Transcriptomic analysis of the innate immune response to in vitro transfection of plasmid DNA

Abstract

The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA sequencing to identify genes that may influence transfection efficiency in four different cell types (PC-3, Jurkat, HEK-293T, and primary T cells). The highest transfection efficiency was observed in HEK-293T cells, which upregulated only 142 genes with no known antiviral functions after transfection with lipofectamine. Lipofection upregulated 1,057 cytokine-stimulated genes (CSGs) in PC-3 cells, which exhibited a significantly lower transfection efficiency. However, when PC-3 cells were transfected in serum-containing media or electroporated, the observed transfection efficiencies were significantly higher while the expression levels of cytokines and CSGs decreased. In contrast, lipofection of Jurkat and primary T cells only upregulated a few genes, but several of the antiviral CSGs that were absent in HEK-293T cells and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain the relatively low Lipofection efficiencies observed with T cells (8%-21% GFP⁺). Indeed, overexpression of one CSG (IFI16) significantly decreased transfection efficiency in HEK-293T cells.

Keywords: IFI16; IKBKE; MT: Oligonucleotides: Therapies and applications; MYD88; STING; cytokine-stimulated genes; cytokines; innate immune response; plasmid.

Graphical abstract

Figure 1

Overview of the innate immune response to foreign DNA pDNA can be recognized by several DNA sensors (yellow), which trigger a cascade of kinases (green) and transcription factors (purple) that culminates in the expression of cytokines that are secreted and bind to cognate receptors (gray) that induce the expression of cytokine stimulated genes (blue) that can trigger apoptosis or inhibit transgene expression in a variety of ways.

Figure 2

Differences in transfection efficiency and transcriptomes between cell lines transfected with lipofectamine LTX in SFMd (A) Transfection efficiency (%GFP⁺ cells) and transgene expression levels (mean GFP) for each cell line at 24 h after transfection with lipofectamine LTX and pEF-GFP pDNA in SFM. Representative flow cytometry histograms and fluorescent microscopy images for each cell line are also shown in Figure S1. Error bars indicate standard deviation of the triplicate samples. (B) Total number of upregulated and downregulated DEGs observed in each cell line after lipofection in SFM. DEGs were defined as having at least a 2-fold change in TPM that was statistically significant (p_adj < 0.05) over the course of three independent experiments. (C–F) Gene expression levels (TPMs) in HEK-293T cells (C), PC-3 cells (D), Jurkat T cells (E), and primary CD3⁺ T cells (F) that were either transfected with lipofectamine LTX (y axis) or not transfected (x axis). All TPM values are averaged from three separate transfections and corresponding mRNA-sequencing experiments. Green triangles indicate genes that were significantly upregulated in transfected cells (p_adj < 0.05), red circles indicate downregulated genes (p_adj < 0.05), and gray squares represent unaffected genes.

Figure 3

Effects of serum and electroporation on transfection efficiency and the PC-3 transcriptome (A) Effects of serum on IFN λ1/3 expression in PC-3 cells. Asterisks (∗) indicate significant (p < 0.05 as determined by Student’s t-test) increases in IFNλ levels after transfection relative to untransfected control cells, while carets (ˆ) indicate significant increases between ELISA measurements at 6 and 24 h after transfection. (B) Transfection efficiencies (green bars, left axis) and the number of significantly upregulated genes (red bars, right axis) observed after transfecting PC-3 cells with lipofectamine in SFM or SCM or electroporation in SCM. Asterisks indicate significant differences in transfection efficiency (∗significantly higher than lipofection in SFM; ∗∗significantly higher than lipofection in SFM and SCM, p < 0.05 as determined by Student’s t-test). (C/D) Gene expression levels (TPM) for PC-3 cells that were lipofected (C) or electroporated (D) in SCM. All TPM values are averaged from three separate transfections and corresponding mRNA-sequencing experiments. Green triangles indicate genes that were significantly upregulated in transfected cells, red circles indicate downregulated genes, and gray squares represent unaffected genes. All error bars indicate the standard deviation of the measurements for each bar.

Figure 4

Effects of pDNA amounts during transfection on the PC-3 transcriptome (A) Transfection efficiencies obtained with PC-3 cells using lipofectamine (2.75 μL/well) and pDNA (0.1 or 1.0 μg), measured at either 1 or 2 days after transfection. Error bars indicate the standard deviation from each triplicate of samples. (B) A summary of the number of genes that were upregulated (green bars) or downregulated (red bars) after each type of transfection. ∗Significant differences in transfection efficiency. (C) Gene expression levels (TPM) for PC-3 cells that were lipofected with either 0.1 or 1.0 μg pDNA. Green triangles indicate genes that were significantly upregulated in the cells that received the higher dose of pDNA, while red circles indicate genes that were downregulated in those cells.

Figure 5

Potential antagonists of transgene expression Expression levels (TPM) of four representative CSGs that were observed to be absent or expressed at low levels in HEK-293T cells, upregulated during lipofection in PC-3 cells, and constitutively expressed at relatively high levels in Jurkat and primary T cell lines. Error brs indicate the standard deviation from each triplicate.

Figure 6

Effects of IFI16 overexpression on pEF-GFP transfection efficiency (%GFP⁺ cells) in HEK-293T cells Untransfected negative control cells were not transfected, while positive control cells were only transfected with pEF-GFP (green box). The blue box represents cells that were transfected with the luciferase expression plasmid pGL4.50 one day prior to transfection with pEF-GFP, while the red box represents cells that were transfected with the IFI16 expression plasmid pIFI16-FL one day before transfection. Horizontal lines within boxes represent the means for each group, while boxes show the interquartile range, and the entire dataset is contained within the whiskers. Letters (a, b, c) indicate samples with significantly different transfection efficiencies (n = 18 for each sample, p < 0.05 using a Wilcoxon rank-sum test).