The HMGB1-RAGE axis mediates traumatic brain injury-induced pulmonary dysfunction in lung transplantation

Abstract

Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE(-/-)) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs' ability to expand), all of which were attenuated in RAGE(-/-) mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE(-/-) TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.

Figure 1

Acute lung injury in C57BL/6 mice 4–24 hours after TBI. Representative H/E histology sections of C57BL/6 mouse lungs after traumatic brain injury at various time points. (A) Control animals with sham TBI. (B) 4 hours after TBI with alveolar hemorrhage. (C) 8 hours after TBI with alveolar hemorrhage. (D) 12 hours after TBI with interstitial neutrophils. (E) 24 hours after TBI with interstitial neutrophils. (F) Acute lung injury scores from a blinded pathologist based on a standardized scoring system from the American Thoracic Society (19). Scores are continuous between 0 and 1 with 0 representing no injury and 1 representing severe acute lung injury. (G) Albumin concentrations in BAL fluid after TBI. (H) Systemic concentrations of HMGB1 24 hours after severe TBI in the sham injured group as well as in C57BL/6 and RAGE −/− mice 24 hours after TBI. n=6–8 per condition, Student’s t test comparison with the reference sham injured group, comparisons versus sham-injured group, *p<0.01.

Figure 2

PaO2/FiO2 ratios and compliance values derived from C57BL/6 and RAGE −/−mice 4–24 hours after TBI. (A) PaO2/FiO2 ratios from controls without TBI and wildtype mice 4–24 hours after TBI. (B) Static compliance values obtained via Pulmonary Function Testing from controls without TBI and wildtype mice 4–24 hours after TBI. (C) PaO2/FiO2 ratios from C57BL/6 and RAGE−/− 24 hours after TBI. Animals were administered either moderate or severe TBI. (D) Compliance values from controls and C57BL/6 and RAGE −/− mice 24 hours after TBI. n=4–7 mice per condition, comparisons versus sham-injured group unless otherwise indicated, *p<0.01 Statistical testing included unpaired t-tests and analysis of variance.

Figure 3

PaO2/FiO2 ratios and compliance values 24 hours after TBI in RAGE −/−, TLR4 −/− mice treated with HMGB1 neutralizing antibody, and mice treated with TAT-4BB decoy peptide. (A) PaO2/FiO2 ratios in control mice and RAGE −/−, TLR4 −/− mice treated with HMGB1 antibody or control antibody, or treated with 4BB (Myd-88) decoy peptide 24 hours after TBI. (B) Compliance values in RAGE −/−, TLR4 −/− mice treated with HMGB1 antibody or control antibody, or with 4BB decoy peptide 24 hours after TBI. n=5–7 per condition, comparisons versus sham-injured group unless otherwise indicated, *p≤0.01. Statistical testing included unpaired t-tests and analysis of variance.

Figure 4

Histology and acute lung injury scores for transplanted left lungs from control donor mice, wildtype TBI donor mice, or RAGE−/− TBI donor mice. Representative H/E histology sections from transplanted lungs 5 days after transplant. (A) Transplanted left lung from healthy donor with sham TBI. (B) Transplanted left lung from wildtype TBI donor. (C) Transplanted left lung from RAGE−/− TBI donor. (D) Acute lung injury scores from transplant recipients who received lungs from three different donors: wildtype animals with sham TBI, wildtype animals with TBI, and RAGE −/− donors with TBI. n=6–8 per condition, comparison versus sham-injured group, *p<0.01. Statistical testing included unpaired t-tests and analysis of variance

Figure 5

BAL cytokine profiles in control and injured donor mice 24 hours after TBI. Cytokine profiling was also performed in left lung transplant recipients of TBI donor lungs 8 hours after transplant. (A–F) Mean Fluorescence Intensity of IL-4, IL-6, IL-17a, IFN-γ, TNF-α, IL-17A, and IL-10. n=5–7 per condition, *p<0.01. Statistical testing included unpaired t-tests and analysis of variance

Figure 6

Representative Western blot of NF-κB activation in rat alveolar type 2 (AT2) cells following exposure to HMGB1. (A) NF-κB p65 and p50 translocation from the cytoplasm to the nucleus demonstrated by stimulating AT2 cells with 0, 5, or 10 μg/mL of HMGB1 for 24 hours. (B–E) Densitometric analysis was performed using GAPDH as a loading control for the cytoplasmic samples and Lamin B1 as a loading control for the intranuclear samples.

Figure 7

PaO2/FiO2 ratios in lung transplant donors and recipients based on donor HMGB1 serum concentrations at the time of harvest. (A) PaO2/FiO2 ratios among all brain dead donors, regardless of mechanism of injury correlated with donor HMGB1 serum concentrations at harvest. (B) PaO2/FiO2 ratios among brain dead donors whose mechanism of injury was TBI, and donor HMGB1 serum concentrations at harvest. (C) Recipient PaO2/FiO2 ratios at 48 hours after transplant correlated with donor HMGB1 serum concentrations at harvest. (D) Recipient PaO2/FiO2 ratios at 48 hours after transplant from brain dead donors whose mechanism of injury was TBI correlated with donor HMGB1 serum concentrations at harvest.