TLR4-independent and PKR-dependent interleukin 1 receptor antagonist expression upon LPS stimulation

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Abstract

Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4neg) DCs and wild type (TLR4pos) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4neg DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.

Introduction

The incidence of sepsis increases by 1.5% annually [1]. Various symptoms of sepsis patients generally come from the activation of the innate immune system by bacterial endotoxin (LPS). LPS activates many types of cells involved in the innate and adaptive immune systems [2], [3]. Dendritic cells (DCs) and macrophages play key roles in the activation of a host’s innate immune responses. They release an array of proinflammatory cytokines and chemokines upon LPS stimulation, such as IL-1 [4], IL-6 [5], [6], TNF-α[7], IL-12 [8], IFN-γ[9], MIP-1 [10] and RANTES [11], etc. Overproduction of these proinflammatory mediators is a hallmark of sepsis. Numerous trials targeting LPS and proinflammatory mediators such as TNF-α, have shown promising outcomes in animal models, but failed for sepsis patients [12]. To ameliorate systemic inflammation, anti-IL-1 antibody has been tried in numerous clinical settings of sepsis [13]. However, this strategy showed low efficacy for the sepsis patients [12]. Better understanding of LPS-mediated immune activation might expedite the development of novel therapeutics for sepsis.

Signal transduction pathways by LPS are mediated by receptor complexes consisting of TLR4, CD14 and MD2 [14]. Interaction of LPS with its receptor complexes results in the production of proinflammatory mediators [15]. In addition, it has been reported that LPS interacts with various surface receptors other than TLR4 (such as CD55, chemokine receptor (CXCR4) and integrin CD11/CD18 as well as heat shock proteins), indicating further complexity of the signal transduction pathways mediated by LPS [16]. Incidentally, we found that expression of I-Ab molecule on TLR4neg DCs was not completely abrogated upon LPS stimulation. This prompted us to investigate molecules modulated by LPS in the absence of TLR4.

Since molecules contaminated in LPS preparation might be responsible for activation of DCs, we used highly purified LPS derived from Escherichia coli and Salmonella. The phenol–chloroform–petroleum ether (PCP) extraction method has been used to isolate LPS from bacteria. However, it was shown that a high percentage of PCP-purified LPS preparations contain significant levels of protein contaminants with endotoxin-like activity [14]. A modified phenol–water extraction procedure was developed to obtain protein-free LPS from the original PCP-extracted preparations [17]. The repurified LPS was known to contain no detectable protein and to stimulate normal C3H/OuJ macrophages to secrete TNF-α[17] and IL-6 [18]. However, it lost >99% of its activity in stimulating TNF-α secretion by TLR4 mutant C3H/HeJ macrophages. Based on the analysis of repurified LPS, contaminants in 1.0 mg/ml of LPS were proteins (0.28%) and nucleic acids (0.62%). Since we used 200 ng/ml of LPS, the proteins and nucleic acids in LPS used in this study were diluted 5000-fold, resulting in lower than 560 pg/ml of proteins and 1.2 ng/ml of nucleic acids. For these reasons, the effect of protein and nucleic acid contaminations might be negligible for TLR4-independent activation of DCs in this study.

Using the repurified LPS, we investigated gene expression profiles of TLR4neg DCs by microarray. Microarray data revealed that TLR4neg DCs have five clusters of genes distinct from TLR4pos DCs upon LPS stimulation. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since it is an endogenous antagonist of IL-1, one of the most critical proinflammatory cytokines in the development of sepsis.

In this study, we report that IL-1rn was transcriptionally and translationally induced by DCs stimulated with LPS in the absence of TLR4, and the TLR4-independent expression was dependent on PKR pathway. These results imply that the balance maintaining the steady-state conditions for DCs might be partly regulated by TLR4-dependent expression of proinflammatory cytokines and TLR4-independent expression of anti-inflammatory cytokines such as IL-1rn.

Section snippets

Animals and cells

C57BL/6 (B6) mice and Rag2neg mice were obtained from Taconic Farms, Inc. (Hudson, NY). TLR4 knockout (TLR4neg) mice were kindly provided by Dr. S. Akira (Osaka University, Osaka, Japan). Rag2 and TLR4 double knockout (Rag2negTLR4neg) mice were generated by breeding Rag2neg mice with TLR4neg mice. In this study, TLRpos DCs were prepared from Rag2neg mice and TLR4neg DCs were from Rag2negTLR4neg mice. Primer sequences used for genotyping are listed in Table 1. Mice were maintained in a specific

Identification of genes modulated by LPS

TLR4, expressed on various cell types involved in the innate immune system, is a well known receptor for LPS [20], [21]. By engaging with LPS, TLR4 complexes lead to the induction of intracellular signal transduction pathways, modulating expression of various genes (Fig. 1). In presence of TLR4, DCs stimulated with LPS result in upregulation of activation markers such as CD80, CD86, and CD40 molecule. In the absence of TLR4, those genes were not upregulated by LPS (Fig. 1). According to the

Discussion

In this study, we investigated whether IL-1rn can be produced by DCs upon LPS stimulation in the absence of TLR4. We used highly purified LPS to rule out the effect of possible contaminated molecules on LPS-mediated signaling in TLR4neg DCs. Microarray analysis showed TLR4neg DCs upregulated proinflammatory genes in response to LPS. This suggests that LPS might interact with other cell surface molecules for the activation of DCs in the absence of TLR4. IL-1rn was of particular interest since

Acknowledgments

This study was supported by a grant from the Ministry of Health and Welfare and Family, Republic of Korea (A060122). We thanks for Dr. Sun-hwa Lee and Ms. Kristy Melton in preparing a manuscript.

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