DNA corona on nanoparticles leads to an enhanced immunostimulatory effect with implications for autoimmune diseases

Abstract

Autoimmune and inflammatory diseases are highly complex, limiting treatment and the development of new therapies. Recent work has shown that cell-free DNA bound to biological microparticles is linked to systemic lupus erythematosus, a prototypic autoimmune disease. However, the heterogeneity and technical challenges associated with the study of biological particles have hindered a mechanistic understanding of their role. Our goal was to develop a well-controlled DNA-particle model system to understand how DNA-particle complexes affect cells. We first characterized the adsorption of DNA on the surface of polystyrene nanoparticles (200 nm and 2 µm) using transmission electron microscopy, dynamic light scattering, and colorimetric DNA concentration assays. We found that DNA adsorbed on the surface of nanoparticles was resistant to degradation by DNase 1. Macrophage cells incubated with the DNA-nanoparticle complexes had increased production of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We probed two intracellular DNA sensing pathways, toll-like receptor 9 (TLR9) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), to determine how cells sense the DNA-nanoparticle complexes. We found that the cGAS-STING pathway is the primary route for the interaction between DNA-nanoparticles and macrophages. These studies provide a molecular and cellular-level understanding of DNA-nanoparticle-macrophage interactions. In addition, this work provides the mechanistic information necessary for future in vivo experiments to elucidate the role of DNA-particle interactions in autoimmune diseases, providing a unique experimental framework to develop novel therapeutic approaches.

Keywords: DNA; autoimmune disease; corona; inflammation; nanoscience.

Fig. 1.

Characterization of NPs and the DNA corona. (A) Preparation of DNA coronas. The structure of the DNA on the NP surface is not known. This schematic is only illustrative. (B) TEM images of NPs with and without DNA coronas comparing short (DNA_S, sheared) and long (DNA_L, unsheared) DNA. (C) Hydrodynamic diameter (left y axis) and polydispersity index (PDI; right y axis) of NPs with DNA_S and DNA_L coronas. Significance was determined using one-way ANOVA with Tukey multiple comparison post hoc. (D) Concentration of DNA_S and DNA_L adsorbed on the surface of NPs. The NP concentration was normalized to 500 pM to allow for comparison between NP diameters. Error bars reflect ±1 SD. Significance was determined using unpaired t tests. ****P < 0.0001, ns = not significant. n = 3 distinct samples.

Fig. 2.

Concentration of DNA present following treatment of DNA–NPs with DNase 1 (0.2 U/µg to 2 U/µg; 30 min, 37 °C). (A) DNA_S (B) DNA_S–NP_{200 nm} (C) DNA_S–NP_{2 µm} (D) DNA_L (E) DNA_L–NP_{200 nm} (F) DNA_L–NP_{2 µm}. Error bars reflect ±1 SD. Significance was measured using one-way ANOVA with Tukey’s multiple comparisons post hoc. **P < 0.01, ***P < 0.001, ****P < 0.0001, nonsignificant comparisons are not displayed. n = 3 distinct samples.

Fig. 3.

Representative TEM images of macrophages incubated with NPs and DNA–NPs (200 nm and 2 µm; 24 h at 37 °C). (A) Cells incubated with 200 nm NPs (bare) or DNA_S and DNA_L coronas. (B) Cells incubated with 2 µm NPs (bare) or DNA_S and DNA_L coronas. (C) i. Representative image of an untreated cell used as a control. ii, 200 nm NPs and iii, 2 µm NPs prepared and stained for TEM using the same embedding, staining, and microtoming method as used for the cells. We also observed internalization of NPs and DNA–NPs using confocal microscopy (SI Appendix, Fig. S4).

Fig. 4.

TNF-α and IL-6 release by macrophages incubated with DNA–NPs (24 h at 37 °C). (A) TNF-α release measured after incubation of cells with short (S; sheared) DNA (green filled circles), DNA_S–NP_{200 nm} (blue filled squares), and DNA_S–NP_{2 μm} (magenta filled triangles), and NPs in the absence of DNA (blue and magenta unfilled squares and triangles). (B) TNF-α release measured after incubation of cells with long (L; unsheared) DNA (green filled circles), DNA_L–NP_{200 nm} (blue filled squares), and DNA_L–NP_{2 μm} (magenta filled triangles), and NPs in the absence of DNA (blue and magenta unfilled squares and triangles). Similar results were observed with human macrophage-like cells (THP-1; SI Appendix, Fig. S5). (C) IL-6 release measured after incubation of cells with short (S; sheared) DNA (green filled circles), DNA_S–NP_{200 nm} (blue filled squares), and DNA_S–NP_{2 μm} (magenta filled triangles), and NPs in the absence of DNA (blue and magenta unfilled squares and triangles). (D) IL-6 release measured after incubation of cells with long (L; unsheared) DNA (green filled circles), DNA_L–NP_{200 nm} (blue filled squares), and DNA_L–NP_{2 μm} (magenta filled triangles), and NPs in the absence of DNA (blue and magenta unfilled squares and triangles). Error bars reflect ±1 SEM. Significance was measured using a two-way ANOVA with Tukey’s multiple comparisons post hoc. ****P < 0.0001, nonsignificant comparisons are not displayed. n = 3 distinct samples.

