Single-Cell Epitope-Transcriptomics Reveal Lung Stromal and Immune Cell Response Kinetics to Nanoparticle delivered RIG-I and TLR4 Agonists

Abstract

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 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 hours after intranasal administration. Within 4 hours, 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-1β. 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.

Multimodal nanoparticle‐containing modified suberoylanilide hydroxamic acid polymer conjugates to mitigate immune dysfunction in severe inflammation

“…Volcano plots were used to identify differentially expressed genes (DEG) between various treatment groups comparing the log2 fold differences. DEGs were identified as genes with at least 0.5/−0.5 log2 fold differences and an unadjusted p ‐value <0.05 41,42 . p values for NanoString data were obtained from nSolver software using a minimum count of 50 as a cutoff 40 .…”

“…DEGs were identified as genes with at least 0.5/À0.5 log2 fold differences and an unadjusted p-value <0.05. 41,42 p values for NanoString data were obtained from nSolver software using a minimum count of 50 as a cutoff. 40 A total of 134 genes met the inclusion criteria and we identified 92 DEGs for LPS versus no LPS treatment (NT), 7 DEGs for iNP (+LPS) versus LPS, 9 DEGs for iNP-SAHA (+LPS) versus LPS, and 4 DEG for iNP-SAHA (+LPS) versus iNP (+LPS).…”