Toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis

Abstract

The high rate of mortality in patients with sepsis results from an inappropriately amplified systemic inflammatory response to infection. Toll-like receptors (TLRs) are important for the activation of innate immunity against microbial pathogens. We demonstrate a critical role of TLR9 in the dysregulated immune response and death associated with sepsis. Compared with wild-type (WT) mice, TLR9(-/-) mice exhibited lower serum inflammatory cytokine levels, higher bacterial clearance, and greater survival after experimental peritonitis induced by cecal ligation and puncture (CLP). Protection of TLR9(-/-) mice after CLP was associated with a greater number of peritoneal dendritic cells (DCs) and granulocytes than in WT controls. Adoptive transfer of TLR9(-/-) DCs was sufficient to protect WT mice from CLP and increased the influx of peritoneal granulocytes. Subsequent experiments with a depleting antibody revealed that granulocytes were required for survival in TLR9(-/-) mice. Remarkably, a single injection of an inhibitory CpG sequence that blocks TLR9 protected WT mice, even when administered as late as 12 h after CLP. Our findings demonstrate that the detrimental immune response to bacterial sepsis occurs via TLR9 stimulation. TLR9 blockade is a potential strategy for the treatment of human sepsis.

Figure 1.

TLR9^−/− mice have less inflammation and increased survival after CLP. (A) The survival rates of WT B6 and TLR9^−/− mice were monitored for 6 d after CLP (15 mice per group, with data pooled from three experiments, each of which had similar statistical significance). Dilutions of blood (B) or peritoneal lavage fluid (C) obtained from TLR9^−/− or WT mice 12 h after CLP were cultured on BHI agar plates, and the number of bacterial colonies was counted (five mice per group). The colony count indicated is of 1 ml of blood and 1 ml of peritoneal fluid. (D) Serum cytokine levels were determined 6 and 12 h after CLP by using a cytometric bead array (five mice per group). Data shown are means of values ± SEM obtained from individual mice and are representative of at least two independent experiments. *, P < 0.05.

Figure 2.

TLR9^−/− mice have increased lymphocyte infiltration and decreased local inflammatory cytokine production after CLP. 12 h after CLP or sham laparotomy, the numbers of viable splenocytes (A) and infiltrating peritoneal lymphocytes (B) were counted on a hemocytometer (BrightLine; Hausser Scientific) after Trypan blue staining (three to five mice per group). Peritoneal cells are expressed per milliliter of peritoneal fluid. (C) Peritoneal cells pooled from three to five mice 12 h after CLP were cultured without restimulation for 24 h, and the supernatant cytokine levels were determined with a cytometric bead array. Peritoneal cells from mice that underwent sham laparotomy secreted a minimal amount of cytokines (not depicted). Data shown are means of values ± SEM obtained from individual mice (except for cells pooled in C) and are representative of at least two independent experiments. *, P < 0.05.

Figure 3.

TLR9^−/− DCs reduce the sepsis-induced lethality of WT mice. (A) 12 h after CLP or sham laparotomy, the number of conventional DCs (CD11c^highMHCII⁺) were determined by flow cytometry in the spleen and peritoneal fluid of WT or TLR9^−/− mice (three to five mice per group). (B) The survival rates of WT mice receiving WT or TLR9^−/− DCs were monitored for 6 d after CLP (23 mice per group, with data pooled from two experiments, each of which had similar statistical significance). (C) 10⁷ Flt3L-expanded WT or TLR9^−/− DCs (CD45.2⁺) were injected i.v. into CD45.1 WT recipients (three to five mice per group). 12 h after transfer, the mice underwent CLP or sham laparotomy, and 12 h later the number of CD45.2⁺ DCs were enumerated from the peritoneal cells by flow cytometry. (D) Dilutions of blood or peritoneal lavage fluid obtained from WT mice pretreated with WT or TLR9^−/− DCs 12 h after CLP were cultured on BHI agar plates, and the number of bacterial colonies was counted (five mice per group). (E) Serum cytokine levels were determined 12 h after CLP by cytometric bead array (five mice per group). Data shown are means of values ± SEM obtained from individual mice and are representative of at least two independent experiments. *, P < 0.05.

Figure 4.

Peritoneal granulocytes are increased by CLP and are necessary for protection. (A) 12 h after CLP or sham laparotomy, the number of peritoneal granulocytes in WT or TLR9^−/− mice (three to five mice per group) was determined by flow cytometry. (B) WT or TLR9^−/− mice received 500 μg anti-GR1 antibody or isotype control 24 and 2 h before CLP, and the survival rates were monitored for 6 d after CLP (15 mice per group, with data pooled from three experiments, each of which had similar statistical significance). (C) 2 × 10⁶ CD45.1 splenic granulocytes were injected i.v. into WT or TLR9^−/− (CD45.2) recipients (three to five mice per group). 12 h after transfer, the mice underwent CLP or sham laparotomy, and 12 h later the number of CD45.1⁺ granulocytes were enumerated from the peritoneal cells by flow cytometry. (D) 12 h after CLP or sham laparotomy, the number of granulocytes (Ly6G⁺) was determined by flow cytometry in the peritoneal fluid of WT mice pretreated with 10⁷ Flt3L-expanded WT or TLR9^−/− DCs (three to five mice per group). Treatment with anti-GR1 antibody achieved >97% depletion of granulocytes at the time of and 24 h after CLP (not depicted). Data shown are means of values ± SEM obtained from individual mice and are representative of at least two independent experiments. *, P < 0.05.

Figure 5.

TLR9 inhibition protects WT mice from CLP. (A) Immediately before CLP, WT mice received 100 μg iCpG or control oligodeoxynucleotide (ODN), and survival rates were monitored for 6 d. (B) 12 and 18 h after CLP, WT mice received 100 μg iCpG or control ODN, and survival rates were monitored for 6 d. Survival curves of mice treated with iCpG at 3 and 6 h were similar to the one shown for the 12-h iCpG group (not depicted). Data shown include 15 mice per group, pooled from three experiments, each of which had similar statistical significance.