Pathogen-Associated Molecules from Gut Translocation Enhance Severity of Cecal Ligation and Puncture Sepsis in Iron-Overload β- Thalassemia Mice

Introduction: Systemic inflammation induced by gut translocation of lipopolysaccharide (LPS), a major component of Gram-negative bacteria, in thalassemia with iron-overload worsens sepsis. However, the impact of (1 -> 3)-beta-D-glucan (BG), a major fungal molecule, in iron-overload thalassemia is still unclear. Hence, the influence of BG was explored in 1) iron-overload mice with sepsis induced by cecal ligation and puncture (CLP) surgery; and 2) in bone marrow-derived macrophages (BMMs). Methods: The heterozygous beta-globin-deficient mice, Hbb(th3/+) mice, were used as representative thalassemia (TH) mice. Iron overload was generated by 6 months of oral iron administration before CLP surgery- induced sepsis in TH mice and wild-type (WT) mice. Additionally, BMMs from both mouse strains were used to explore the impact of BG. Results: Without sepsis, iron-overload TH mice demonstrated more severe intestinal mucosal injury (gut leakage) with higher LPS and BG in serum, from gut translocation, when compared with WT mice. With CLP in iron-overload mice, sepsis severity in TH mice was more severe than WT as determined by survival analysis, organ injury (kidney and liver), bacteremia, endotoxemia, gut leakage (FITC-dextran) and serum BG. Activation by LPS plus BG (LPS+BG) in BMIVIs and in peripheral blood-derived neutrophils (both WT and TH cells) demonstrated more prominent cytokine production when compared with LPS activation alone. In parallel, LPS+BG also prominently induced genes expression of Ml macrophage polarization (iNOS, TNF-alpha and IL-1 beta) in both WT and TH cells in comparison with LPS activation alone. In addition, LPS+BG activated macrophage cytokine production was enhanced by a high dose of ferric ion (800 mM), more predominantly in TH macrophages compared with WT cells. Moreover, LPS+BG induced higher glycolysis activity with similar respiratory capacity in RAW264.7 (a macrophage cell line) compared with LPS activation alone. These data support an additive pro-inflammatory effect of BG upon LPS. Conclusion: The enhanced-severity of sepsis in iron-overload TH mice was due to 1) increased LPS and BG in serum from iron-induced gut-mucosal injury; and 2) the pro-inflammatory amplification by ferric ion on LPS+BG activation.