Flightless I Homolog Negatively Modulates the TLR Pathway

Abstract

To date, much of our knowledge about the signaling networks involved in the innate immune response has come from studies using nonphysiologic model systems rather than actual immune cells. In this study, we used a dual-tagging proteomic strategy to identify the components of the MyD88 signalosome in murine macrophages stimulated with lipid A. This systems approach revealed 16 potential MyD88-interacting partners, one of which, flightless I homolog (Fliih) was verified to interact with MyD88 and was further characterized as a negative regulator of the TLR4-MyD88 pathway. Conversely, a reduction in endogenous Fliih by small-interfering RNA enhanced the activation of NF-κB, as well as cytokine production by LPS. Results from immunoprecipitation and a two-hybrid assay further indicated that Fliih directly interfered with the formation of the TLR4-MyD88 signaling complex. These results in turn suggest a new basis for the regulation of the TLR pathway by Fliih.

Nucleoredoxin Negatively Regulates Toll-like Receptor 4 Signaling via Recruitment of Flightless-I to Myeloid Differentiation Primary Response Gene (88)

“…NRX Links Fli-I to MyD88-Recent reports have shown the involvement of Fli-I in the TLR4/MyD88 pathway (17,20). As we identified Fli-I as a novel binding partner of NRX, we hypothesized that NRX may affect the TLR4/MyD88 pathway.…”

“…On the basis of the results of co-immunoprecipitation analyses, Wang et al (20) showed that MyD88 and Fli-I form a complex; however, whether the interaction is direct or not was not determined. Using purified recombinant proteins, we clearly showed that the association between MyD88 and Fli-I is not direct but requires NRX as a link (Fig.…”

“…It is reported that co-immunoprecipitation between Fli-I and MyD88 was enhanced upon LPS stimulation (20). Therefore, we performed immunoprecipitation analyses with LPS-treated WT or NRX Ϫ/Ϫ MEFs to see the complex formation between endogenous MyD88 and Fli-I in these cells.…”

“…Signaling Pathway with Fli-I-Because NRX is required for Fli-I⅐MyD88 complex formation, we next performed NF-B reporter assays to evaluate any functional role of NRX in the TLR4/MyD88 signaling pathway, which is reportedly suppressed by Fli-I (20). Ectopic expression of either Fli-I or NRX alone resulted in a marginal suppression of LPS-induced NF-B activation.…”

“…TLR4 recognizes lipopolysaccharide (LPS) and recruits IRAK1 and IRAK4 through MyD88, resulting in the activation of transcription factor NF-B. Fli-I co-immunoprecipitates with MyD88 and inhibits the LPS-stimulated activation of the TLR4/MyD88 pathway (20), but its molecular detail is poorly characterized.…”