Mitochondrial DNA induces inflammation and increases TLR9/NF-κB expression in lung tissue

Abstract

Mitochondrial DNA (mtDNA) contains unmethylated CpG motifs that exhibit immune stimulatory capacities. The aim of this study was to investigate whether mtDNA activates the Toll-like receptor 9 (TLR9)/nuclear factor-κB (NF-κB) pathway, thereby contributing to post-traumatic systemic inflammatory response syndrome (SIRS) and lung injury in rats. The effects of mtDNA on macrophage culture were examined in order to elucidate the putative cellular mechanisms. Rats and macrophage cultures were treated with phosphate-buffered saline, nuclear DNA, or mtDNA for 2, 4, 8 and 24 h. Histological analysis of lung tissue was undertaken following hematoxylin and eosin staining, and cytokine levels were assessed by ELISA. NF-κB and IκB-α phosphorylation levels, as well as TLR9 protein expression were determined by western blot analysis; NF-κB, IκB-α and TLR9 mRNA levels were analyzed by RT-PCR. A greater degree of inflammation and lung injury was observed in response to mtDNA. In addition, mtDNA increased serum tumor necrosis factor-α, interleukin (IL)-6 and IL-10 levels in vivo and increased their secretion by cultured macrophages (p<0.05). In lung tissue, mtDNA increased NF-κB, IκB-α and TLR9 mRNA levels (p<0.05); it also increased phosphorylated NF-κB p65 and TLR9 protein levels in the macrophage cultures. Thus, mtDNA may be part of the danger-associated molecular patterns, contributing to the initiation of sterile SIRS through the activation of the TLR9/NF-κB pathway and the induction of pro-inflammatory cytokine production.

Figure 1

Histological analysis of lung tissue. Representative images of lung tissue from (A) the phosphate-buffered saline (PBS) group treated with intravenous sterile PBS alone, (B) the nuclear DNA (nDNA) group treated with 10 μg nDNA, and (C) the mitochondrial DNA (mtDNA) group treated with 10 μg mtDNA after 8 h. (D) Histological score among the treatment groups at each time point. ^*Significantly different from the PBS group (p<0.001). ^†Significantly different from the nDNA group (p<0.001).

Figure 2

Effect of mitochondrial DNA (mtDNA) on serum cytokine levels in vivo. Serum levels of (A) tumor necrosis factor-α (TNF-α), (B) interleukin (IL)-6 and (C) IL-10 were determined at each time point using ELISA. (A) With the exception of the 24-h time point, mtDNA induced significantly higher serum TNF-α levels in vivo (p<0.001). (B) Significantly higher serum IL-6 levels were observed in the mtDNA group in vivo at 2, 4, 8 and 24 h (p≤0.002, 0.001, 0.001 and 0.02, respectively). (C) Serum IL-10 levels were significantly higher in the mtDNA group compared with the PBS and nDNA groups at 2, 8 and 24 h (p≤0.035, 0.037 and 0.008). ^*Significantly different from the phosphate-buffered saline (PBS) group. ^†Significantly different from the nuclear DNA (nDNA) group.

Figure 3

Effects of mitochondrial DNA (mtDNA) on cytokine secretion by macrophage cultures. (A) Tumor necrosis factor-α (TNF-α), (B) interleukin (IL)-6 and (C) IL-10 concentrations were determined in the cell culture supernatant at each time point using ELISA. (A) In vitro TNF-α levels were significantly higher in the mtDNA group than the other treatment groups at all time points analyzed (all p<0.001). (B) mtDNA significantly enhanced the release of IL-6 by macrophages in vitro after 4, 8 and 24 h as compared to the other groups (p=0.011, 0.001 and 0.001, respectively). (C) The levels of IL-10 in culture were significantly higher in the mtDNA group at each time point (0.004, 0.001, 0.001 and 0.001, respectively). ^*Significantly different from the phosphate-buffered saline (PBS) group. ^†Significantly different from the nuclear DNA (nDNA) group.

Figure 4

Protein expression profiles of phosphorylated (p)-nuclear factor-κB (NF-κB), p-IκB-α, and Toll-like receptor 9 (TLR9) in macrophage cultures upon stimulation with mitochondrial DNA (mtDNA). Protein levels were determined by western blot analysis. (A) Temporal profiles of p-NF-κB p65 and p-IκB-α protein expression. (B) Temporal profile of TLR9 protein expression. GAPDH served as the loading control.

Figure 5

Effects of mitochondrial DNA (mtDNA) on nuclear factor-κB (NF-κB), IκB-α, and Toll-like receptor 9 (TLR9) mRNA expression in lung tissues. (A) NF-κB, (B) IκB-α, and (C) TLR9 mRNA expression over time was determined in rat lung tissue by RT-PCR. (A) NF-κB mRNA expression in the lung tissues was significantly higher in the mtDNA group as compared with the PBS control group at 2 h (p=0.025); at 4 and 8 h, it was higher than both the PBS and nDNA groups (p≤0.004 and 0.003, respectively). No significant differences were observed at 24 h. (B) IκB-α mRNA levels were significantly higher in the mtDNA group compared with the PBS group at each time point (all p≤0.017). IκB-α mRNA levels were also significantly higher in the mtDNA group than the nDNA group at 8 h (p=0.038). (C) TLR9 mRNA levels were also significantly higher in the mtDNA group compared with PBS and nDNA groups at each time point (all p<0.001). ^*Significantly different from the phosphate-buffered saline (PBS) group. ^†Significantly different from the nuclear DNA (nDNA) group.