After recognition of microbial products by members of the Toll-like receptor (TLR) family, the adaptor molecule MYD88 initiates intracellular signalling cascades that result in the activation of host defence mechanisms. However, recent studies have shown a MYD88-independent signalling pathway downstream of TLR3 and TLR4 that is regulated by the adaptor molecule TRIF (also known as TICAM1) and leads to the activation of interferon regulatory factor 3 (IRF3), as well as signals that sustain activation of nuclear factor-κB (NF-κB). Now, three independent studies have identified TRAM (also known as TICAM2) as an adaptor molecule that is a key link in the TRIF-dependent TLR4, but not TLR3, signalling cascade.

Previously identified TLR adaptor molecules contain Toll–interleukin 1 receptor (TIR) domains and each of the three groups identified TRAM using database searches for new TIR-domain-containing molecules. Oshiumi et al. and Fitzgerald et al. used a yeast two-hybrid assay and/or co-immunoprecipitation studies to show that TRAM interacts directly with both TRIF and TLR4, but not TLR3, implicating TRAM as a specific component of the TLR4-signalling cascade.

To determine the role of TRAM in TLR4 signalling, all three groups used the same approach — analysing the effect of ectopic overexpression of TRAM — and all observed activation of IRF3 and NF-κB, as well as activation of the promoter for the gene encoding interferon-β (IFN-β).

Oshiumi et al. and Fitzgerald et al. went on to generate a series of TRAM mutants, which they used to show that TRAM is crucial for IRF3 and NF-κB activation after ligation of TLR4 by lipopolysaccharide (LPS), but not TLR3 triggering with double-stranded RNA. Further evidence for the importance of TRAM in the TLR4-signalling pathway was provided by their demonstration that marked reduction of the level of TRAM by small interfering RNA impaired IRF3 and NF-κB activation in response to TLR4 triggering, but not TLR3 ligation.

Yamamoto et al. examined the physiological role of TRAM in the TLR-signalling cascade by generating Tram-knockout mice. Tram-deficient cells responded normally to signalling through TLR3; however the production of pro-inflammatory cytokines and IFN-β was substantially impaired after LPS triggering of TLR4, as was the activation of B cells. These functional defects coincided with inefficient activation of signalling molecules downstream of TRAM, including IRF3, and an inability to sustain NF-κB activation. By contrast, activation of the MYD88-signalling pathway was intact in the TRAM-deficient cells. So, in vivo, TRAM is essential for the MYD88-independent signalling cascade after LPS ligation of TLR4. This cascade induces B-cell activation and the production of IFN-β; however, TRAM and MYD88 signals are required for the secretion of pro-inflammatory cytokines in response to LPS.

These studies provide invaluable insight into the TRIF-regulated MYD88-independent response generated after triggering of both TLR3 and TLR4. Ligation of these molecules induces distinct host responses and this specificity might be a result of TLR4 using the adaptor molecule TRAM to initiate signalling through TRIF, whereas TLR3 signals directly to TRIF.