Spite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 calls for iron chelation instead of the Ent Lcn2 complicated itself and also could be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression NADPH Oxidase Inhibitor medchemexpress microarrays, quantitative PCR, and cytokine immunoassays. Ent triggered HIF-1 protein stabilization, induced the expression of genes regulated by hypoxia-inducible aspect 1 (HIF-1 ), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly enhanced Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1 was sufficient to improve Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production.ue to its capability to assume many oxidative states, iron is an crucial element in quite a few human cellular processes, such as DNA replication, oxygen metabolism, and electron transfer (1, two). Iron homeostasis represents a distinctive challenge, due to the fact free ferric iron (Fe3 ) is insoluble and ferrous iron (Fe2 ) may be toxic to cells. Therefore, ferric iron is transported while complexed to transferrin, keeping serum iron concentrations at 10 24 M (3?). Bacteria demand 10 six M iron in their cytosol for cellular processes, a considerably greater concentration of iron than is readily readily available (3). To acquire the iron needed for development inside the ironlimiting conditions of your human body, Gram-negative pathogens for instance Escherichia coli and Klebsiella pneumoniae secrete the siderophore enterobactin (Ent). Ent is really a prototypical catecholate siderophore using the highest identified affinity for iron (three, four, 6). To counter the iron-scavenging effects of Ent, neutrophils and host mucosal cells secrete lipocalin 2 (Lcn2; neutrophil gelatinaseassociated lipocalin [NGAL]; also known as siderocalin or 24p3) (7). Lcn2 binds Ent in its binding pocket, either in its ferric (FeEnt) or aferric form, thereby disrupting bacterial iron acquisition and inhibiting bacterial replication (7?0). Lcn2 is crucial for host defense, as Lcn2-deficient mice rapidly succumb to infection with E. coli and K. pneumoniae isolates that rely on Ent for iron acquisition (7, 11?3). As an evasion mechanism, some strains of K. pneumoniae along with other Gram-negative bacteria secrete siderophores that are not bound by Lcn2, like salmochelin and yersiniabactin (Ybt). Salmochelin is glycosylated Ent (GlyEnt), which cannot be bound by Lcn2 because of steric hindrance from added glucose groups (3). In addition, the glucose groups reduce the membrane partitioning ability of Ent, potentially altering the potential of Na+/Ca2+ Exchanger Molecular Weight GlyEnt to access cellu-Dlar iron (14). Ybt is actually a phenolate siderophore with high iron affinity that’s structurally distinct from Ent and promotes pneumonia regardless of the presence of Lcn2 (3, 13, 15). Production of either GlyEnt or Ybt by strains of K. pneumoniae is sufficient for bacterial growth for the duration of nasal colonization and pneumonia (8, 13). The interaction in between siderophores and Lcn2 can modulate the inflammatory response to infection. Ent and Lcn2 each induce secretion of your neutrophil chemoattractant interleukin-8 (IL-8) by cultured respiratory epithelial.