Selective TRIF-dependent signaling by a synthetic toll-like receptor 4 agonist

Abstract

In response to ligand binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation-2 (MD-2) receptor complex, two major signaling pathways are activated that involve different adaptor proteins. One pathway depends on myeloid differentiation marker 88 (MyD88), which elicits proinflammatory responses, whereas the other depends on Toll-IL-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF), which elicits type I interferon production. Here, we showed that the TLR4 agonist and vaccine adjuvant CRX-547, a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic lipid A mimetics, displayed TRIF-selective signaling in human cells, which was dependent on a minor structural modification to the carboxyl bioisostere corresponding to the 1-phosphate group on most lipid A types. CRX-547 stimulated little or no activation of MyD88-dependent signaling molecules or cytokines, whereas its ability to activate the TRIF-dependent pathway was similar to that of a structurally related inflammatory AGP and of lipopolysaccharide from Salmonella minnesota. This TRIF-selective signaling response resulted in the production of substantially less of the proinflammatory mediators that are associated with MyD88 signaling, thereby potentially reducing toxicity and improving the therapeutic index of this synthetic TLR4 agonist and vaccine adjuvant.

Fig. 1

CRX-547 stimulates production of similar amounts of TRIF-dependent cytokines and chemokines by human primary monocytes and monocyte-derived DCs to those stimulated by CRX-527, but reduced amounts of MyD88-dependent cytokines. (A) Structures of the lipid A mimetics CRX-527 and CRX-547. The molecules differ only in the configuration of the aglycon stereocenter (indicated by an arrow): CRX-527 has the _L configuration, whereas CRX-547 has the _D configuration. (B and C) The abilities of CRX-527, CRX-547, and S. minnesota Re595 LPS to stimulate the production of MyD88-dependent (TNF-α) and TRIF-dependent (RANTES and IP-10) cytokines and chemokines were compared in (B) human PBMC–derived monocytes and (C) monocyte-derived DCs. When data from three independent experiments with monocytes and monocyte-derived DCs were analyzed, the difference in induction of TNF-α between CRX-527 and CRX-547 was significantly higher than the difference in the induction of RANTES and IP-10 [P < 0.01; analysis of variance (ANOVA) with Tukey post hoc testing]. Graphs are from one donor and are representative of three independent donors with three replicate samples from each.

Fig. 2

Dominant-negative adaptor protein mutants inhibit MyD88- and TRIF-dependent cytokine and chemokine production by human macrophages. THP-1 cells were differentiated into macrophages in culture and then were mock-transfected (UNTRFX) or were transfected with an empty plasmid control (PC), a plasmid expressing dominant-negative MyD88 (MyD88-DN), or a plasmid encoding dominant-negative TRIF (TRIF-DN). (A and B) The transfected cells were then treated with vehicle, CRX-527 (1 μM), or CRX-547 (1 μM) for 14 hours and then supernatants were analyzed by Luminex for the presence of (A) TNF-α and (B) RANTES. Data are means ± SEM from three independent experiments with three replicates each and were analyzed by ANOVA with Tukey post hoc testing (*P < 0.05; **P < 0.01).

Fig. 3

Stimulation of RANTES production by CRX-547 depends on endocytosis. THP-1 cells were differentiated into macrophages in culture, pretreated with the endocytosis (dynamin) inhibitor Dynasore (80 μM) for 60 min in serum-free medium with rhLBP (20 ng/ml), and then treated with the indicated range of concentrations of CRX-527, CRX-547, or vehicle for 14 hours. (A and B) Supernatants were collected and analyzed by Luminex for the presence of (A) TNF-α and (B) RANTES. Data are means ± SEM from three replicates from three independent experiments and were analyzed by ANOVA with Tukey post hoc testing (*P < 0.05; **P < 0.01).

Fig. 4

CRX-547 induces less nuclear translocation of NF-κB than CRX-527 in MonoMac6 cells. (A) Gating strategy used for the analysis of NF-κB nuclear translocation (top panels). Live cells were distinguished from dead cells by the presence of a single, well-formed DRAQ5-containing nucleus (Live Cells) after which we gated on live cells (R1) and NF-κB–containing cells (Focused R1) (bottom panels). Nuclear translocation was defined by the colocalization of NF-κB (green) with the nuclear stain DRAQ5 (red), which is indicated by a bright yellow nucleus in the overlaid image of the R1- and NF-κB–containing (Focused R1) cells. (B) Analysis of the nuclear translocation of NF-κB in MonoMac6 cells after incubation with increasing concentrations of CRX-527, CRX-547, vehicle (VEH), or medium for 5, 15, 30, or 120 min. Data are representative of three separate experiments with 3000 to 5000 live focused cells recorded in each experiment.

Fig. 5

CRX-547 stimulates less NF-κB–inducible promoter activity in HEK 293–hTLR4/hMD-2/hCD14 cells than does CRX-527 or S. minnesota Re595 LPS. Stably transfected HEK 293 cells were transiently transfected with a plasmid encoding an NF-κB–inducible reporter gene (encoding SEAP) and were treated with the indicated concentrations of CRX-527, CRX-547, or LPS for 24 hours. Supernatants were assayed for normalized SEAP activity. Data are means ± SEM for three replicates from three independent experiments and were analyzed by ANOVA with Tukey post hoc analysis (*P < 0.05).

Fig. 6

CRX-547, but not CRX-527, selectively activates TRIF-dependent rather than MyD88-dependent signaling proteins. (A and B) Lysates from human primary PBMC-derived, IFN-γ–prestimulated monocytes that were treated with CRX-547, CRX-527, or vehicle for 4 hours were analyzed by Western blotting for the presence of pIRF3 and total IRF3 (TRIF pathway) as well as for IRAK1 (MyD88 pathway). (A) Samples from CRX-547–treated cells showed similar kinetics and extent of activation of IRF3, as assessed by measurement of pIRF3 amounts, but less disappearance of IRAK1 than did samples from CRX-527–stimulated cells. Densitometric analysis (ImageJ) was used to calculate (B) the fold increase in the amount of pIRF3 as a percentage of total IRF3 (pIRF + IRF) and (C) the fold decrease in IRAK1 after treatment with CRX-527 and CRX-547. Data are the average ± SEM from three independent donors and were analyzed by one-way ANOVA with Tukey post hoc analysis (*P < 0.05).

Fig. 7

CRX-547 is a competitive inhibitor of CRX-527 for signaling through the TLR4-MyD88 pathway. Human primary monocytes were treated with a range of concentrations of CRX-527 after they were pretreated for 15 min with one of three concentrations (0.016, 4, or 100 nM) of CRX-547. (A and B) The concentrations of (A) TNF-α and (B) RANTES in the culture media were determined by Luminex analysis. (C) A Schild regression analysis plot of the shift in log EC₅₀ (DR-1) for each concentration of CRX-547 versus the log of the concentration of CRX-547 was used to calculate the affinity of CRX-547 on the basis of inhibition of CRX-527–induced TNF-α production (K_b,app).