DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance

Qisheng Peng,Jason L O’Loughlin,Mary Beth Humphrey,Stefan Bereswill

doi:10.1371/journal.pone.0039967

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.

Highlights

Toll-like receptors (TLRs) are pattern recognition receptors used by cells of the innate immune system to detect pathogenassociated molecular patterns (PAMPs), including the bacterial cell wall component lipopolysaccharide (LPS), which combines with the small molecule MD2 to activate TLR4 [1]
DOK1 expression was partially reduced at the highest dose of LPS in a manner similar to peritoneal macrophages [17]; whereas DOK2 expression remained stable at all doses (Figure 1B)
Ligands for TLR3 or TLR2 failed to alter DOK3 expression, CpG triggered decreases in DOK3 expression with maximal loss at 60 min (Figure 1B) indicating that DOK3 is regulated downstream of TLR4 and TLR9 signaling in Bone Marrow Macrophages (BMM)

Summary

Introduction

Toll-like receptors (TLRs) are pattern recognition receptors used by cells of the innate immune system to detect pathogenassociated molecular patterns (PAMPs), including the bacterial cell wall component lipopolysaccharide (LPS), which combines with the small molecule MD2 to activate TLR4 [1]. LPS, in turn, initiates downstream intracellular signaling events, including the activation of NF-kB and of the mitogen activated protein kinases (MAPKs) ERK, JNK, and p38 [1,2,3,4] Activation of these signaling components leads to the production of pro-inflammatory cytokines, including tumor necrosis factor a (TNFa), interleukin1b (IL-1b), and interleukin-6 (IL-6). The neutrophils, monocytes, and macrophages of septic patients develop a refractory state to subsequent LPS exposure and become incapable of producing cytokines at levels comparable to those prior to sepsis [7]. This state of unresponsiveness is termed endotoxin tolerance and prevents overstimulation from continuous exposure to LPS. Significant morbidity occurs in hospitalized patients due to super-infections; determining mechanisms that regulate endotoxin tolerance is critical to identify therapeutics that could reverse LPS-induced immune paralysis

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Conclusion