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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

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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.


Iron and UPEC together activate induce IL‐6 production in pMacs. qPCR for Il‐6 relative to uninfected pMacs without iron supplementation (A), Western Blot for IL‐6, ferritin, and actin, (B), and an ELISA for IL‐6 (C) in pMACs infected with UPEC after iron supplementation (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-0002: Iron and UPEC together activate induce IL‐6 production in pMacs. qPCR for Il‐6 relative to uninfected pMacs without iron supplementation (A), Western Blot for IL‐6, ferritin, and actin, (B), and an ELISA for IL‐6 (C) in pMACs infected with UPEC after iron supplementation (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: Host iron homeostasis is known to be regulated by the pro‐inflammatory cytokine IL‐6, which induces hepcidin production by liver cells 23, thereby promoting iron retention. We reasoned that pMacs may use IL‐6 signaling in response to UPEC to regulate iron availability, since macrophages and bladder epithelial cells produce IL‐6 as a first response to UPEC infection 20, 21. We therefore infected iron‐supplemented and control pMacs with UPEC and determined IL‐6 production. We found, as expected, that UPEC induced Il‐6 mRNA expression (Fig. 2A), IL‐6 protein production (Fig. 2B), and IL‐6 protein export (Fig. 2C). Interestingly, we found that iron supplementation was sufficient to activate IL‐6 production even in the absence of a bacterial stimulus (Fig. 2B), consistent with previously published data 33. However, iron supplementation did not augment IL‐6 export (Fig. 2C). Taken together, these data suggests that iron promotes the IL‐6 production pathway in response to UPEC.


Macrophagic control of the response to uropathogenic E. coli infection by regulation of iron retention in an IL ‐ 6 ‐ dependent manner
Iron and UPEC together activate induce IL‐6 production in pMacs. qPCR for Il‐6 relative to uninfected pMacs without iron supplementation (A), Western Blot for IL‐6, ferritin, and actin, (B), and an ELISA for IL‐6 (C) in pMACs infected with UPEC after iron supplementation (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-0002: Iron and UPEC together activate induce IL‐6 production in pMacs. qPCR for Il‐6 relative to uninfected pMacs without iron supplementation (A), Western Blot for IL‐6, ferritin, and actin, (B), and an ELISA for IL‐6 (C) in pMACs infected with UPEC after iron supplementation (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: Host iron homeostasis is known to be regulated by the pro‐inflammatory cytokine IL‐6, which induces hepcidin production by liver cells 23, thereby promoting iron retention. We reasoned that pMacs may use IL‐6 signaling in response to UPEC to regulate iron availability, since macrophages and bladder epithelial cells produce IL‐6 as a first response to UPEC infection 20, 21. We therefore infected iron‐supplemented and control pMacs with UPEC and determined IL‐6 production. We found, as expected, that UPEC induced Il‐6 mRNA expression (Fig. 2A), IL‐6 protein production (Fig. 2B), and IL‐6 protein export (Fig. 2C). Interestingly, we found that iron supplementation was sufficient to activate IL‐6 production even in the absence of a bacterial stimulus (Fig. 2B), consistent with previously published data 33. However, iron supplementation did not augment IL‐6 export (Fig. 2C). Taken together, these data suggests that iron promotes the IL‐6 production pathway in response to UPEC.

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.