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Macrophagic control of the response to uropathogenic E. coli infection by regulation of iron retention in an IL ‐ 6 ‐ dependent manner

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Uropathogenic Escherichia coli (UPEC), the causative agent of over 85% of urinary tract infections (UTIs), elaborate a number of siderophores to chelate iron from the host. On the other hand, the host immune imperative is to limit the availability of iron to the bacteria. Little is known regarding the mechanisms underlying this host‐iron‐UPEC interaction. Our objective was to determine whether macrophages, in response to UPEC infection, retain extracellular siderophore‐bound and free iron, thus limiting the ability of UPEC to access iron.

Methods: Quantitative PCR, immunoblotting analysis, and gene expression analysis of wild type and IL‐6‐deficient macrophages was performed.

Results: We found that (1) macrophages upon UPEC infection increased expression of lipocalin 2, a siderophore‐binding molecule, of Dmt1, a molecule that facilitates macrophage uptake of free iron, and of the intracellular iron cargo molecule ferritin, and decreased expression of the iron exporter ferroportin; (2) bladder macrophages regulate expression of genes involved in iron retention upon UPEC infection; (3) IL‐6, a cytokine known to play an important role in regulating host iron homeostasis as well as host defense to UPEC, regulates this process, in part by promoting production of lipocalin 2; and finally, (4) inhibition of IL‐6 signaling genetically and by neutralizing antibodies against the IL‐6 receptor, promoted intra‐macrophagic UPEC growth in the presence of excess iron.

Conclusions: Together, our study suggests that macrophages retain siderophore‐bound and free iron in response to UPEC and IL‐6 signaling is necessary for macrophages to limit the growth of UPEC in the presence of excess iron. IL‐6 signaling and iron regulation is one mechanism by which macrophages may mediate UPEC clearance.

No MeSH data available.


Related in: MedlinePlus

pMacs retain iron in response to UPEC, and limit UPEC utilization of iron for UPEC survival. (A) Summary of procedure for infecting pMacs with UPEC. Peritoneal macrophages (“pMacs”) were pre‐treated for 24 h with or without ferric ammonium citrate (FAC) as a source of iron for iron supplementation. The pMacs were infected with UTI89 at an MOI of 0.1 for 3 or 6 h. Intracellular CFU analysis in pMacs uninfected or infected with UTI89 at an MOI of 0.1 for 3 or 6 h in the presence of iron supplementation before infection (B) or during infection (C). qPCR analysis on mRNA expression of for lipocalin 2 (D), TFR1 (E), Dmt1 (F), ferroportin (G), and hepcidin (H). Western Blots for ferritin and actin (I), IRP1, IRP2, and actin (J) in pMacs (three biological replicates; qPCR values relative to uninfected pMacs without iron supplementation; compared by ANOVA; bars represent ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001).
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iid3123-fig-0001: pMacs retain iron in response to UPEC, and limit UPEC utilization of iron for UPEC survival. (A) Summary of procedure for infecting pMacs with UPEC. Peritoneal macrophages (“pMacs”) were pre‐treated for 24 h with or without ferric ammonium citrate (FAC) as a source of iron for iron supplementation. The pMacs were infected with UTI89 at an MOI of 0.1 for 3 or 6 h. Intracellular CFU analysis in pMacs uninfected or infected with UTI89 at an MOI of 0.1 for 3 or 6 h in the presence of iron supplementation before infection (B) or during infection (C). qPCR analysis on mRNA expression of for lipocalin 2 (D), TFR1 (E), Dmt1 (F), ferroportin (G), and hepcidin (H). Western Blots for ferritin and actin (I), IRP1, IRP2, and actin (J) in pMacs (three biological replicates; qPCR values relative to uninfected pMacs without iron supplementation; compared by ANOVA; bars represent ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001).

Mentions: UPEC exploits host iron for its survival 5, 15, 17, including by chelating iron from within bladder epithelial cells 18, 19. We recently showed that high iron levels promote UPEC growth within bladder epithelial cells 19. UPEC can also persist within macrophages 30, 31. It remains unclear, however, whether UPEC exploits macrophages as a source of iron under high iron conditions and homeostatic iron conditions that mimic in vivo infection 4. We therefore sought to investigate the macrophage response to UPEC in the context of homeostatic levels of iron (“control”) or following iron supplementation (“iron supplemented”) resulting from the addition of ferric ammonium citrate (FAC) as a source of iron at a concentration consistent with previous publications 7, 32. We infected control and iron‐supplemented peritoneal macrophages (pMacs) with UPEC for 3 and 6 h (see schematic in Fig. 1A) and measured intracellular and extracellular bacterial CFUs. Interestingly, there was no significant difference in intracellular UPEC load within control pMacs in comparison to iron‐supplemented pMacs (Fig. 1B), nor was there a significant difference in extracellular UPEC load (data not shown). In addition, UPEC had no significant effect on pMac survival at these time‐points (data not shown). We have previously shown that iron alone promotes UPEC growth in minimal media and in bladder epithelial cells 19; thus, the UPEC growth restriction observed in macrophages is attributable to macrophage function.


