Receptor for Advanced Glycation End Products (RAGE) Serves a Protective Role during Klebsiella pneumoniae - Induced Pneumonia

Abstract

Klebsiella species is the second most commonly isolated gram-negative organism in sepsis and a frequent causative pathogen in pneumonia. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory responses. We here aimed to investigate the role of RAGE in the host response to Klebsiella (K.) pneumoniae pneumonia and intransally inoculated rage gene deficient (RAGE-/-) and normal wild-type (Wt) mice with K. pneumoniae. Klebsiella pneumonia resulted in an increased pulmonary expression of RAGE. Furthermore, the high-affinity RAGE ligand high mobility group box-1 was upregulated during K. pneumoniae pneumonia. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE-/- mice. RAGE-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. Relative to Wt mice, RAGE-/- mice demonstrated similar lung inflammation, and slightly elevated-if any-cytokine and chemokine levels and unchanged hepatocellular injury. In addition, RAGE-/- mice displayed an unaltered response to intranasally instilled Klebsiella lipopolysaccharide (LPS) with respect to pulmonary cell recruitment and local release of cytokines and chemokines. These data suggest that (endogenous) RAGE protects against K. pneumoniae pneumonia. Also, they demonstrate that RAGE contributes to an effective antibacterial defense during K. pneumoniae pneumonia, at least partly via its participation in the phagocytic properties of professional granulocytes. Additionally, our results indicate that RAGE is not essential for the induction of a local and systemic inflammatory response to either intact Klebsiella or Klebsiella LPS.

Fig 1. Pulmonary expression of receptor for advanced glycation end products and of its ligand high mobility group box-1 (HMGB1) during Klebsiella pneumoniae pneumonia.

Representative view of a lung from a normal, uninfected wild-type mouse (A) displaying ubiquitous expression of RAGE on the surface of endothelium. Arrow indicates bronchial epithelium, being negative for RAGE staining. B, Absence of RAGE positivity in the lung of a RAGE^-/- mouse. C, Lungs from a wild-type mouse 48 h after the inoculation of K. pneumoniae. Original magnification, x10. D, Western blot was performed for HMGB1 in brochoalveolar lavage fluid (BALF) from wild-type mice at 0, 6, 24 and 48 h after K. pneumoniae intranasal inoculation (n = 3 mice per time point).

Fig 2. Increased mortality of receptor for advanced glycation end products deficient (RAGE^-/-) mice during Klebsiella pneumoniae pneumonia.

Survival of wild-type and RAGE^-/- mice after intranasal inoculation with 1 x 10⁴ CFUs K. pneumoniae. Mortality was assessed for 14 days (n = 13–14 mice per genotype).

Fig 3. Receptor for advanced glycation end products deficiency enhances local bacterial outgrowth and dissemination during Klebsiella pneumoniae pneumonia in vivo and reduces in vitro phagocytosis of Klebsiella pneumoniae by neutrophils.

Bacterial loads in lung homogenate (A), blood (B), liver (C) and spleen (D) were determined in wild-type and RAGE^-/- mice 24 and 48 h after intranasal inoculation 1 x 10⁴ CFUs K. pneumoniae. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation of 8–10 mice per genotype at each time point. E, Phagocytosis of growth-arrested viable Alexa647-SE labeled Klebsiella pneumoniae of neutrophils from wild type and RAGE^-/- mice at 37°C or 4°C. Phagocytosis was quantified as described in the Methods section. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (of 4 mice for 4°C and 8 mice 37°C) per genotype. * p < 0.05, compared with wild-type mice.

Fig 4. Unchanged lung inflammation during Klebsiella pneumonia.

Wild-type and RAGE^-/- mice were inoculated intranasally with 1 x 10⁴ CFUs K. pneumoniae. Representative hematoxylin-eosin stainings of lung tissue at 24 (A and B) and 48 (C and D) h post inoculation in wild-type (A and C) and RAGE^-/- (B and D) mice. Original magnification, x20. E, Graphical representation of the degree of lung inflammation at 24 and 48 h. F, Myeloperoxidase (MPO) levels in lung tissues. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation of 8–10 mice per genotype at each time point. * p < 0.05, compared with wild-type mice.

Fig 5. Hepatocellular injury during Klebsiella pneumoniae pneumonia.

Wild-type and RAGE^-/- mice were inoculated intranasally with 1 x 10⁴ CFUs K. pneumoniae and sacrificed after 24 and 48 h. Aspartate aminotransferase (AST, A) and alanine aminotransferase (ALT, B) in plasma of wild-type and RAGE^-/- mice. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation of 8–10 mice per genotype at each time point.