Abstract
Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN-β, IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid-β by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer's disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.
Highlights
Astrocytes constitute 19-40% of brain glial cells and are the key component responsible for homeostasis and immune and oxidative stress defense in the central nervous system (CNS) [1, 2]
To study whether the observed changes have a reflection in protein distribution at the cellular level following stimulation and better understand the connection between Toll-like receptor 3 (TLR3), endosomal sorting complex required for transportation-0 (ESCRT-0), and Spleen tyrosine kinase (Syk) in astrocytes, we examined the localization of TLR3, Syk, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and signal transducing adaptor molecule (STAM) in C8-D1A cells stimulated with poly(I:C) (10 μg/ml) for 5 min, 4 h, and 24 h
Our findings indicate that TLR3, STAM, Syk, and Hrs are highly expressed in murine astrocytes (Figures 7(a)–7(d))
Summary
Astrocytes constitute 19-40% of brain glial cells and are the key component responsible for homeostasis and immune and oxidative stress defense in the CNS [1, 2]. Amyloid-β (Aβ) plaques, AD’s hallmark, activate cerebral glial cells and cause neuroinflammation resulting in neuronal cell death [6]. Such a process is decisive for the progression of AD. One of the factors leading to neuroinflammation, which may contribute to astrocyte reprogramming and enhanced astrocytic secretion of Aβ, is herpes simplex type 1 virus (HSV-1) infection in the brain. Aβ accumulates in HSV-1-infected cell cultures, while viral particles, as well as viral nucleic acid were found in the vicinity of amyloid plaques in the brains of mice and humans [9]. Toll-like receptor 3 (TLR3) plays an essential role in the innate immune control of cerebral HSV-1 infection. It is likely that infected astrocytes detect the virus through TLR3, activating them and contributing to production of Aβ
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