Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex

Abstract

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-β expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.

Boosting mTOR-dependent autophagy via upstream TLR4-MyD88-MAPK signalling and downstream NF-κB pathway quenches intestinal inflammation and oxidative stress injury

“…To uncover the potential pathways that orchestrate LPS-induced mTOR activation and autophagy suppression in the intestinal epithelium, we focused on toll-like receptors (TLRs), which provoke a quick activation of innate immunity by inducing secretion of pro-inflammatory cytokines. TLR4, in particular, is a membrane receptor for LPS and abundantly expressed in various epithelial cells [ 23 , 24 ]. Notably, treatment with LPS in Caco-2 and HT-29 cells significantly enhanced the protein levels of TLR4, MyD88, p-p38 MAPK, p-JNK, p-ERK and p-NF-κB p65 ( Fig.…”

Homeostatic and pathogenic roles of GM 3 ganglioside molecular species in TLR 4 signaling in obesity

“…The EMBO Journal 39: e101732 | 2020 TLR4 ligands (Wang et al, 2016;Chan et al, 2017;Okamoto et al, 2017). Utilization of naturally occurring GM3 species may prevent production of autoantibodies (Bowes et al, 2002).…”

The Potential Role of RP105 in Regulation of Inflammation and Osteoclastogenesis During Inflammatory Diseases

“…Although the RP105 signaling pathway depends on TLR4, TLR2, and TLR9, the RP105 is being increasingly considered as a negative regulator of downstream signaling pathways of these receptors, including NF-κB, MAPKs, and Akt-PI3K in monocytes and immune cells ( Park et al, 2017 ). RP105 acts as a physiological and endogenous inhibitor of TLR4 in macrophages ( Yu et al, 2015 ; Carreras-Gonzalez et al, 2018 ), DCs ( Gorczynski et al, 2006 ; Tada et al, 2008 ; Okamoto N. et al, 2017 ), and monocytes ( Honda et al, 2007 ; Wezel et al, 2015 ; Zhang et al, 2019 ). RP105 negatively regulates TLR4-mediated IFN-β expression ( Okamoto N. et al, 2017 ), signaling elements such as p38MAPK ( Kikuchi et al, 2018 ), kinase phosphorylation levels of c-Jun/AP1 ( Dong et al, 2019 ) and Akt ( Yu et al, 2015 ), and production of proinflammatory cytokines such as IL-6 ( Wezel et al, 2016 ), IL-1β ( Chen et al, 2019 ), and TNF-α ( Louwe et al, 2014 ; Carreras-Gonzalez et al, 2018 ).…”

“…RP105 acts as a physiological and endogenous inhibitor of TLR4 in macrophages ( Yu et al, 2015 ; Carreras-Gonzalez et al, 2018 ), DCs ( Gorczynski et al, 2006 ; Tada et al, 2008 ; Okamoto N. et al, 2017 ), and monocytes ( Honda et al, 2007 ; Wezel et al, 2015 ; Zhang et al, 2019 ). RP105 negatively regulates TLR4-mediated IFN-β expression ( Okamoto N. et al, 2017 ), signaling elements such as p38MAPK ( Kikuchi et al, 2018 ), kinase phosphorylation levels of c-Jun/AP1 ( Dong et al, 2019 ) and Akt ( Yu et al, 2015 ), and production of proinflammatory cytokines such as IL-6 ( Wezel et al, 2016 ), IL-1β ( Chen et al, 2019 ), and TNF-α ( Louwe et al, 2014 ; Carreras-Gonzalez et al, 2018 ). Targeted inhibition of MD-1 stimulates the TLR4/MyD88/NF-κB signaling axis in colitis-induced mice ( Chen et al, 2019 ), which also proves the role of RP105 as a TLR4 inhibitor.…”