High-mobility group box 1 and the receptor for advanced glycation end products contribute to lung injury during Staphylococcus aureus pneumonia

Abstract

Introduction: Staphylococcus (S.) aureus has emerged as an important cause of necrotizing pneumonia. Lung injury during S. aureus pneumonia may be enhanced by local release of damage associated molecular patterns such as high-mobility group box 1 (HMGB1). In the current study we sought to determine the functional role of HMGB1 and its receptors, toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE), in the injurious host response to S. aureus pneumonia.

Methods: Pneumonia was induced in wild type (Wt), TLR4 deficient (tlr4-/-) and RAGE deficient (rage-/-) mice by intranasal inoculation of 1 × 107 colony-forming units (CFU) of a USA300 S. aureus. In a separate set of experiments, Wt mice were injected intraperitoneally with a monoclonal anti-HMGB1 antibody or an isotype matched control antibody immediately before and every 24 hours after intranasal infection of S. aureus. Mice were sacrificed at 6, 24, 48 or 72 hours after infection for harvesting of blood and organs.

Results: S. aureus pneumonia was associated with HMGB1 release in the bronchoalveolar compartment peaking after 24 hours. Anti-HMGB1 attenuated lung pathology and protein leak and reduced interleukin-1β release 6 hours after infection, but not at later time points. RAGE deficiency more modestly attenuated lung pathology without influencing protein leak, while TLR4 deficiency did not impact on lung injury.

Conclusion: These data suggest that HMGB1 and RAGE, but not TLR4, contribute to lung injury accompanying the early phase of S. aureus pneumonia.

Figure 1

High mobility group box 1 (HMGB1) concentrations in mouse bronchoalveolar lavage (BAL) fluid during S. aureus pneumonia. Wt mice were intranasally infected with 1 × 10⁷ colony forming units (CFU) S. aureus and euthanized after 6, 24, 48 and 72 hours. HMGB1 concentrations in BAL fluid were quantified by densometric analysis of HMGB1 western blots and expressed as a percentage of the mean density of control BAL fluid samples (0 hours). Data represent the means ± standard error of the mean (n = 5 mice per time point). ^*P <0.05 versus naïve mice (0 hours).

Figure 2

Anti-high mobility group box 1 (HMGB1)-treated mice show reduced lung pathology early after induction of S. aureus pneumonia. Representative slides of lung H&E staining of control treated (A) and anti-HMGB1 treated mice (C), original magnification × 2. The boxed areas are also shown at a higher magnification for controls (B) and anti-HMGB1-treated mice (D), original magnification × 10. Scale bars indicate 200 μm. Total pathology scores were determined at the indicated time points post infection in control treated (gray) and anti-HMGB1 treated mice (white) according to the scoring system described in the Methods section (E). Total protein was measured in BAL fluid from control (grey) and anti-HMGB1 treated (white) mice (F). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (7 to 8 mice per group at each time point). ^**P <0.01 versus control treated mice at the same time point.

Figure 3

Receptor for advanced glycation end products (Rage) ^−/−but not Toll-like receptor (tlr)4^−/−mice show reduced lung pathology during S. aureus pneumonia. Representative slides of lung HE staining of wild-type (Wt) (A), tlr4^−/−(C) and rage^−/− mice (E), original magnification × 2. The boxed areas are also shown at a higher magnification for Wt (B)tlr4^−/−(D) and rage^−/− mice (F), original magnification × 10. Scale bars indicate 200 μm. Total pathology scores at indicated time points after intranasal infection with S. aureus in Wt (dark gray), tlr4^−/− (light gray) and rage^−/− mice (white) were determined according to the scoring system described in the Methods section (G). Total protein was measured in bronchoalveolar lavage (BAL) fluid from Wt (dark gray), tlr4^−/− (light gray) and rage^−/− mice (white) at the indicated time points after intranasal S. aureus infection (H). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (7 to 8 mice per group at each time point). ^*P <0.05, ^**P <0.01 versus Wt mice at the same time point.

Figure 4

Anti-high mobility group box 1 (HMGB1) treatment reduces IL-1β and keratinocyte-derived chemokine (KC) levels in bronchiolar lavage (BAL) fluid after intranasal infection with S. aureus. Cytokine (TNF-α, IL-6 and IL-1β) (A-C) and chemokine (KC and macrophage inflammatory protein (MIP)-2) (D-E) levels in BAL fluid at different time points after intranasal infection of 1 × 10⁷ colony-forming units (CFU) S. aureus in mice treated with control (gray) and anti-HMGB1 antibodies (white). Data are expressed as box-and-whisker diagrams depicting the median, the smallest observation, lower quartile, median, upper quartile and largest observation (7 to 8 mice per group at each time point). ^*P <0.05, ^**P <0.01 versus Wt mice at the same time point.

Figure 5

Receptor for advanced glycation end products (Rage)^−/−mice show lower levels of TNF-α and IL-6 in bronchiolar lavage (BAL) fluid during S. aureus pneumonia. Cytokine (TNF-α, IL-6 and IL-1β) (A-C) and chemokine (keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2) (D-E) levels 6, 24, 48 and 72 hours after intranasal S. aureus infection in Wt (dark gray) and tlr4^−/− (light gray) and rage^−/− mice (white). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (7 to 8 mice per group at each time point). ^*P <0.05, ^**P <0.01 versus Wt mice.

Figure 6

Bacterial outgrowth in Receptor for advanced glycation end products (Rage)^−/−mice is reduced early after intranasal infection with S. aureus. Bacterial loads after intranasal infection with 1 × 10⁷ colony-forming units (CFU) S. aureus in bronchiolar lavage (BAL) fluid (A) of mice treated with control (gray) and anti-HMGB1 antibodies (white) and in BAL fluid (B) of Wt (dark gray), Toll-like receptor (tlr)4^−/− (light gray) and rage^−/− mice (white) at 6, 24, 48 and 72 hours after infection. Data are expressed as box-and-whisker diagrams depicting the median, the smallest observation, lower quartile, median, upper quartile and largest observation (n = 7 to 8 mice per group at each time point). ^**P <0.01 versus Wt mice at the same time point.