Syk and Hrs Regulate TLR3-Mediated Antiviral Response in Murine Astrocytes

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.

Figure 1

TLR3 signaling in astrocytes. Upon dsRNA recognition in the endosomal compartment, TLR3 undergoes dimerization and interacts with the TRIF adaptor molecule. TRIF activation is followed by TRAF6 and TRAF3 recruitment. TRAF6 conducts the signal via RIP-1 and RIP-3 kinases which facilitate NEMO, IKK-α, and IKK-β complex formation, followed by NF-κB phosphorylation and translocation into the nucleus. TRAF3 engages TBK1 and IKK-i/Ɛ for IRF3 and IRF7 activation, followed by their dimerization and translocation into the nucleus. This leads to the induction of type I IFNs and proinflammatory cytokine gene expression. The dotted arrows highlight possible roles of ESCRT-0 in TLR3 transport from the ER to the endosome, as well as the role of Hrs and Syk in TLR3 degradation.

Figure 2

Poly(I:C) treatment of murine astrocytes induces Syk and Hrs phosphorylation and Syk-Hrs interaction. Hrs interacts with the N-terminal cleaved form of TLR3. (a) After poly(I:C) or poly(I:C)/LyoVec stimulation for 1, 2, 5, 10, and 15 min, the phosphorylation of Syk was analyzed by Western blot. The density level of phosphorylated Syk was normalized to GAPDH. Data was obtained from three independent experiments and presented as mean ± SD. ^∗p ≤ 0.05 and ^∗∗p ≤ 0.01. (b) After poly(I:C) or poly(I:C)/LyoVec stimulation for 5, 8, 12, and 15 min, C8-D1A cells were lysed and Hrs was immunoprecipitated using the anti-Hrs antibody. Phosphotyrosine (P-Tyr), Syk, and TLR3 were then detected by Western blot. (c) Following transfection with Syk siRNA, cells were stimulated with poly(I:C) for 5, 8, 12, and 15 min and lysed and Hrs was immunoprecipitated using the anti-Hrs antibody. Phosphotyrosine (P-Tyr) was detected by Western blot. For all immunoprecipitation experiments, 0 min presents untreated cells and mouse IgG were used as a negative control. EL: immunoprecipitation eluate; FT: immunoprecipitation flow-through; CN: control cell lysate. GAPDH was used as protein loading control. (d) Syk silencing efficiency was visualized by immunoblotting with anti-Syk antibodies.

Figure 3

NF-κB nuclear translocation is downregulated in poly(I:C)-treated astrocytes with silenced Syk and Hrs. C8-D1A cells were untreated or treated with poly(I:C) or poly(I:C)/LyoVec for 5 min, 8 min, 12 min, 15 min, 30 min, and 60 min. In advance to the stimulation, astrocytes were not transfected (a) or transfected with siRNA pools for TLR3 (b), Syk (c), and Hrs (d). Following poly(I:C) or poly(I:C)/LyoVec treatment, cytoplasmic and nuclear extracts were immunoblotted with anti-NF-κB p65, -IRF3, -IRF7, -GAPDH, and -PARP antibodies. (e) TLR3 and Hrs silencing efficiency was visualized by immunoblotting with anti-TLR3 and -Hrs antibodies.

Figure 4

Knockdown of Syk and Hrs expression by siRNA upregulates poly(I:C)-induced IFN-β, IL-6, and CXCL-8 production. C8-D1A cells were transfected with control siRNA-A or siRNA pools for TLR3, Syk, Hrs, and STAM. Following the transfection, astrocytes were treated with poly(I:C) (10 μg/ml) for 24 h. IFN-β (a), IL-6 (b), and CXCL-8 (c) were measured in culture supernatants by ELISA. Because Syk transfection lasted 48 h, in each experiment, supernatants from untreated (not treated (Syk)), poly(I:C)-treated (poly(I:C) (Syk)), and poly(I:C)-treated cells with silenced Syk (siRNA Syk) were tested in the group independent from cells with silenced TLR3, Hrs, and STAM, where transfection lasted 72 h. (d) STAM silencing efficiency was visualized by immunoblotting with anti-STAM antibodies. Data was obtained from three (IFN-β, CXCL-8) or five (IL-6) independent experiments and presented as mean ± SD. ^∗p ≤ 0.05 and ^∗∗p ≤ 0.01.

