Gas6 was lost in these same tissues from Axl-/? mice, and Gas6 did not accumulate in serum. As Gas6 is the sole ligand for Axl, the data suggest that Axl and Gas6 are co-dependent (18). This is a unique role for a ligand/receptor interaction, and thus is integral for understanding Gas6/Axl downstream signaling. Shown in Figure 1 is a model proposed by Zagorska et al. (18) in which following proinflammatory stimuli, Axl is increased resulting in Axl-dependent phagocytosis and resolution of inflammation that results in shedding of Axl from the cell surface (soluble Axl). They further suggest that Mertk is more involved in homeostatic phagocytosis of circadian tolerogenic macrophages during the maintenance of tissue homeostasis. In vivo intravenous (IV) injection of activating anti-Axl to mice found spleen Axl kinase activity was activated 15 min later, with Axl activity back to baseline 24h later; no change in Mertk was detected. IgG had no effect activating Axl or Mertk. Similar results were observed with a Mertk activating antibody (R&D) with activated Mertk detected after 1h in vivo; however, Mertk remained at the membrane and no soluble Mertk was detected (18). Our findings (7,11,12) support and validate the proposed Model. The rationale for studying the beneficial effect of Axl and Mertk antibodies administered during myelin oligodendrocyte glycoprotein (MOG)- induced experimental autoimmune encephalopathy (EAE) is due to their longer half-life in vivo versus the short half-life of Gas6. Even with its short half-life we observe beneficial effects of icv administration of Gas6 during cuprizone toxicity and MOG-induced (11, 41, see enclosed manuscript). Whether in vivo treatment with activating Axl antibody or Mertk antibody can be anti-inflammatory and beneficial during EAE aiding in the clearance of apoptotic debris and protective to the CNS will be a focus of this study. There is evidence for antibodies crossing the BBB in individuals with neurodegenerative diseases including Alzheimer's disease (22-26). Figure 1: Model for proposed differences in regulation and action of Axl and Mertk (Mer in figure) in inflammatory and homeostatic environments respectively (18).
Specific Aim 1 will determine the best route of antibody administration using IP and icv injections to administer either IgG or activating anti-Axl antibody during MOG-induced EAE and PLP-induced EAE to WT mice. We predict that WT mice (n=5/group) receiving anti-Axl antibody will have a less severe disease course with reduced proinflammatory cytokines and fewer axonal spheroids than IgG. We will demonstrate specificity using Axl-/- mice and predict the clinical course will be worse than WT mice treated with IgG and thus not be beneficial.
Specific Aim 2 will determine whether prior to the onset clinical scores a single IP injection of activating or inhibiting Mertk antibody can beneficially ater the EAE clinical course relative to IgG and PBS, and maintain the reduced clinical scores and preserve axonal integrity that is lost during chronic EAE. These studies will further characterize the role of Axl and Mertk during EAE using activating antibodies as a possible therapeutic approach to target proinflammatory signaling molecules. This more refined approach will target two of the TAMs since Gas6 activates all three TAMs with a relative affinity of Gas6 for its receptors Axl>Tyro3>Mertk. We will not study Tyro3 as there is no evidence Tyro3 changes during inflammation, EAE or in MS lesions. This more focused approach may provide neuroprotection against axonal damage and loss found in chronic MS CNS tissue, and perhaps is beneficial as a therapy for individuals with progressive MS.
This is an exploratory, high risk study to test whether IP or icv administration of antibodies to activate the receptor tyrosine kinase Axl will reduce the clinical course of EAE and be considered as a beneficial therapy for patients with multiple sclerosis (MS). We reported that in MS lesions, soluble Axl, and soluble and full- length Mertk are significantly increased in both chronic active and chronic silent lesions; that treatment with Gas6 reduces the clinical course of EAE; that Gas6 is effective at reducing axonal damage during chronic EAE, and when administered to Axl-/- mice has no beneficial effects, suggesting that Gas6 activation of Axl and downstream signaling is important for the reduction in clinical disease and protecting against axonal dystrophy. Gas6 has a short half-life, and antibodies can remain in the periphery and brain for as long as one week; thus, we will determine if treatment with an activating Axl antibody is more beneficial than Gas6, and the effect of antibody activation and inhibition of Mertk during EAE.
|Ray, Alex K; DuBois, Juwen C; Gruber, Ross C et al. (2017) Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure. Glia 65:2051-2069|