Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). Combinations of PAMPs can trigger synergistic immune responses, but the underlying molecular mechanisms of synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with monophosphoryl lipid A (MPLA) and CpG as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for dual adjuvant immune responses. PLP-based co-delivery of MPLA and CpG to GM-CSF–driven mouse bone marrow–derived antigen-presenting cells (BM-APCs) elicited synergistic interferon-β (IFN-β) and interleukin-12p70 (IL-12p70) responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that MyD88 and interferon regulatory factor 5 (IRF5) were necessary for IFN-β and IL-12p70 production, while TRIF signaling was required for the synergistic response. Both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling drove the synergy. These results identify the key mechanisms of synergistic Toll-like receptor 4 (TLR4)–TLR9 co-signaling in mouse BM-APCs and underscore the critical role of signaling kinetics and biophysical properties on the integrated response to combination adjuvants.

对病原体的先天免疫反应是由多种病原体相关分子模式 (PAMP) 的共同呈现驱动的。 PAMP 的组合可以触发协同免疫反应，但协同作用的潜在分子机制尚不清楚。在这里，我们使用共同负载单磷酰脂质 A (MPLA) 和 CpG 作为病原体样颗粒 (PLP) 的合成颗粒载体来剖析负责双重佐剂免疫反应的信号通路。基于 PLP 的 MPLA 和 CpG 共同递送至 GM-CSF 驱动的小鼠骨髓源性抗原呈递细胞 (BM-APC) 引起协同干扰素-β (IFN-β) 和白介素-12p70 (IL-12p70) 反应，这受到 PLP 的生物物理特性的强烈影响。从机制上讲，我们发现 MyD88 和干扰素调节因子 5 (IRF5) 对于 IFN-β 和 IL-12p70 的产生是必需的，而 TRIF 信号传导是协同反应所必需的。下游 TRAF6 和 IRF5 信号传导的动力学和强度都推动了协同作用。这些结果确定了小鼠 BM-APC 中 Toll 样受体 4 (TLR4)-TLR9 协同信号传导的关键机制，并强调了信号动力学和生物物理特性对组合佐剂的综合反应的关键作用。