DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance

Abstract

Innate immune activation via Toll-like receptors (TLRs), although critical for host defense against infection, must be regulated to prevent sustained cell activation that can lead to cell death. Cells repeatedly stimulated with lipopolysaccharide (LPS) develop endotoxin tolerance making the cells hypo-responsive to additional TLR stimulation. We show here that DOK3 is a negative regulator of TLR signaling by limiting LPS-induced ERK activation and cytokine responses in macrophages. LPS induces ubiquitin-mediated degradation of DOK3 leading to SOS1 degradation and inhibition of ERK activation. DOK3 mice are hypersensitive to sublethal doses of LPS and have altered cytokine responses in vivo. During endotoxin tolerance, DOK3 expression remains stable, and it negatively regulates the expression of SHIP1, IRAK-M, SOCS1, and SOS1. As such, DOK3-deficient macrophages are more sensitive to LPS-induced tolerance becoming tolerant at lower levels of LPS than wild type cells. Taken together, the absence of DOK3 increases LPS signaling, contributing to LPS-induced tolerance. Thus, DOK3 plays a role in TLR signaling during both naïve and endotoxin-induced tolerant conditions.

DOK3 Is Required for IFN-β Production by Enabling TRAF3/TBK1 Complex Formation and IRF3 Activation

“…In contrast, we demonstrate here a positive role for DOK3 in the induction of IFN-b production during virus infection and poly(I:C) stimulation of macrophages. We observed that DOK3 did not undergo degradation upon poly(I:C) stimulation, consistent with previous findings (12). Its presence facilitates TBK1 and TRAF3 interaction during virus infection and likely enables maximal transduction of downstream signaling for optimal IFN-b production.…”

“…This is in contrast to the demonstrated degradation of DOK1-3 proteins in LPS-stimulated macrophages (12), suggesting that DOK3 could have a different role in TLR3 activation in macrophages. In this study, we examined the involvement of DOK3 in TLR3 signaling and showed that the adaptor was phosphorylated upon poly(I:C) stimulation and played a critical role in TLR3-triggered IFN-b production.…”

“…Previously, DOK3 has largely been characterized in the context of B cell receptor (32) and FcgRIIb (33) signaling where it serves as a negative regulator by interacting with Grb2 and the inhibitor SHIP to restrict the intensity of Ca 2+ mobilization (7) and limit JNK activation (8). While this manuscript was being prepared, a report was published describing DOK3 as a negative regulator of TLR4 signaling in the same manner as DOK1 and 2 by inhibiting ERK as it was degraded upon LPS but not poly(I:C) stimulation (12). In contrast, we demonstrate here a positive role for DOK3 in the induction of IFN-b production during virus infection and poly(I:C) stimulation of macrophages.…”

“…DOK1 and 2 have been shown to participate in TLR4 signaling by negatively regulating ERK activation and TNF-a production in LPS-stimulated macrophages (11). Recently, DOK3 was also demonstrated to negatively regulate LPS-induced ERK activation and the production of inflammatory cytokines in macrophages (12). Thus, it seems that DOK1-3 act similarly to inhibit TLR4 signaling in macrophages.…”

Epigenetic Regulation of Tolerance to Toll-Like Receptor Ligands in Alveolar Epithelial Cells

“…Suppressor of cytokine signaling (SOCS) proteins are important negative regulators of the TLR signaling pathway, and have been shown to play a role in the induction of tolerance in macrophages (28,29). The expression of suppressor of cytokine signaling 1 was measured in LPS-tolerized AECs, and was found to significantly increase after secondary LPS ( Figure 5A).…”