LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF

Abstract

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Figure 1.

LPS and dsRNA activate IRF-3 and IRF-7. (a) BM-derived macrophages from WT and MyD88-deficient mice were stimulated with LPS (0.1–100 ng/ml), Malp-2 (5 nM), and dsRNA (1–100 μg/ml) for 12 h. The concentration of RANTES was measured by ELISA. (b) Nuclear extracts were isolated from WT and MyD88-deficient macrophages stimulated with LPS (10 ng/ml), Malp-2 (5 nM), and dsRNA (50 μg/ml) for 1 h and subjected to EMSA using a ³²P-labeled ISRE consensus sequence (ISG-15) as a probe. Activated complexes were visualized by autoradiography. Activated ISRE DNA-binding complexes were preincubated with polyclonal antibody to IRF-3 or two control antibodies before incubation with the ISRE probe (right). (c) TLR3 and TLR4/MD2-expressing HEK293 cell lines were transfected with a luciferase reporter gene containing the Gal4 upstream activation sequence and with Gal4-DBD, Gal4–IRF-3, or Gal4–IRF-7 (40 ng). After 24 h, cells were stimulated with LPS (10 ng/ml), dsRNA (50 μg poly IC/ml), IL-1β (10 ng/ml), or left untreated for ∼8 h, and luciferase reporter gene activity was measured.

Figure 2.

TRAM activates IRF-3 and IRF-7. (a) Alignment of TIR domains of TRAM, TRIF, MyD88, and Mal with TLR1, TLR2, TLR3, and TLR4. The amino acid colors are based on their physico-chemical properties where yellow = small, green = hydrophobic, turquoise = aromatic, blue = positively charged, and red = negatively charged. (b) HEK293 cells were transfected as in Fig. 1 c and cotransfected with 40 ng of TRAM or TRIF. After 24 h, luciferase reporter gene activity was measured.

Figure 3.

TRAM interacts with IRF-3 and CBP and signals via IKKɛ and TBK1. (a) IRF-3–GFP–expressing HEK293 cells were plated on 35-mm glass-bottom sterile tissue culture dishes and transiently transfected with 1 μg of Flag-tagged TRAM, TRIF, or pCDNA3.1 and visualized 24 h later by confocal microscopy. (b) 293T cells were transfected with 4 μg of Flag-TRAM with or without a plasmid encoding IRF-3 (untagged) as indicated. 24 h later, whole cell lysates were immunoprecipitated with anti–IRF-3, anti-Flag, or anti-CBP, and the immunoprecipitated complexes were immunoblotted for Flag-tagged TRAM and IRF-3. Whole cell lysates (WCL) were also analyzed for Flag-tagged proteins. (c) HEK293 cells were transfected with the RANTES luciferase reporter gene and TRAM (20 ng) and cotransfected with increasing concentrations of IKKɛ-k38a, TBK1-k38a, or IRF3-ΔN from 10, 20, 30, 40, 60, and 80 ng. Luciferase reporter gene activity was measured 24 h after transfection.

Figure 4.

TRAM mediates the TLR4 pathway to IRF-3 and IRF-7. (a) HEK293 cells were transfected with 40 ng of a RANTES reporter construct and cotransfected with TRAM, TRAM-TIR, TRAM-C113H, or TRAM-P112H. (b) TLR4/MD2- and TLR3-expressing HEK293 cell lines were transfected with a luciferase reporter gene containing the Gal4 upstream activation sequence and cotransfected with Gal4–DBD, Gal4–IRF-3, or Gal4–IRF-7 or the RANTES luciferase reporter gene as well as TRAM-C113H or TRIF-ΔNΔC. The next day, cells were stimulated with LPS (10 ng/ml) or dsRNA (50 μg/ml poly IC) or left untreated for ∼8 h, and luciferase reporter gene activity was measured.

Figure 5.

TRAM activates NF-κB and is specific to the TLR4 pathway. (a) HEK293 cells were transfected with 40 ng of an NF-κB reporter construct and cotransfected with TRAM, TRAM-TIR, TRAM-C113H, and TRAM-P112H. (b) TLR-expressing HEK293 stable cell lines were transfected with 40 ng of an NF-κB reporter gene and cotransfected with increasing concentrations of TRAM-C113H. 1 d after transfection, TLR-expressing cells were stimulated with Malp-2 (2 nM), dsRNA (100 μg/ml poly IC), LPS (10 ng/ml), R-848 (10 μM), IL1β (10 ng/ml) or TNFα (10 ng/ml) or left untreated or 8 h, and luciferase reporter gene activity was measured.

Figure 6.

TRAM signaling requires the expression of TRIF. (a) HEK293 cells were transfected with the RANTES luciferase reporter gene, TRAM or TRIF (40 ng), and cotransfected with TRIF-ΔNΔC or TRAM-C113H. Luciferase reporter gene activity was measured 24 h later. (b) 293T cells were transfected with 4 μg of TRAM-CFP or TRIF-CFP and cotransfected with Flag-Mal, Flag-Mal-P125H, or Flag-TRIF. Whole cell lysates were harvested 48 h later and immunoprecipitated with anti-GFP antibody (which also immunoprecipitates cyan fluorescent protein [CFP] or yellow fluorescent protein [YFP]). Immunoprecipitated complexes were resolved by SDS-PAGE and immunoblotted for Flag-tagged adapters. Whole cell lysates (WCL) were also analyzed for CFP- and Flag-tagged proteins by immunoblotting. (c) Stable TLR4^YFP- or TLR3^YFP-expressing HELA cells were transfected with 4 μg of plasmid encoding Flag-Mal, Flag-TRAM, or Flag TRAM-C113H. 48 h later, whole cell lysates were immunoprecipitated with anti-GFP antibody, and immunoprecipitated complexes were immunoblotted for Flag-tagged adapters. Western blotting of lysates demonstrates expression of stable TLRs and transfected adaptor proteins.

Figure 7.

TRAM and TRIF are required for RANTES activation by LPS. (a) 293T cells plated in 24-well plates were transfected with 1 μg of plasmids encoding TRAM-CFP, TRIF-CFP, or Mal-CFP and cotransfected with 50 nM siRNA-TRAM or lamin A/C as indicated. 24 h later, CFP fluorescence was measured by flow cytometry using a 405 nm laser for excitation of CFP. The siRNA-TRAM nearly completely abolished expression of the subpopulation of cells that express CFP. (b) U373–CD14 or TLR3-expressing HEK293 cells were transfected with a RANTES reporter gene and cotransfected with siRNA duplexes as indicated for 36 h. Cells were then stimulated for 8 h with LPS or dsRNA, and luciferase reporter gene activity was measured.