Figure 5

TLR3 of murine astrocytes is cleaved upon stimulation of cells with poly(I:C). Representative western blots of TLR3 expression in C8D1A cells treated with various concentrations of poly(I:C) (0, 0.1, 1, 2, 5, and 10 μg/ml) (a) or poly(I:C)-LyoVec (0, 0.1, 1, 2, and 5 μg/ml) (b) and lysed 24 h after stimulation. TLR3 expression was also analyzed in cells treated with poly(I:C) at concentration 10 μg/ml (c), or with poly(I:C)-LyoVecat concentration 1 μg/ml (d), and lysed at various times of stimulation (0, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h). TLR3 FL: full-length TLR3; TLR3 N: cleaved N-terminal TLR3 form; GAPDH: protein loading control. Densitometry analysis of TLR3 forms was performed in cells treated with indicated poly(I:C) concentrations for 24 h (a), indicated poly(I:C)/LyoVecconcentrations for 24 h (b), 10 μg/ml poly(I:C) for indicated time points (c), or 1 μg/ml poly(I:C)/LyoVec for indicated time points (d). The density level of each protein was normalized to GAPDH. Data was obtained from three independent experiments and presented as mean ± SD. ^∗p ≤ 0.05 and ^∗∗p ≤ 0.01.

Figure 6

Stimulation of murine astrocytes with poly(I:C) leads to the time-dependent increase in Syk and Hrs expression, while the expression of STAM does not significantly change after stimulation of cells with the TLR3 ligand. Representative western blots of Hrs, Syk, and STAM expression in C8D1A cells treated with various concentrations of poly(I:C) (0, 0.1, 1, 2, 5, and 10 μg/ml) (a), or poly(I:C)-LyoVec (0, 0.1, 1, 2, and 5 μg/ml) (b), and lysed 24 h after stimulation. Hrs, Syk, and STAM expression was also analyzed in cells treated with poly(I:C) at concentration 10 μg/ml (c), or with poly(I:C)-LyoVecat concentration 1 μg/ml (d), and lysed at various times of stimulation (0, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h). GAPDH was used for evaluating protein loading control. Densitometry analysis of Hrs, Syk, and STAM was performed in cells treated with indicated poly(I:C) concentrations for 24 h (a), indicated poly(I:C)/LyoVecconcentrations for 24 h (b), 10 μg/ml poly(I:C) for indicated time points (c), or 1 μg/ml poly(I:C)/LyoVec for indicated time points (d). The density level of each protein was normalized to GAPDH. Data was obtained from three independent experiments and presented as mean ± SD. ^∗p ≤ 0.05 and ^∗∗p ≤ 0.01.

Figure 7

Immunostains of TLR3, Syk, Hrs, and STAM expression and localization in C8D1A cells after treatment with poly(I:C). C8-D1A murine astrocytes were not treated or treated with poly(I:C) (10 μg/ml) for 5 min, 4 h, and 24 h, fixed and immunostained with specific antibodies. Selected images present intracellular distribution of TLR3 (a), Syk (b), Hrs (c), and STAM (d) (red fluorescence). To visualize colocalization of ER with TLR3 or STAM, following poly(I:C) stimulation at the indicated time points, cells were double stained with anti-TLR3 (red) and anti-PDI (green) antibodies (e), or with anti-STAM (red) and anti-PDI (green) antibodies (f). Nuclear DNA was stained with Hoechst 33342 (blue fluorescence). Scale bar = 10 μm, N.T. = no treatment.

Figure 8

Poly(I:C) treatment of murine astrocytes induces TLR3 tyrosine phosphorylation and promotes interaction with Hrs. (a) After poly(I:C) or poly(I:C)/LyoVec stimulation for 5, 8, 12, 15, and 30 min, C8-D1A cells were lysed and TLR3 was immunoprecipitated using the anti-TLR3 antibody. Phosphotyrosine (P-Tyr), Hrs, and STAM were then detected by Western blot. (b) Following poly(I:C) or poly(I:C)/LyoVec stimulation for 5, 8, 12, andn 15 min, C8-D1A cells were lysed and TLR3 was immunoprecipitated using the anti-TLR3 antibody. Ubiquitin was detected by Western blot. Blue arrows indicate ubiquitinated TLR3. (c) Following poly(I:C) stimulation for 5, 8, 12, and 15 min, murine astrocytes were lysed and STAM and Hrs were immunoprecipitated using anti-STAM and anti-Hrs antibodies, respectively. Hrs and STAM were detected by Western blot. For all immunoprecipitation experiments, 0 min presents untreated cells and mouse IgG were used as a negative control. EL: immunoprecipitation eluate; FT: immunoprecipitation flow through; CN: control cell lysate. GAPDH was used as protein loading control.