Exposure to bacterial DNA before hemorrhagic shock strongly aggravates systemic inflammation and gut barrier loss via an IFN-gamma-dependent route

Abstract

Objective: To investigate the role of bacterial DNA in development of an excessive inflammatory response and loss of gut barrier loss following systemic hypotension.

Summary background data: Bacterial infection may contribute to development of inflammatory complications following major surgery; however, the pathogenesis is not clear. A common denominator of bacterial infection is bacterial DNA characterized by unmethylated CpG motifs. Recently, it has been shown that bacterial DNA or synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) are immunostimulatory leading to release of inflammatory mediators.

Methods: Rats were exposed to CpG-ODN prior to a nonlethal hemorrhagic shock. The role of interferon-gamma (IFN-gamma) was investigated by administration of anti IFN-gamma antibodies.

Results: Exposure to CpG-ODN prior to hemorrhagic shock significantly augmented shock-induced release of IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) (P < 0.05), interleukin (IL)-6 (P < 0.05), and nitrite levels (P < 0.05), while there was a defective IL-10 response (P < 0.05). Simultaneously, expression of Toll-like receptor (TLR) 4 in the liver was markedly enhanced. Furthermore, intestinal permeability for HRP significantly increased and bacterial translocation was enhanced in hemorrhagic shock rats pretreated with CpG-ODN. Interestingly, inhibition of IFN-gamma in CpG-treated animals reduced TNF-alpha (P < 0.05), IL-6 (P < 0.05), nitrite (P < 0.05), and intestinal permeability following hemorrhagic shock (P < 0.05) and down-regulated expression of TLR4.

Conclusion: Exposure to bacterial DNA strongly aggravates the inflammatory response, disrupts the intestinal barrier, and up-regulates TLR4 expression in the liver following hemorrhagic shock. These effects are mediated via an IFN-gamma-dependent route. In the clinical setting, bacterial DNA may be important in development of inflammatory complications in surgical patients with bacterial infection.

FIGURE 1. Preexposure to CpG-ODN results in significantly higher plasma IFN-γ levels after hemorrhagic shock. Plasma IFN-γ levels were measured in (nonexposed) controls and rats preexposed to non-CpG or CpG-ODN at 18 hours after exposure and at 4 hours after hemorrhagic shock (n = 7 per group). Administration of CpG-ODN alone resulted in detectable, significantly increased plasma IFN-γ (34 ± 2 pg/mL, ^†P = 0.003) compared with nonexposed control rats after 18 hours. Hemorrhagic shock caused a rise of circulating IFN-γ levels at 4 hours after shock in control (1.3 ± 0.1 ng/mL, *P < 0.01) and non-CpG treated rats (0.9 ± 0.1 ng/mL, ^†P < 0.01) compared with nonexposed control rats not subjected to shock. Preexposure to CpG-ODN followed by hemorrhagic shock doubled circulating plasma levels of IFN-γ at 4 hours after shock (2.6 ± 0.5 ng/mL, ^#P < 0.05) compared with control rats and non-CpG treated rats (^#P < 0.05) subjected to hemorrhagic shock.

FIGURE 2. Preexposure to CpG-ODN significantly elevated TNF-α, IL-6, and nitrite (NO2−) and down-regulated IL-10 via an IFN-γ-dependent route. TNF-α, IL-6, nitrite, and IL-10 levels were determined in plasma of controls and CpG-ODN-exposed rats at 18 hours after exposure and at 4 hours after hemorrhagic shock (n = 7 per group). A, Administration of CpG-ODN alone enhanced plasma TNF-α (14 ± 6 pg/mL, ^†P < 0.05). CpG-ODN preexposure together with hemorrhagic shock elevated TNF-α (68 ± 13 pg/mL, *P < 0.001). Anti-IFN-γ reduced TNF-α after hemorrhagic shock in CpG-ODN-treated rats (27 ± 8 pg/mL, ^#P < 0.05). B, Preexposure to CpG-ODN strongly enhanced hemorrhagic shock-induced IL-6 (171 ± 33 pg/mL, *P < 0.01), which was reduced by administration of anti IFN-γ (52 ± 20 pg/mL, ^#P < 0.05). C, Administration of CpG-ODN alone increased nitrite (NO2−) (91± 7 μmol/L, ^†P < 0.01). Hemorrhagic shock caused a marked increase in NO2− compared with controls (97 ± 6 μmol/L, **P < 0.01). CpG-ODN preexposure elevated hemorrhagic shock-induced NO2− (169 ± 38 μmol/L, *P< 0.05). Treatment with anti IFN-γ reduced nitrite levels following hemorrhagic shock in CpG-ODN rats (83 ± 12 μmol/L, ^#P < 0.05). D, Administration of CpG-ODN alone enhanced plasma IL-10 levels (68 ± 12 pg/mL, ^†P < 0.006). Hemorrhagic shock caused a marked increase of plasma IL-10 (148 ± 13 pg/mL, *P < 0.01) in control and non-CPG-treated rats (133 ± 7 pg/mL, *P < 0.01). CpG-ODN preexposure together with hemorrhagic shock caused a defective IL-10 response that was restored by administration of anti IFN-γ (109 ± 12 pg/mL, ^#P < 0.01): **,^†compared with control; *compared with control hemorrhagic shock; #compared with CpG-ODN shock.

FIGURE 3. Preexposure to CpG-ODN followed by hemorrhagic shock enhanced intestinal permeability for horseradish peroxidase (HRP) via an IFN-γ-dependent route. Exposure to CpG-ODN increased permeability for HRP (5.5 ± 0.5 μg/mL, ^†P < 0.01) compared with control rats (1.0 ± 0.1 μg/mL). Hemorrhagic shock caused a substantial leakage of HRP (36 ± 3 μg/mL, **P < 0.01). Preexposure to CpG-ODN followed by hemorrhagic shock strongly aggravated intestinal permeability for HRP (60 ± 11 μg/mL, *P < 0.05). Administration of anti IFN-γ markedly reduced permeability for HRP in both control (^‡P < 0.05) and CpG-ODN-treated rats (^#P < 0.01) subjected to hemorrhagic shock: **,^†compared with control; *compared with control hemorrhagic shock; ^#compared with CpG-ODN shock; ‡compared with control hemorrhagic shock.

FIGURE 4. Exposure to CpG-ODN up-regulates expression of TLR4 in macrophages and in the liver leading to increased intracellular signaling via an IFN-γ-dependent route. Cell lysates and nuclear extracts were isolated from peritoneal macrophages of rats exposed to non-CpG, CpG-ODN or CpG-ODN and anti-IFN-γ before and after stimulation with LPS. Exposure to CpG-ODN alone caused enhanced expression of TLR4 in vivo in the liver, which was strongly enhanced 90 minutes following hemorrhagic shock in an IFN-γ-dependent manner (A). Expression of TLR4 and translocation of NF-κB to the nucleus in peritoneal macrophages following stimulation were markedly enhanced following CpG-ODN treatment (B, C). This enhanced expression of TLR4 and translocation of NF-κB to the nucleus were reduced after administration with anti IFN-γ. Stimulation of peritoneal macrophages from rats exposed to CpG-ODN with LPS (1 and 10 ng/mL) caused a significantly increased release of TNF-α (D), (*P < 0.01 compared with control).