Bacterial siderophores that evade or overwhelm lipocalin 2 induce hypoxia inducible factor 1α and proinflammatory cytokine secretion in cultured respiratory epithelial cells

Abstract

Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself and also can 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 microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1α (HIF-1α), 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 increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1α was sufficient to enhance 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.

FIG 1

Enterobactin and Ent+Lcn2 induce distinct gene expression patterns. Heat maps of relative gene expression by A549 respiratory cells in response to combinations of 50 μM enterobactin (Ent) and 50 μM ferric ammonium citrate (Fe) alone (A) or with 25 μM lipocalin 2 (Lcn2) (B), as measured by microarray, are shown. Red indicates upregulation of gene expression and green indicates downregulation of gene expression relative to the median for each experiment. Stimulations were repeated independently, and gene expression was validated by qPCR of NDRG1 (C), IL1R1 (D), VEGFA (E), IL-8 (F), CCL20 (G), and IL-6 (H) in response to combinations of Fe, Ent, and Lcn2. Data are shown as means ± standard errors of the means (SEM) from 3 replicate samples and are representative of at least 2 independent experiments. Statistics were calculated using ANOVA (#, P < 0.001 for the indicated comparisons).

FIG 2

In combination, Ent and Lcn2 strongly induce cytokine production in A549 respiratory cells. Cells were stimulated for 16 h with combinations of 50 μM FAC (Fe), 50 μM Ent, or 25 μM Lcn2. IL-8 (A), CCL20 (B), and IL-6 (C) secretion were measured by ELISA. Values shown are means ± SEM from 3 replicate samples and are representative of at least 2 independent experiments. Statistics were calculated using one-way ANOVA (***, P < 0.0001 induction relative to PBS; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 for the indicated comparison).

FIG 3

Unbound Ent in combination with Lcn2 is required for synergistic IL-8 and IL-6 secretion in A549 cells. Combinations of 50 μM Ent (669 Da) and 25 μM Lcn2 (20.5 kDa) were spun, as indicated, through a 10,000-MWCO column, and cells were stimulated with the retentate, containing Lcn2 or Ent bound by Lcn2, for 16 h. IL-8 (A) and IL-6 (B) secretion were measured by ELISA. Values shown are means ± SEM from 3 replicate samples and are representative of at least 2 independent experiments. Statistics were calculated using one-way ANOVA (***, P < 0.0001; ns, P > 0.05).

FIG 4

Ybt and Ent, but not GlyEnt, induce expression of the iron starvation gene NDRG1 and calcein fluorescence. (A) Cells were stimulated for 16 h with combinations of 50 μM FAC (Fe), 50 μM Ent, 50 μM Ybt, or 50 μM GlyEnt. NDRG1 expression, a marker associated with iron chelation, was measured by qPCR. (B) Cells were stimulated for 16 h with combinations of 100 μM FAC (Fe), 100 μM Ent, 100 μM Ybt, or 100 μM GlyEnt, and calcein fluorescence was examined. Values shown are means ± SEM from 3 replicate samples and are representative of at least 2 independent experiments. Statistics were calculated using one-way ANOVA (*, P < 0.0001 for induction relative to PBS; #, P < 0.0001 for the indicated comparison).

FIG 5

Ybt+Lcn2 and DFO+Lcn2 induce chemokine release by A549 respiratory cells. Cells were stimulated for 16 h with combinations of 50 μM Ybt, 50 μM GlyEnt, 200 μM DFO, or 25 μM Lcn2, and ELISA was used to measure IL-8 (A), IL-6 (B and E), and CCL20 (C) secretion. Relative NDRG1 expression (D and F) was assayed using qPCR. Values shown are means ± SEM from 3 replicate samples and are representative of at least 2 independent experiments. Statistics were calculated using one-way ANOVA (**, P < 0.01 relative to PBS; ##, P < 0.01; ###, P < 0.001 for the indicated comparison; ns, P > 0.05).

FIG 6

Ent stabilizes HIF-1α in A549 respiratory epithelial cells, which is sufficient to enhance Lcn2-dependent IL-6 secretion. Cells were stimulated for 16 h with combinations of 50 μM Ent, 3 mM DMOG, or 25 μM Lcn2, and Western blotting or ELISA was used to measure HIF-1α stabilization (A, B, and C), IL-8 secretion (D), or IL-6 secretion (E). Western blot data are representative of 2 independent experiments. ELISA values shown are means ± SEM from 3 replicate samples and are representative of at least 2 independent experiments. Statistics were calculated using unpaired two-tailed t tests (**, P < 0.01; ns, P > 0.05).

FIG 7

Lcn2 acts as a sensor by modulating airway epithelial cell inflammatory cytokine secretion in response to iron chelation by unbound Ent and Ybt. (A) Small amounts of Ent can be bound and neutralized by Lcn2, leading to a low level of Lcn2-induced cytokine secretion in the airway. Large amounts of Ent (B) or Ybt (C) evade Lcn2 binding, leading to altered host iron status and HIF-1α stabilization. The combination of cellular iron depletion and Lcn2 signaling increases production of inflammatory cytokines, such as IL-8, IL-6, and CCL20.