TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation

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.

TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). PAMPs and PAMP-analogs are also used as immune-adjuvants to enhance vaccine efficacy by activating various Pattern Recognition Receptors (PRRs), like Toll-like receptors (TLRs). Various combinations of PAMP adjuvants can trigger synergistic immune responses, but the underlying molecular mechanisms driving that synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with MPLA (TLR4-adjuvant) and CpG (TLR9-adjuvant) as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for the integrated, dual-adjuvant immune response. PLP-based co-presentation of MPLA and CpG to mouse bone marrow derived antigen-presenting cells (BM-APCs) elicited synergistic Type-I Interferon (IFN-β) and IL-12p70 responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that the adapter protein MyD88 and the Interferon-Regulatory-Factor-5 (IRF-5), but not the canonical factors IRF-3 or IRF-7, were necessary for production of both IFN-β and IL12p70. TRIF signaling was required to elicit the synergistic response; the absence of TRIF abolished synergy. Importantly, both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling (TRIF-TRAF-IRF5 pathway kinetics) drove the observed synergy. These results identify not only the key signaling mechanism that cooperates to generate a combinatorial response to MPLA-CpG dual engagement in BM-APCs, but they also underscore the critical role that signaling kinetics and biophysical presentation plays in integrated responses to combination adjuvants.

Overcoming the Limits of Flash Nanoprecipitation: Effective Loading of Hydrophilic Drug into Polymeric Nanoparticles with Controlled Structure

Flash nanoprecipitation (FNP) is a widely used technique to prepare particulate carriers based on various polymers, and it was proven to be a promising technology for the industrial production of drug loaded nanoparticles. However, up to now, only its application to hydrophobic compounds has been deeply studied and the encapsulation of some strongly hydrophilic compounds, such as caffeine, remains a challenge. Caffeine loaded poly-ε-caprolactone (PCL) nanoparticles were produced in a confined impinging jet mixer using acetone as the solvent and water as the antisolvent. Caffeine was dissolved either in acetone or in water to assess the effects of two different process conditions. Nanoparticles properties were assessed in terms of loading capacity (LC%), encapsulation efficiency (EE%), and in vitro release kinetics. Samples were further characterized by dynamic light scattering, scanning electron microscopy, X-ray photo electron spectroscopy, and infrared spectroscopy to determine the size, morphology, and structure of nanoparticles. FNP was proved an effective technique for entrapping caffeine in PCL and to control its release behavior. The solvent used to solubilize caffeine influences the final structure of the obtained particles. It was observed that the active principle was preferentially adsorbed at the surface when using acetone, while with water, it was embedded in the matrix structure. The present research highlights the possibility of extending the range of applications of FNP to hydrophilic molecules.