Systemic inflammation and liver injury following hemorrhagic shock and peripheral tissue trauma involve functional TLR9 signaling on bone marrow-derived cells and parenchymal cells

Abstract

Hemorrhagic shock due to trauma (HS/T) induces an inflammatory response that can contribute to end-organ injury. The pathways involved in the initiation and propagation of HS/T-induced inflammation are incompletely understood. Here, we hypothesized that the DNA sensor TLR9 would have a role in inflammatory signaling after HS/T. Using mice expressing a nonfunctional, mutant form of TLR9, we identified a role of TLR9 in driving the initial cytokine response and liver damage in a model of hemorrhagic shock and bilateral femur fracture. Circulating DNA levels were found to correlate with the degree of tissue damage. Experiments using chimeric mice show that TLR9 on both bone marrow-derived cells and parenchymal cells are important for the TLR9-mediated liver and tissue damage, as well as systemic inflammation after HS/T. These data suggest that release of DNA may be a driver of the inflammatory response to severe injury as well as a marker of the extent of tissue damage. One of the sensors of DNA in the setting of HS/T seems to be TLR9.

Fig. 1

Measurements of systemic cell-free DNA, inflammation, and liver damage following HS. C57BL/6 mice underwent hemorrhagic shock (HS), or sham procedure. Plasma double-stranded (ds) DNA (A) and single-stranded (ss) DNA (B) levels were measured for analysis of cell-free plasma DNA release. Plasma IL-6 (C) and IL-10 (D) levels were measured for analysis of systemic inflammation. Plasma alanine aminotransferase (ALT, E) and aspartate aminotransferase (AST, F) levels were analyzed for analysis of liver damage. (*, P<0.05 vs. baseline) Data represent means ± SEM; n = 3 mice for each time point.

Fig. 2

Circulating IL-6 and IL-10 levels are lower in TLR9 mutant mice after HS/T. WT and TLR9 mice were subjected to bilateral femur fracture and hemorrhagic shock (HS/T), or sham procedure. Plasma IL-6 (A) and IL-10 (B) levels were measured to assess the systemic inflammatory response. TLR9 mutant mice subjected to HS/T exhibited lower IL-6 and IL-10 levels as compared with WT mice subjected to HS/T (*, P<0.05; **, P<0.01). Data represent means ± SEM; n = 6–8 mice group.

Fig. 3

TLR9 mutant mice exhibit less liver damage and apoptosis after HS/T. WT and TLR9 mice were subjected to bilateral femur fracture and hemorrhagic shock (HS/T), or sham procedure. Plasma ALT (A) and AST (B) levels were analyzed for analysis of liver damage. TLR9 mutant mice demonstrated significantly lower ALT and AST levels as compared with WT mice after HS/T. The number of In situ TdT-mediated dUTP nick-end labelling (TUNEL) positive nuclei in liver (C) of TLR9 mutant mice, expressed as a percent of the total number of nuclei, was reduced compared with WT mice after HS/T (*, P<0.05; **, P<0.01). Data represent means ± SEM; n = 6–8 mice group. H&E (D, magnification × 400) liver tissue histology of TLR9 mutant mice showed significantly less necrosis (→) compared with WT following fracture. Data shown represent 1 of 3 independent experiments yielding similar results.

Fig. 4

Chloroquine reduces liver damage following HS/T. Serum ALT levels were analyzed for liver damage after HS/T. Chloroquine (30mg/kg i.p.) 14 h and 1 h before trauma significantly prevented HS/T induced increases in plasma ALT, which was shown in the phosphate buffered saline (PBS) controlled mice (*, P<0.05; **, P<0.01). Data represent means ± SEM; n = 4–6 mice group.

Fig. 5

Circulating DNA levels are lower in TLR9 mutant mice following HS/T. WT and TLR9 mutant mice were subjected to bilateral femur fracture and hemorrhagic shock (HS/T), or sham procedure. Plasma double-stranded (ds) DNA (A) and single-stranded (ss) DNA (B) levels were measured for analysis of DNA release following injury. TLR9 mutant mice subjected to HS/T showed significantly lower cell-free plasma dsDNA and ssDNA levels as compared with WT (*, P<0.05; **, P<0.01). Data represent means ± SEM; n = 6–8 mice group.

Fig. 6

Cell-free DNA release and liver injury following HS/T require functional TLR9 on bone marrow (BM)-derived cells and non-BM-derived cells. Chimeric mice underwent bilateral femur fracture and hemorrhagic shock (HS/T), or sham procedure. Plasma dsDNA (A) and ssDNA (B) levels were analyzed to assess cell-free DNA release after trauma. Plasma IL-6 (C) levels were analyzed for inflammatory response and ALT (D) for liver injury. WT/WT mice (recipient/donor) demonstrated significant increases in plasma ssDNA and dsDNA, Il-6, and ALT after HS/T, which was not seen in WT/Mu, Mu/WT, or Mu/Mu mice (A and B, **, P<0.01). (C and D, *, P<0.05). Data represent means ± SEM; n = 4–6 mice group. ssDNA, single stranded DNA; dsDNA, double stranded DNA; WT, wild type; Mu, TLR9 mutant.