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Mucosal lipocalin 2 has pro-inflammatory and iron-sequestering effects in response to bacterial enterobactin - PubMed

Mucosal lipocalin 2 has pro-inflammatory and iron-sequestering effects in response to bacterial enterobactin

Michael A Bachman et al. PLoS Pathog. 2009 Oct.

Abstract

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. K. pneumoniae colonizes the nasopharynx of C57BL/6 mice.

C57BL/6 mice were inoculated intranasally with 2×106 cfu of an overnight LB culture of KPPR1 in 10 µL PBS. At each time point, 5 mice were sacrificed, nasopharyngeal lavage was performed, and lavage fluid was plated for quantitative culture. Colony forming unit (cfu) counts are shown as a scatter plot where the bar represents the median; the dashed line is the lower limit of detection (20 cfu/ml).

Figure 2
Figure 2. K. pneumoniae colonization elicits an acute inflammatory infiltrate.

Mice colonized with wild-type K. pneumoniae KPPR1 were sacrificed at day 1 post-inoculation. Inflammatory infiltrates are seen adjacent to the turbinates of the nasopharynx (A, 20×, arrows) containing neutrophils (B, 600×, arrowheads). To enumerate the neutrophil influx, mice were sacrificed at day 3 post-inoculation for nasopharyngeal lavage, and 100 µL of lavage fluid was analyzed by flow cytometry. Neutrophils (CD45+, Ly6G+ and CD11b+) from a representative KPPR1 (C, D) or PBS mock-colonized (E, F) mouse lavage sample are shown. Compiled neutrophil counts are shown as a scatter plot; the bars represent median numbers of CD45+, Ly6G+, CD11b+ events/ml for each animal (G). p<0.01 by Mann-Whitney test.

Figure 3
Figure 3. Neutrophils inhibit K. pneumoniae colonization and prevent bacteremia.

Mice were treated with RB6-8C5 rat mAb to murine Ly6G or control rat IgG one day prior to inoculation with 2×106 cfu of wild-type K. pneumoniae KPPR1 were sacrificed at day 1 post-inoculation. Nasopharyngeal lavage cfu/ml (A) and spleen cfu/gm (B) are shown as a scatter plot with the bar at the median; dashed line represents lower limit of detection. ** p<0.01 by Mann-Whitney test; * p<0.05 by Fisher's Exact Test for presence of detectable bacteria in the spleen.

Figure 4
Figure 4. K. pneumoniae requires enterobactin or yersiniabactin, but not both, to colonize the nasopharynx efficiently.

Colonization density was determined in C57BL/6 mice (n = 5 per group) at day 3 after intranasal inoculation with 2×106 cfu of the K. pneumoniae mutants indicated. Box and whiskers graph shows the median and interquartile ranges; the dashed line is the lower limit of detection (20 cfu/ml). Siderophores encoded by each mutant are indicated by a plus (+). *** p<0.001 by Mann-Whitney test.

Figure 5
Figure 5. The iroA locus protects against growth inhibition by lipocalin 2.

Overnight growth in Lcn2-deficient mouse serum with or without 1.6 µM recombinant Lcn2 was determined for the K. pneumoniae mutants indicated (A). For complementation studies, growth in serum was determined for the iroA and iroA ybtS mutant transformed with the vector control (pACYC184) or pIroA (pACYC184::iroBCDN) (B). Mean±SEM for at least three independent experiments is shown as log10 CFU/ml. Siderophores encoded by each mutant are indicated by a plus (+). *** p<0.001 comparing each condition to WT plus rLcn2, and # p<0.001 comparing pIroA to vector control, as determined by one-way ANOVA with Tukey's multiple comparison test. To determine if wild-type K. pneumoniae produces Lcn2-resistant and sensitive Ent, growth of the entB mutant in serum with or without 5 µM recombinant Lcn2 was compared between vehicle control, 16 nM of purified Ent or Gly-Ent (Salmochelin S4), or wild-type culture supernatant (1∶640 dilution) (C). Mean ± standard deviation for two independent experiments is shown as log10 CFU/ml.

Figure 6
Figure 6. Enterobactin-dependent K. pneumoniae is defective in colonization.

Colonization density at day 3 after intranasal inoculation with 2×106 cfu of the K. pneumoniae mutants indicated was determined in C57BL/6 mice (n≥5 mice per group). Box and whiskers graph shows the median and interquartile ranges; the dashed line is the lower limit of detection (20 cfu/ml). Siderophores encoded by each mutant are indicated by a plus (+). *** p<0.001 as determined by Kruskall-Wallis Test with Dunn's post test.

Figure 7
Figure 7. Mucosal lipocalin 2 is required to inhibit colonization by enterobactin-dependent K. pneumoniae.

Colonization density of Lcn2+/+ (shaded symbols) and Lcn2−/− mice (open symbols) on day 3 after intranasal inoculation with 2×106 cfu of the K. pneumoniae mutants indicated (A). Box and whiskers graph shows the median and interquartile ranges for ≥10 mice per group; the dashed line is the lower limit of detection (20 cfu/ml). Siderophores encoded by each mutant are indicated by a plus (+). * p<0.05, ** p<0.01, ns p>0.05 as determined by Kruskall-Wallis Test with Dunn's post test. To remove the contribution of neutrophil-produced Lcn2, colonization density on day 1 was determined in Lcn2+/+ (n = 6) and Lcn2−/− mice (n = 8) treated with RB6-8C5 rat mAb to murine Ly6G one day prior to inoculation with 2×106 cfu of iroA ybtS mutant K. pneumoniae (B). * p<0.05 as determined by Mann-Whitney Test.

Figure 8
Figure 8. Lipocalin 2 does not stimulate IL-8 release from A549 respiratory cells in response to glycosylated enterobactin.

IL-8 release from cultured A549 respiratory cells was measured by ELISA after overnight stimulation with 50 µM Ent or Gly-Ent (Salmochelin S4) and/or 25 µM recombinant Lcn2 (shaded bars). Mean±SEM of fold-increase above vehicle control from at least three independent experiments is shown. ** p<0.01 comparing Ent-Lcn2 to all other stimuli as determined by one-way ANOVA with Tukey's multiple comparison test.

Figure 9
Figure 9. Lipocalin 2 promotes increased neutrophil influx in response to enterobactin-producing K. pneumoniae.

Intraluminal neutrophil counts (PMNs) were measured by flow cytometry of 100 µL nasopharyngeal lavage fluid on day 3 after intranasal inoculation of C57BL/6 mice with 2×106 cfu of the following combinations of K. pneumoniae: wild-type KPPR1 vs. iroA mutant (A), or iroA mutant vs. entB mutant (B). *p<0.05 by the Mann-Whitney test. To determine if neutrophil influx to Ent-producing strains is Lcn2-dependent, Lcn2+/+ and Lcn2−/− littermates were colonized with 2×106 cfu of iroA mutant K. pneumoniae (C). * p<0.05 as determined by the Wilcoxon matched pair test. Data points represent CD45+, Ly6G+, CD11b+ events and dotted lines connect littermates (n≥5 per group). Siderophores encoded by each mutant are indicated by a plus (+).

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