Abstract Introduction Pneumonia is a common and dangerous infection characterized by alveolar filling with inflammatory exudate and, in severe cases, dissemination of pathogens into the bloodstream. Klebsiella, an facultative anaerobic Gram-negative bacillus, is an important cause of hospital-acquired pneumonia. Heme, a molecule composed of a porphyrin ring containing a covalently-bound iron atom, is a component of hemoproteins such as hemoglobin. Bacteria, such as Klebsiella, can utilize heme as a source of iron; in addition, extracellular labile heme is an alarmin and has complex effects on the host. The host scavenges extracellular heme with the acute-phase protein, hemopexin, but the role of hemopexin in pneumonia has not been investigated to date.Hypothesis: We hypothesize that hemopexin-mediated clearance of heme protects the host during Klebsiella pneumonia. Materials and Methods We used murine models of pneumonia and bacteremia, induced by intra-pulmonary or intravenous administration of Klebsiella pneumoniae in wildtype C57Bl/6 mice and hemopexin-knockout mice. We quantified lung and blood bacterial content by dilution and culture, measured hemopexin transcription by qRT-PCR and protein using a commercial ELISA, quantified extracellular heme concentration using a spectroscopic assay and quantified protein hemoglobin using a commercial ELISA. We assessed the growth of Klebsiella in vitro on non-nutrient agarose containing mouse plasma with heme or tin protoporphyrin. Results During experimental pneumonia, hemopexin was induced in the liver, resulting in increasing concentrations of hemopexin protein in plasma and lower levels in the lungs. As compared to wildtype animals, hemopexin-deficient mice had markedly increased mortality associated with increased bacteremia but not lung bacterial burden. Hemopexin deficiency resulted in an increase in the plasma concentration of both extracellular heme and cell-free hemoglobin in infected animals. In vitro, Klebsiella did not form colonies in media containing wildtype plasma unless the media was supplemented by heme. In contrast, hemopexin-deficient plasma supported the formation of Klebsiella colonies, and colony formation was abrogated with the reconstitution of hemopexin-deficient plasma with recombinant hemopexin protein. Conclusion Our data indicated that hemopexin protects against Klebsiella infection by inhibiting bacteremia and hemolysis, and that hemopexin in plasma inhibits bacterial growth by sequestering heme. We speculate that hemopexin prevents a positive feedback loop in which labile heme in plasma leads to overactivation of the complement system during pneumonia and subsequent further hemolysis, and that heme serves to promote the growth of the pathogen in the bloodstream. Supported by: NIH R01AI135128 and R01HL169974 This abstract is funded by: NHLBI
Qi et al. (Fri,) studied this question.