Mycobacterium tuberculosis ESAT6 Drives the Activation and Maturation of Bone Marrow-Derived Dendritic Cells via TLR4-Mediated Signaling

6-kDa early secretory antigenic target (ESAT6), a virulent factor of Mycobacterium tuberculosis, is involved in immune regulation. However, the underlying mechanism behind the activation and maturation of dendritic cells (DCs) by ESAT6 remains unclear. In this study, we investigated the effect on TLRs signaling on the regulation of ESAT6-induced activation and maturation of DCs. ESAT6 induced production of IL-6, TNF-α, and IL-12p40 in bone marrow-derived dendritic cells (BMDCs) from wild-type and TLR2-deficient mice, with this induction abolished in TLR4-deficient cells. NF-κB is essential for the ESAT6-induced production of the cytokines in BMDCs. TLR4 was also required for ESAT6-induced activation of NF-κB and MAPKs in BMDCs. ESAT6 additionally upregulated the expression of surface molecules CD80, CD86, and MHC-II, and also promoted the ability of CD4⁺ T cells to secrete IFN-γ via the TLR4-dependent pathway. Our findings suggest that TLR4 is critical in the activation and maturation of DCs in response to ESAT6.

p62 Negatively Regulates TLR4 Signaling via Functional Regulation of the TRAF6-ECSIT Complex

Sequestosome 1 (SQSTM1, p62), a ubiquitin binding protein, plays a role in cell signaling, oxidative stress, and autophagy. However, its functional role in inflammatory signaling is controversial. Recent studies have shown that p62 is negatively implicated in inflammatory responses. But, the precise molecular mechanisms by which p62 regulates inflammatory responses remain unclear. In this study, we report on a new regulatory role for p62 in TLR4-mediated signaling. p62 overexpression led to the suppression of NF-κB activation and the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β in response to TLR4 stimulation. In contrast, p62(−/−) mouse embryonic fibroblast (MEF) cells exhibited marked enhancement of NF-κB activation and production of pro-inflammatory cytokines by TLR4 stimulation, compared to p62(+/+) MEF cells. Additionally, the TLR4-induced activation of signal transduction was significantly augmented in p62(−/−) MEF cells, indicating that p62 was negatively implicated in TLR4-mediated signaling. Biochemical studies revealed that p62 interacted with the internal domain of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which is critical for associating with the TNF receptor associated factor 6 (TRAF6)-ECSIT complex to activate NF-κB in TLR4 signaling. Interestingly, p62-ECSIT interaction inhibited the interaction between TRAF6 and ECSIT and attenuated the ubiquitination of ECSIT. Furthermore, upon LPS challenge, the mortality of p62(−/−) (p62-knockout) mice was markedly enhanced compared to p62(+/+) (p62 wild-type) mice. Taken together, our data demonstrate that p62 negatively regulated TLR4 signaling via functional regulation of the TRAF6-ECSIT complex.