Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response

Abstract

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon-β (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Bruton's tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-β upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to D-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling. T oll-like receptors (TLRs) recognize pathogen-associated molecular patterns and activate signaling pathways that induce the expression of host immune and inflammatory genes (1). All TLRs contain extracellular leucine-rich domains for ligand recognition and a cytoplasmic Toll/IL-1R (TIR) domain for signaling. Most TLRs signal via the adapter myeloid differentiating factor 88 (MyD88) with the exception of TLR4, which also uses a few other adapters and TLR3, which uses solely TIRcontaining adaptor inducing interferon-β (TRIF) (2). Hence, TLR3 provides a unique system to study innate signaling.TLR3 is involved in antiviral response. Its engagement by viral double-stranded RNA (dsRNA) or the synthetic analog polyinsosinic: polyribocytidylic acid (p(I:C)) triggers the secretion of IFN-β and other inflammatory cytokines such as IL-6 and TNF-α. Mechanistically, TLR3 stimulation leads to the recruitment of TRIF and subsequent activation of the transcription factor IFN regulatory factor-3 (IRF3) (3). The N-terminal of TRIF binds tumor necrosis factor receptor-associated factor 3 (TRAF3)-TANK-binding kinase 1 (TBK1) complex that leads to IRF3 phosphorylation, dimerization, and translocation to nucleus to induce IFN-β expression (4). The C-terminal of TRIF binds receptor-interacting protein 1 (RIP1) and induces the ubiquitination of RIP1 (5). TLR3 signaling also activates ERK, JNK, p38 MAPKs, and phosphoinositide 3-kinase (PI3K) (6). MAPKs activate the AP-1 family of transcription factors, which induces proinflammatory cytokine genes (7). On the other hand, TLR3-induced PI3K signaling contributes to the full activation of IRF3 (8). TRIF signaling leading to TNF-α production also acts in a feed-forward manner to further and fully activate NFκB signaling (9, 10). Although the major signaling pathways associated with TLR3/TRIF activation have...

Macrophages eat cancer cells using their own calreticulin as a guide: Roles of TLR and Btk

“…To understand further the mechanism by which the activation of TLR signaling in macrophages promoted tumor cell phagocytosis, we treated macrophages by combining TLR agonists with various inhibitors targeting key molecules that positively (MAPK, Btk) (23)(24)(25)(26)(27) or negatively (PI3K, caspase-1) (28,29) regulate TLR signaling. Blockers of MAPK, PI3K, and caspase-1 showed no effect on phagocytosis of cancer cells.…”

The Inducible Nitric-oxide Synthase (iNOS)/Src Axis Mediates Toll-like Receptor 3 Tyrosine 759 Phosphorylation and Enhances Its Signal Transduction, Leading to Interferon-β Synthesis in Macrophages

“…Interestingly, the implication of EGF receptor-recruited Src in TLR3 Tyr-759 phosphorylation has been shown in constitutive Src-expressing cells (29), supporting the importance of Src in TLR3-induced signaling as we reported in this study. Recently, the role of BTK has been implicated in TLR3 Tyr-759 phosphorylation and initiating antiviral responses in dsRNA-treated macrophages (28). Given that BTK was activated within 30 min after dsRNA stimulation, BTK might be the kinase responsible for the early TLR3 Tyr-759 phosphorylation in macrophages.…”

“…signaling (18,26,28), the essential role of iNOS and Src in dsRNA-mediated TLR3 Tyr-759 phosphorylation and IFN-␤ production has not been investigated in the past. In this study, we demonstrated that dsRNA-stimulation induced a biphasic (early versus late) TLR3 Tyr-759 phosphorylation in macrophages.…”

“…Remarkably, in addition to NF-B-mediated gene induction in dsRNA-treated cells (27), phosphorylation of TLR3 at both Tyr-759 and Tyr-858 independently mediates PI3K and TBK1 activation, leading to the phosphorylation and activation of IRF3 (18). Recently, the importance of tyrosine kinases BTK (28) and Src (29) has been implicated in dsRNA-mediated TLR3 Tyr-759 phosphorylation in different types of cells. However, whether Src is involved in TLR3 Tyr-759 phosphorylation and antiviral response in dsRNA-stimulated macrophages is still unclear.…”

Bruton's Tyrosine Kinase Is Required for Apoptotic Cell Uptake via Regulating the Phosphorylation and Localization of Calreticulin

“…Recent studies, however, have focused on its role in regulating innate immune responses in myeloid cells, acting downstream of TLR4 and the antiviral TLRs, TLR3, -7, and -9, to regulate proinflammatory cytokine and type I IFN production (6)(7)(8)(9)(10)(11). Btk has also been shown to interact with and become activated following ligation of TLR8 and -9 in the monocytic cell line THP-1 (8).…”