Ligands of the receptor for advanced glycation end products, including high-mobility group box 1, limit bacterial dissemination during Escherichia coli peritonitis

Abstract

Objective: The receptor for advanced glycation end products mediates a variety of inflammatory responses. Soluble receptor for advanced glycation end products has been suggested to function as a decoy abrogating cellular activation. High-mobility group box 1 is a high-affinity binding ligand for the receptor for advanced glycation end products with cytokine activities and plays a role in sepsis.

Design: Controlled, in vivo laboratory study.

Setting: Research laboratory of a health sciences university.

Subjects: C57BL/6 mice.

Interventions: Peritonitis was induced by intraperitoneal injection of Escherichia coli. Mice received soluble receptor for advanced glycation end products or anti-high-mobility group box 1 immunoglobulin G, or the appropriate control treatment.

Measurements and main results: Soluble receptor for advanced glycation end products-treated mice demonstrated an enhanced bacterial dissemination to liver and lungs, accompanied by increased hepatocellular injury and exaggerated systemic cytokine release, 20 hrs after intraperitoneal administration of Escherichia coli. Soluble receptor for advanced glycation end products administration in healthy, uninfected mice did not induce an immune response. Remarkably, lung inflammation was unaffected. Furthermore, high-mobility group box 1 release was enhanced during peritonitis and anti-high-mobility group box 1 treatment was associated with higher bacterial loads in liver and lungs.

Conclusions: These data are the first to suggest that receptor for advanced glycation end products ligands, including high-mobility group box 1, limit bacterial dissemination during Gram-negative sepsis.

Figure 1

Soluble receptor for advanced glycation end product (sRAGE)-treated mice demonstrate an enhanced dissemination. Number of Escherichia coli colony-forming units (CFUs) in peritoneal lavage fluid (PLF) (A), blood (B), liver (C), and lungs (D) at 20 hrs after intraperitoneal injection of 5 × 10⁴ CFUs of Escherichia coli in mice treated with either vehicle (white bars) or sRAGE (black bars) (n = 8–10 mice/group). Data are mean ± SEM; *p < .05 and ***p < .005 vs. vehicle-treated mice.

Figure 2

Soluble receptor for advanced glycation end product (sRAGE) worsens Escherichia coli (E. coli) sepsis-associated liver damage. Mice were treated with either vehicle or sRAGE intraperitoneally at 0.5 hrs after 5 × 10⁴ colony-forming units E. coli injection. Representative hematoxylin-eosin stainings of liver tissue at 20 hrs after E. coli infection in vehicle-(A) and sRAGE-treated (B) mice. Original magnification ×10, scale bars are shown in yellow. Arrow points out thrombus. Graphical representation of the degree of liver inflammation (C) and of fibrin and thrombi (G) determined according to the scoring system described in the Materials and Methods section. Control mice received vehicle or sRAGE in the absence of E. coli infection (“uninfected”) and “0 hrs” indicates baseline levels (C). Plasma aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) levels (D). Representative fibrin(ogen) immunostaining of liver tissue of mice administered with either vehicle (E) or sRAGE (F) after injection with E. coli. Original magnification ×10, scale bars are shown in yellow. White bars represent vehicle-treated and black bars indicate sRAGE-treated mice (n = 8–10 mice/group). Data are mean ± SEM; *p < .05 vs. vehicle-treated mice.

Figure 3

Soluble receptor for advanced glycation end product (sRAGE) increases hepatic neutrophil influx during Escherichia coli sepsis. Mice were treated with either vehicle or sRAGE intraperitoneally at 0.5 hrs after 5 × 10⁴ colony-forming units Escherichia coli injection. Representative leukocyte antigen-6 stainings of liver tissue at 20 hrs after Escherichia coli infection in vehicle-(A) and sRAGE-treated (B) mice. Original magnification ×10, scale bars are shown in yellow. Myeloperoxidase (MPO; C), keratinocyte-derived chemokine (KC; D), and macrophage inflammatory protein 2 (MIP-2; E) levels in liver homogenate in mice treated with control (white bars) or sRAGE (black bars) (n = 8–10 mice/group). Data are mean ± SEM; **p < .01 vs. vehicle-treated mice, ***p < .005 vs. vehicle-treated mice.

Figure 4

Treatment of soluble receptor for advanced glycation end product (sRAGE) elevates cytokine concentrations in the liver in Escherichia coli (E. coli)-induced septic mice. Mice were treated with either vehicle or sRAGE intraperitoneally at 0.5 hrs after 5 × 10⁴ colony-forming units E. coli injection. Control mice received either vehicle or sRAGE without E. coli infection (“uninfected”) and “0 hrs” indicates baseline levels. Tumor necrosis factor (TNF)-α (A), interleukin (IL)-6 (B), monocyte chemoattractant protein (MCP)-1 (C), and IL-10 (D) levels of liver homogenates in mice treated with vehicle (white bars) or sRAGE (black bars) (n = 8–10 mice/group). Data are mean ± SEM; *p < .05 vs. vehicle-treated mice, ***p < .001 vs. vehicle-treated mice.

Figure 5

Influence of soluble receptor for advanced glycation end product (sRAGE) on pulmonary inflammation. Mice were treated with either vehicle or sRAGE intraperitoneally at 0.5 hrs after 5 × 10⁴ colony-forming units Escherichia coli (E. coli) injection. Representative hematoxylin-eosin stainings of lung tissue at 20 hrs after E. coli infection in vehicle- (A) and sRAGE-treated (B) mice. Original magnification ×20, scale bars are shown in yellow. Graphical representation of the degree of lung inflammation (C) determined according to the scoring system described in the Materials and Methods section and myeloperoxidase (MPO) levels in lung homogenate (D) in mice treated with vehicle (white bars) or sRAGE (black bars) (n = 8–10 mice/group). Control mice received vehicle or sRAGE in the absence of E. coli infection (“uninfected”) and “0 hrs” indicates baseline levels (C). Representative leukocyte antigen-6 stainings of lung tissue at 20 hrs after E. coli infection in vehicle-(E) and sRAGE-treated (F) mice. Original magnification ×10, scale bars are shown in yellow. Data are mean ± SEM; *p < .05 vs. vehicle treated-mice.

Figure 6

Effects of treatment of anti-high-mobility group box 1 (HMGB1) immunoglobulin G (IgG) during Escherichia coli (E. coli)-induced sepsis. Wild-type mice were inoculated intraperitoneally with 5 × 10⁴ colony-forming units (CFUs) of E. coli and euthanized after 20 hrs. Local (peritoneal lavage fluid [PLF]; A) HMGB1 levels. Bacterial loads in PLF (B), blood (C), liver (D), and lungs (E) were determined in wild-type mice treated with anti-HMGB1 IgG or control IgG. Data are mean ± SEM (n = 9–10 mice/group); *p < .05 vs. control-treated mice.