Single-cell epitope-transcriptomics reveal lung stromal and immune cell response kinetics to nanoparticle-delivered RIG-I and TLR4 agonists.

Lung-resident and circulatory lymphoid, myeloid, and stromal cells, expressing various pattern recognition receptors (PRRs), detect pathogen- and danger-associated molecular patterns (PAMPs/DAMPs), and defend against respiratory pathogens and injuries. Here, we report the early responses of murine lungs to nanoparticle-delivered PAMPs, specifically the retinoic acid-inducible gene I (RIG-I) agonist poly-U/UC (PUUC), with or without the TLR4 agonist monophosphoryl lipid A (MPLA). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), we characterized the responses at 4 and 24 h after intranasal administration. Within 4 h, ribosome-associated transcripts decreased in both stromal and immune cells, followed by widespread interferon-stimulated gene (ISG) expression. Using RNA velocity, we show that lung-neutrophils dynamically regulate the synthesis of cytokines like CXCL-10, IL-1α, and IL-1ß. Co-delivery of MPLA and PUUC increased chemokine synthesis and upregulated antimicrobial binding proteins targeting iron, manganese, and zinc in many cell types, including fibroblasts, endothelial cells, and epithelial cells. Overall, our results elucidate the early PAMP-induced cellular responses in the lung and demonstrate that stimulation of the RIG-I pathway, with or without TLR4 agonists, induces a ubiquitous microbial defense state in lung stromal and immune cells. Nanoparticle-delivered combination PAMPs may have applications in intranasal antiviral and antimicrobial therapies and prophylaxis.

TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells in vitro and generate unique cellular immune responses in influenza vaccination.

Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection - partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-κB and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4-T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also, in the spleen, T lymphopenia, especially after in vitro restimulation with dual adjuvants, was observed, indicating highly antigen-reactive T cells. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination.

Conjunctive measurement of aquatic exercise in an office setting.

The Hydrotrack, a treadmill immersed in a water bath, is designed to provide an in-office approach to aquatic therapy. 24 clinical clients in an outpatient sports medicine practice were invited to use the Hydrotrack as part of their rehabilitation treatment. Our purpose was to describe the way(s) in which the Hydrotrack is useful in rehabilitation treatment. Exercise protocols designed by a physical therapist were used for each subject. We monitored an external criterion, heart rate, to measure the subjects' exertion during the exercise process. Self-reported ratings of exercise efficacy and exertion were also used to quantify the subjects' performance on the Hydrotrack. Heart-rate values and self-report ratings supported the usefulness of this device in physical therapy.