In this study we investigated the functional and immunological effects of IC100, a monoclonal antibody against the inflammasome protein ASC, in the EAE animal model of MS. Here we show that ASC plays a critical role in the development of EAE, and that, by blocking ASC, the adaptive and innate immune response that sustain EAE are significantly reduced. Indeed, IC100 decreased the number of CD4+, CD8+ T and CD11b+MHCII+ cells trafficking into the CNS, while reducing the number and activation state of CNS resident microglia. This is the first report to show that targeting ASC with a monoclonal antibody in MOG-induced EAE significantly alters innate and adaptive immune responses leading to improved clinical outcomes. Importantly, this work constitutes a novel approach to identify new drugs targeting the inflammasome that could be useful for the treatment of MS.
Our previous studies have shown that ASC is a reliable disease biomarker found in serum of patients with MS [9], traumatic brain injury [32, 33] and depression [25], as well as in extracellular vesicles isolated from individuals with stroke [11]. It is possible that IC100 works extracellularly to prevent ASC oligomerization into highly cross-linked macromolecular assemblies. On the other hand, ASC or ASC-IC100 complexes may be taken up by cells and internalized to prevent recruitment of ASC into the inflammasome complex. Previous studies in our laboratory have shown that anti-ASC is taken up by spinal cord neurons after trauma [10], and by the human monocytic leukemia cell line (THP-1 cells, unpublished data). It has been shown that monoclonal antibodies enter cells by receptor- mediated endocytosis through binding of the Fc domain to Fc-gamma-receptors (FcgRs) expressed on many immune cells, including monocytes, macrophages, microglia, myeloid progenitor cells and dendritic cells [34, 35]. However, it is generally accepted that low affinity FcgRs differ from other receptors in that their activation requires cross-linking of polyvalent immune complexes, such as when multiple antibodies are bound to a protein aggregate [36, 37]. Multiple studies have suggested a role for microglia-mediated clearance of aggregated proteins as a-synuclein [38], Ab aggregates (31, 34) and Tau [15]. The later study also observed a clear size dependence of the microglial clearance pathway. Since ASC is secreted and oligomerizes into ASC specks, it is possible that IC100 may identify ASC assemblies and then are taken up by immune cells by receptor-mediated endocytosis. Therefore, establishing the precise specific mechanism of IC100 activity will help to design and optimize efficacy in vivo.
IL-1b/IL-18 secreted after inflammasome activation induce neutrophils and macrophages to engulf pathogens, promote inflammatory cell recruitment, and further amplify inflammatory responses [39, 40]. Apart from its impact on innate immunity, inflammasome activation also initiates adaptive immunity through the modulation of T helper subsets, skewing T cell differentiation in favor of Th1 and Th17 phenotypes [41, 42]. IL-1b also activates IL-1R signaling, which has a substantial influence on Th17 cell differentiation and related immune responses [43]. Several studies with ASC-/- mice have suggested inflammasome-independent roles of ASC in the adaptive immune response. A novel microbial pathogen-induced mechanism for ASC in activating chemokine expression through MAPK activation, independent of the conventional caspase-1 inflammasome, has been reported [44]. Two studies of antigen-induced murine arthritis show dependence on ASC, but caspase-1, NLRP3, and NLRC4 independence [17]. A requirement for ASC, but not NLRP3 or caspase-1, was also demonstrated for antigen-specific humoral immunity after vaccination with MF59-adjuvented influenza [16]. In addition, ASC-/- mice have reduced numbers of MOG-specific T cells in the lymph nodes and CNS, resulting in protection from EAE [19]. Lastly, genetic evidence shows an unexpected role of ASC in regulating motility of T lymphocytes and B-lymphocytes and antigen uptake by professional antigen-presenting cells independently of inflammasomes [17]. In this study, ASC was shown to regulate the mRNA stability and expression of Dock2, a guanine nucleotide-exchange factor that mediates Rac-dependent signaling in immune cells [17].
The blood-brain barrier (BBB) presents a great impediment for brain drug delivery in that it is questioned whether sufficient intralesional drug concentrations can be reached after systemic administration. This problem is especially evident for biologicals such as monoclonal antibodies (mAbs) that are relatively large molecules. To overcome the presumed drug delivery obstacle in the brain, many treatment strategies for CNS diseases are directed at disrupting, passing, or bypassing the effective BBB [46, 47]. Disruption has been tried chemically, by drugs that influence passive diffusion (e.g., bradykinin, mannitol, regadenoson, and borneol) or active transport mechanisms (e.g., elacridar), or by radiotherapy, ultrasound, or microwaves. Moreover, via viral vectors, nanoparticles, liposomes, exosomes, and transporter or receptor ligands have been tried to bypass the BBB [46,47].
However, the BBB is disrupted under various pathological conditions of diseases such as stroke, diabetes, seizures, hypertensive encephalopathy, acquired immunodeficiency syndrome, traumatic brain injuries, multiple sclerosis, Parkinson’s disease (PD) and Alzheimer disease (AD) [47]. It appears that in some pathological conditions, remodeling of the protein complex in interendothelial junctions is an important reason for the BBB breakdown [47]. Therefore, in CNS disease conditions associated with a leaky BBB, mAbs have been widely explored because of their specificity and high affinity for critical disease targets. Additionally, other important areas that deserve further investigation are the influence of aging [47] and major depressive disorder [48] on BBB dysfunctions and to discover whether mAbs may be used as possible approaches to deliver drugs into the brain to treat these conditions.