Ligand-regulated Chimeric Receptor Approach Reveals Distinctive Subcellular Localization and Signaling Properties of the Toll-like Receptors

Tadashi Nishiya,Anthony L Defranco

doi:10.1074/jbc.m311618200

Abstract

Toll-like receptors (TLRs) are sensors for the detection of invading infectious agents and can initiate innate immune responses. Because the innate immune system induces an appropriate defense against different pathogens, different TLR signaling domains may have unique properties that are responsible for eliciting distinctive responses to different types of pathogens. To test this hypothesis, we created ligand-regulated TLR chimeric receptors composed of the extracellular region of TLR4 and the transmembrane and cytoplasmic regions of other TLRs and expressed these chimeras in macrophages lacking endogenous TLR4. Interestingly, the chimeras between TLR4 and either TLR3, TLR7, or TLR9 were localized completely intracellularly whereas other chimeras were expressed on the cell surface. Lipopolysaccharide (LPS), a ligand for these chimeras, induced the activation of nuclear factor kappa B and mitogen-activated protein kinases and the subsequent production of pro-inflammatory cytokines in macrophages expressing TLR4, TLR4/TLR5, or TLR4/TLR8 chimeras but not in macrophages expressing TLR4/TLR1, TLR4/TLR2, or TLR4/TLR6 chimeras. Co-expression of unresponsive chimeras in some combinations (chimeras with TLR1+TLR2 or TLR2+TLR6 but not TLR1+TLR6) resulted in LPS responsiveness, indicating functional complementarity. Furthermore, the pair of TLR2+TLR6 chimera required approximately 10-fold less LPS to induce the same responses compared with the TLR1+TLR2 pair. Finally, LPS induced effective interferon-beta production and subsequent Stat1 phosphorylation in macrophages expressing full-length TLR4 but not other cell surface TLR chimeras. These results suggest that the functions of TLRs are diversified not only in their extracellular regions for ligand recognition but also in their transmembrane and cytoplasmic regions for subcellular localization and signaling properties.

Highlights

Vertebrates have developed innate and adaptive immune systems to combat infections by bacteria, fungi, and viruses [1]
Type I IFNs are more critical for the elimination of many viral infections. Because these immune responses are mediated by Toll-like receptors (TLRs) in innate immune cells, such as macrophages and dendritic cells, we hypothesized that different TLRs may have different properties which allow innate immune cells to tailor their responses to individual pathogens by producing specific patterns of cytokines and inflammatory mediators
Ligand-regulated Chimeric Toll-like Receptors—To investigate the signaling properties and biological functions of different TLRs in a ligand-dependent manner, we established a new system in which chimeric receptors were composed of the extracellular region of TLR4 fused with the transmembrane and cytoplasmic region of other TLRs and expressed in primary macrophages lacking endogenous TLR4 (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Mice, and Cell Culture—Highly purified LPS from Salmonella minnesota R595 (Re) and peptidoglycan from Staphylococcus aureus were purchased from List Biological Laboratories (Campbell, CA) and Sigma, respectively. Anti-mouse IFN-␤ monoclonal antibody (clone 7F-D3, a rat IgG1) was purchased from Seikagaku America (Falmouth, MA). Murine TLR3 and TLR7 were amplified by PCR from the mouse spleen cDNA library. Murine TLR5 and TLR8 were amplified by PCR from mouse liver cDNA and IFN-␥-treated RAW 264.7 macrophage cDNA, respectively. Viral Production and Infection—The TLR chimeras were transduced into bone marrow-derived macrophages by retroviral gene transfer. The concentration of TNF, IL-6, and IL-12 p40 in cell culture medium was measured by enzymelinked immunosorbent assay according to the instructions from the manufacturer (BD Pharmingen). For the intracellular TNF staining, cells were stained with PE-conjugated anti-mouse TNF-␣ monoclonal antibody (BD Pharmingen) after cells were treated with Fc Block, fixed, and permeabilized as described above. Primers for IFN-␤ and HPRT were described by Alexopoulou et al [18]

RESULTS

26 Ϯ 2 548 Ϯ 32

DISCUSSION