Epigenetic modulation of the source neutrophils by ITA-LNPs. (A) Schematic of the in vivo–in vitro experiments yielding epigenetically modulated neutrophils from the bone marrow. (B) Cytokines in cell culture supernatants from experiments in panel A. (C) RNA sequencing was performed on neutrophils obtained as in panel A after 1 hour of LPS stimulation. Principal component analysis is shown. (D) GO analysis from RNA-sequencing data sets in neutrophils obtained as in panel C. (E) Volcano plot indicating significantly differentially expressed genes in ITA-LNP– vs Ctrl-LNP–treated neutrophils from aforementioned experiments. (F) TF enrichment analysis using ChEA3 examining co-occurrence of TFs from multiple orthogonal omics data sets. (G) Immunoblotting for various ac histone targets in lysates from primary neutrophils treated with Ctrl- or ITA-LNPs and 100 ng/mL LPS in culture for 3 hours at 1 μM. (H) Densitometry quantification of blots in panel G, n = 2 per group, mean value is depicted. (I) Immunoblotting for H4K12ac from the same experiments as in panel G. (J) Assay for transposase-accessible chromatin with sequencing (ATACseq) heat maps from experiments in panel G. (K) Genome browser tracks for selected gene targets from ATACseq experiments in panel J. (L) hypergeometric optimization of motif enrichment analysis from peak calling. Most significant motifs with target to background ratios are shown. Statistical analysis is through a 2-tailed t test in panel B comparing only LNP treatments in the LPS group. ac, acetylated; bp, base pairs; Cbf1, C-repeat/DRE binding factor 1; CXCL1, C-X-C motif chemokine ligand 1; NFY, nuclear transcription factor Y; PBS, phosphate-buffered saline; PC1/2, principal component 1 and 2; RUNX2, runt-related transcription factor 2; Sfpi1, Spi-1 proto-oncogene; TBR, target to background ratio; WT, wild type.

Epigenetic modulation of the source neutrophils by ITA-LNPs. (A) Schematic of the in vivo–in vitro experiments yielding epigenetically modulated neutrophils from the bone marrow. (B) Cytokines in cell culture supernatants from experiments in panel A. (C) RNA sequencing was performed on neutrophils obtained as in panel A after 1 hour of LPS stimulation. Principal component analysis is shown. (D) GO analysis from RNA-sequencing data sets in neutrophils obtained as in panel C. (E) Volcano plot indicating significantly differentially expressed genes in ITA-LNP– vs Ctrl-LNP–treated neutrophils from aforementioned experiments. (F) TF enrichment analysis using ChEA3 examining co-occurrence of TFs from multiple orthogonal omics data sets. (G) Immunoblotting for various ac histone targets in lysates from primary neutrophils treated with Ctrl- or ITA-LNPs and 100 ng/mL LPS in culture for 3 hours at 1 μM. (H) Densitometry quantification of blots in panel G, n = 2 per group, mean value is depicted. (I) Immunoblotting for H4K12ac from the same experiments as in panel G. (J) Assay for transposase-accessible chromatin with sequencing (ATACseq) heat maps from experiments in panel G. (K) Genome browser tracks for selected gene targets from ATACseq experiments in panel J. (L) hypergeometric optimization of motif enrichment analysis from peak calling. Most significant motifs with target to background ratios are shown. Statistical analysis is through a 2-tailed t test in panel B comparing only LNP treatments in the LPS group. ac, acetylated; bp, base pairs; Cbf1, C-repeat/DRE binding factor 1; CXCL1, C-X-C motif chemokine ligand 1; NFY, nuclear transcription factor Y; PBS, phosphate-buffered saline; PC1/2, principal component 1 and 2; RUNX2, runt-related transcription factor 2; Sfpi1, Spi-1 proto-oncogene; TBR, target to background ratio; WT, wild type.

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