Fig. 5.

TNF-α and IL-6 release by macrophages incubated (24 h at 37 °C) with NPs and DNA added separately (DNA + NPs) to cells. (A) TNF-α release measured after incubation of cells with short (S; sheared) DNA_S (green filled circles), DNA_S + NP_{200 nm} (blue filled squares), and DNA_S + NP_{2 µm} (magenta filled triangles). (B) TNF-α release measured after incubation of cells with long (L; unsheared) DNA_L (green filled circles), DNA_L + NP_{200 nm} (blue filled squares) and DNA_L + NP_{2 µm} (magenta filled triangles). (C) IL-6 release measured after incubation of cells with DNA_S (green filled circles), DNA_S + NP_{200 nm} (blue filled squares), and DNA_S + NP_{2 µm} (magenta filled triangles). (D) IL-6 release measured after incubation of cells with DNA_L (green filled circles), DNA_L + NP_{200 nm} (blue filled squares), and DNA_L + NP_{2 µm} (magenta filled triangles). Error bars reflect ±1 SEM. Significance was measured using a two-way ANOVA with Tukey’s multiple comparisons post hoc. ****P < 0.0001, nonsignificant comparisons are not displayed. n = 3 distinct samples.

Fig. 6.

Comparison of TNF-α release by cells treated with DNA + NPs (3 µg/mL DNA) separately, DNA–NPs (3 µg/mL DNA), and DNA (3 µg/mL). Data is a repeat of Figs. 4 and 5. Error bars reflect ±1 SEM. Significance was measured using two-way ANOVA with Tukey’s multiple comparisons post hoc. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. n = 3 distinct samples.

Fig. 7.

TNF-α release by macrophages in response to DNA and DNA–NPs treated with DNase 1 (0 to 5 U/µg) (24 h at 37 °C). (A) DNA_S and DNA_S–NPs (1 µg/mL). (B) DNA_L and DNA_L–NPs (1 µg/mL). Error bars reflect ±1 SEM. Significance was measured using one-way ANOVA with Sidak’s multiple comparisons post hoc. **P < 0.01, ***P < 0.001, ns = not significant. n = 3 distinct samples.

Fig. 8.

TNF-α release by macrophages incubated with free DNA (1 µg/mL) and DNA–NPs (1 µg/mL) in the presence of ODN 2088 (0.1 µM), a TLR9 inhibitor. (A) DNA_S and DNA_S–NPs. (B) DNA_L and DNA_L–NPs. Error bars reflect ±1 SEM. Significance was measured using two-way ANOVA with Sidak’s multiple comparisons post hoc. **P < 0.01, nonsignificant comparisons are not displayed. n = 3 distinct samples.

Fig. 9.

TNF-α release by macrophages incubated with free DNA (1 µg/mL) and DNA–NPs (1 µg/mL) (24 h at 37 °C) in the presence of cGAS-STING inhibitors [RU.521 (10 µg/mL) and H-151 (5 µM)]. The inhibitors were added to cells 24 h prior to the DNA or DNA–NPs and remained present during experiments. (A) DNA_S and DNA_S–NPs with RU.521. (B) DNA_L and DNA_L–NPs with RU.521. (C) DNA_S and DNA_S–NPs with H-151. (D) DNA_L and DNA_L–NPs with H-151. Error bars reflect ±1 SEM. Significance was measured using two-way ANOVA with Sidak’s multiple comparisons post hoc. ***P < 0.001, ****P < 0.0001. n = 3 distinct samples.

Fig. 10.

Schematic representation of DNA and DNA–NPs stimulating cells via the cGAS-STING and TLR9 pathways. DNA can be internalized into endosomes and stimulate immune cells through a TLR9-dependent process or enter the cytosol and interact through a cGAS-STING-dependent pathway. Our data suggests that DNA–NPs, stimulate cells though a cGAS-STING-dependent pathway. The stimulation of cells results in the release of cytokines such as TNF-α and IL-6.