Abstract
Toll-like receptors (TLRs) play a key role in the activation of innate immune cells, in which their engagement leads to production of cytokines and co-stimulatory molecules. TLRs signaling requires recruitment of toll/IL-1R (TIR) domain-containing adaptors, such as MyD88 and/or TRIF, and leads to activation of several transcription factors, such as NF-κB, the AP1 complex, and various members of the interferon regulatory factor (IRF) family, which in turn results in triggering of several cellular functions associated with these receptors. A role for Src family kinases (SFKs) in this signaling pathway has also been established. Our work and that of others have shown that this type of kinases is activated following engagement of several TLRs, and that this event is essential for the initiation of specific downstream cellular response. In particular, we have previously demonstrated that activation of SFKs is required for balanced production of pro-inflammatory cytokines by monocyte-derived dendritic cells after stimulation with R848, an agonist of human TLRs 7/8. We also showed that TLR7/8 triggering leads to an increase in interferon regulatory factor 1 (IRF-1) protein levels and that this effect is abolished by inhibition of SFKs, suggesting a critical role of these kinases in IRF-1 regulation. In this study, we first confirmed the key role of SFKs in TLR7/8 signaling for cytokine production and accumulation of IRF-1 protein in monocytes and in B lymphocytes, two other type of antigen-presenting cells. Then, we demonstrate that TLR7 triggering leads to an increase of K63-linked ubiquitination of IRF-1, which is prevented by SFKs inhibition, suggesting a key role of these kinases in posttranslational regulation of IRF-1 in the immune cells. In order to understand the mechanism that links SFKs activation to IRF-1 K63-linked ubiquitination, we examined SFKs and IRF-1 possible interactors and proved that activation of SFKs is necessary for their interaction with TNFR-associated factor 6 (TRAF6) and promotes the recruitment of both cIAP2 and IRF-1 by TRAF6. Collectively, our data demonstrate that TLR7/8 engagement leads to the formation of a complex that allows the interaction of cIAP2 and IRF-1 resulting in IRF-1 K63-linked ubiquitination, and that active SFKs are required for this process.
Highlights
The innate immune system represents the first defense mechanism engaged by the host organism against pathogen infections and is required to activate adaptive immune responses
We have previously demonstrated that Src family kinases (SFKs) activity is required for the release of pro-inflammatory cytokines by human monocyte-derived dendritic cells (MoDCs) stimulated with the imidazoquinoline compound R848 (Resiquimod), an agonist for human TLR7 and TLR8 [16]
To understand whether this mechanism is shared by other immune cells, which are targets for a TLR7/8 agonist vaccine adjuvant, we extended our analysis to monocytes and B-lymphocytes
Summary
The innate immune system represents the first defense mechanism engaged by the host organism against pathogen infections and is required to activate adaptive immune responses. Following recognition of highly conserved pathogen-associated molecular patterns [5, 6], TLRs activate a signaling cascade that in turn leads to the production of pro-inflammatory cytokines and co-stimulatory molecules, which are required for the response to pathogens [4, 7] Due to their critical role in the activation of the innate immune response TLRs are preferential targets of new vaccine adjuvants based on small molecules [8]. TLR7 has been shown to be a good target for a recently described vaccine adjuvant [11, 12] Engagement of these two TLRs by appropriate agonists triggers an intracellular signaling pathway that involves recruitment of the adaptor protein Myeloid differentiation primary response 88 (MyD88), which in turn binds interleukin 1 (IL-1) receptor-associated kinase family of protein kinases. We pre viously demonstrated that pharmacological inhibition of SFKs with PP2 in human monocyte-derived dendritic cells (MoDCs) impaired TLR7/8-mediated release of several pro-inflammatory cytokines by interfering with the accumulation of IRF-1, suggesting an involvement of SFKs in IRF-1 regulation and TLR7/8 signaling [16]
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