Macrophagic control of the response to uropathogenic E. coli infection by regulation of iron retention in an IL ‐ 6 ‐ dependent manner
pMacs retain iron in response to UPEC, and limit UPEC utilization of iron for UPEC survival. (A) Summary of procedure for infecting pMacs with UPEC. Peritoneal macrophages (“pMacs”) were pre‐treated for 24 h with or without ferric ammonium citrate (FAC) as a source of iron for iron supplementation. The pMacs were infected with UTI89 at an MOI of 0.1 for 3 or 6 h. Intracellular CFU analysis in pMacs uninfected or infected with UTI89 at an MOI of 0.1 for 3 or 6 h in the presence of iron supplementation before infection (B) or during infection (C). qPCR analysis on mRNA expression of for lipocalin 2 (D), TFR1 (E), Dmt1 (F), ferroportin (G), and hepcidin (H). Western Blots for ferritin and actin (I), IRP1, IRP2, and actin (J) in pMacs (three biological replicates; qPCR values relative to uninfected pMacs without iron supplementation; compared by ANOVA; bars represent ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001).
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iid3123-fig-0001: pMacs retain iron in response to UPEC, and limit UPEC utilization of iron for UPEC survival. (A) Summary of procedure for infecting pMacs with UPEC. Peritoneal macrophages (“pMacs”) were pre‐treated for 24 h with or without ferric ammonium citrate (FAC) as a source of iron for iron supplementation. The pMacs were infected with UTI89 at an MOI of 0.1 for 3 or 6 h. Intracellular CFU analysis in pMacs uninfected or infected with UTI89 at an MOI of 0.1 for 3 or 6 h in the presence of iron supplementation before infection (B) or during infection (C). qPCR analysis on mRNA expression of for lipocalin 2 (D), TFR1 (E), Dmt1 (F), ferroportin (G), and hepcidin (H). Western Blots for ferritin and actin (I), IRP1, IRP2, and actin (J) in pMacs (three biological replicates; qPCR values relative to uninfected pMacs without iron supplementation; compared by ANOVA; bars represent ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001).
Mentions: UPEC exploits host iron for its survival 5, 15, 17, including by chelating iron from within bladder epithelial cells 18, 19. We recently showed that high iron levels promote UPEC growth within bladder epithelial cells 19. UPEC can also persist within macrophages 30, 31. It remains unclear, however, whether UPEC exploits macrophages as a source of iron under high iron conditions and homeostatic iron conditions that mimic in vivo infection 4. We therefore sought to investigate the macrophage response to UPEC in the context of homeostatic levels of iron (“control”) or following iron supplementation (“iron supplemented”) resulting from the addition of ferric ammonium citrate (FAC) as a source of iron at a concentration consistent with previous publications 7, 32. We infected control and iron‐supplemented peritoneal macrophages (pMacs) with UPEC for 3 and 6 h (see schematic in Fig. 1A) and measured intracellular and extracellular bacterial CFUs. Interestingly, there was no significant difference in intracellular UPEC load within control pMacs in comparison to iron‐supplemented pMacs (Fig. 1B), nor was there a significant difference in extracellular UPEC load (data not shown). In addition, UPEC had no significant effect on pMac survival at these time‐points (data not shown). We have previously shown that iron alone promotes UPEC growth in minimal media and in bladder epithelial cells 19; thus, the UPEC growth restriction observed in macrophages is attributable to macrophage function.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Uropathogenic Escherichia coli (UPEC), the causative agent of over 85% of urinary tract infections (UTIs), elaborate a number of siderophores to chelate iron from the host. On the other hand, the host immune imperative is to limit the availability of iron to the bacteria. Little is known regarding the mechanisms underlying this host&#8208;iron&#8208;UPEC interaction. Our objective was to determine whether macrophages, in response to UPEC infection, retain extracellular siderophore&#8208;bound and free iron, thus limiting the ability of UPEC to access iron.

Methods: Quantitative PCR, immunoblotting analysis, and gene expression analysis of wild type and IL&#8208;6&#8208;deficient macrophages was performed.

Results: We found that (1) macrophages upon UPEC infection increased expression of lipocalin 2, a siderophore&#8208;binding molecule, of Dmt1, a molecule that facilitates macrophage uptake of free iron, and of the intracellular iron cargo molecule ferritin, and decreased expression of the iron exporter ferroportin; (2) bladder macrophages regulate expression of genes involved in iron retention upon UPEC infection; (3) IL&#8208;6, a cytokine known to play an important role in regulating host iron homeostasis as well as host defense to UPEC, regulates this process, in part by promoting production of lipocalin 2; and finally, (4) inhibition of IL&#8208;6 signaling genetically and by neutralizing antibodies against the IL&#8208;6 receptor, promoted intra&#8208;macrophagic UPEC growth in the presence of excess iron.

Conclusions: Together, our study suggests that macrophages retain siderophore&#8208;bound and free iron in response to UPEC and IL&#8208;6 signaling is necessary for macrophages to limit the growth of UPEC in the presence of excess iron. IL&#8208;6 signaling and iron regulation is one mechanism by which macrophages may mediate UPEC clearance.

No MeSH data available.


Related in: MedlinePlus