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Hfe deficiency impairs pulmonary neutrophil recruitment in response to inflammation.

Benesova K, Vujić Spasić M, Schaefer SM, Stolte J, Baehr-Ivacevic T, Waldow K, Zhou Z, Klingmueller U, Benes V, Mall MA, Muckenthaler MU - PLoS ONE (2012)

Bottom Line: The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types.As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections.In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Hematology and Immunology, University Hospital of Heidelberg, Heidelberg, Germany.

ABSTRACT
Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe(-/-) mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe(-/-) and wild-type mice by intratracheal instillation of 20 µg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe(-/-) mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe(-/-) and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.

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mRNA expression of selected inflammatory mediators in lung samples of female wild-type and Hfe−/− mice.qPCR results are shown as relative mRNA expression normalized to GAPDH mRNA expression. n = 5–7 mice per group. The affiliation to functional annotation groups is indicated by brackets. An overlap of bracket indicates the affiliation of the respective inflammatory mediators to more than one functional annotation group. Genes that differed significantly in expression between wild-type and Hfe−/− mice in either vehicle- or LPS-treated groups are highlighted in grey and bold letters. ‡P<0.05 and ‡‡P≤0.001 versus WT control mice; ★P<0.05 and ★★P≤0.005 versus WT control mice; †P<0.05 and ††P≤0.005 versus Hfe−/− control mice; ⧫P<0.05 and ⧫⧫P≤0.005 versus LPS-treated WT mice.
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pone-0039363-g005: mRNA expression of selected inflammatory mediators in lung samples of female wild-type and Hfe−/− mice.qPCR results are shown as relative mRNA expression normalized to GAPDH mRNA expression. n = 5–7 mice per group. The affiliation to functional annotation groups is indicated by brackets. An overlap of bracket indicates the affiliation of the respective inflammatory mediators to more than one functional annotation group. Genes that differed significantly in expression between wild-type and Hfe−/− mice in either vehicle- or LPS-treated groups are highlighted in grey and bold letters. ‡P<0.05 and ‡‡P≤0.001 versus WT control mice; ★P<0.05 and ★★P≤0.005 versus WT control mice; †P<0.05 and ††P≤0.005 versus Hfe−/− control mice; ⧫P<0.05 and ⧫⧫P≤0.005 versus LPS-treated WT mice.

Mentions: Inappropriately low hepatic hepcidin mRNA levels in Hfe−/− mice (Fig. S2) cause iron depletion of macrophages by deregulating ferroportin levels. To assess whether iron levels are depleted in AM we analyzed intracellular ferric iron deposits by computerized analysis of Prussian blue (PB) staining. We show that AM of female Hfe−/− mice contained significantly less PB-stained precipitate in the cytoplasm compared to wild-type control mice (Fig. 4A). Our results extend previous observations that show iron depletion in peritoneal macrophages of Hfe−/− mice [9], [10], [11]. In principle this finding can be explained by low hepatic hepcidin expression (Fig. S2). It is however of interest that pulmonary hepcidin levels are reduced in Hfe−/− compared to wild-type mice (Fig. 5). Thus, the impairment of a pulmonary hepcidin-controlled ferroportin autoregulatory loop in Hfe−/− mice may contribute to increased iron export from AM. By contrast, intracellular PB-stained iron deposits in AM of HfeLysMCre (+) and HfeLysMCre (−) mice did not differ significantly (Fig. 4C), suggesting that macrophage Hfe is not required to control iron levels in AM. Consistent with earlier reports of inflammation-triggered iron sequestration in macrophages [30], [31], LPS-instillation significantly increased the cytoplasmic PB-stained precipitates in AM of wild-type and Hfe−/− mice, HfeLysMCre (−) and HfeLysMCre (+) mice (Fig. 4A, 4C). To exclude that differences in the size of the cell area analyzed account for the iron depletion in Hfe−/− control mice, we additionally measured the cell size of the AM. While the size of the AM of vehicle-treated Hfe−/− was not different from the one of wild-type mice, we observed a significant increase in AM size in all mouse strains studied following pulmonary LPS instillation. Our finding is consistent with previous observations that relate AM size to inflammatory activity (Fig. 4B, 4D) [32]. Taken together, our analyses suggest that the reduced macrophage iron content in AM of Hfe−/− mice may contribute to attenuated neutrophil recruitment (Fig. 1). It is further of note that BAL neutrophils of LPS-instilled Hfe−/− mice showed elevated iron levels compared to LPS-treated wild-type controls (0.54±0.08% vs. 1.10±0.09%; P<0.01). To interpret these data measurements of iron levels in BAL neutrophils of untreated mice would be required. However, due to the low numbers of BAL neutrophils in untreated mice accurate assessment of iron levels was not possible.


Hfe deficiency impairs pulmonary neutrophil recruitment in response to inflammation.

Benesova K, Vujić Spasić M, Schaefer SM, Stolte J, Baehr-Ivacevic T, Waldow K, Zhou Z, Klingmueller U, Benes V, Mall MA, Muckenthaler MU - PLoS ONE (2012)

mRNA expression of selected inflammatory mediators in lung samples of female wild-type and Hfe−/− mice.qPCR results are shown as relative mRNA expression normalized to GAPDH mRNA expression. n = 5–7 mice per group. The affiliation to functional annotation groups is indicated by brackets. An overlap of bracket indicates the affiliation of the respective inflammatory mediators to more than one functional annotation group. Genes that differed significantly in expression between wild-type and Hfe−/− mice in either vehicle- or LPS-treated groups are highlighted in grey and bold letters. ‡P<0.05 and ‡‡P≤0.001 versus WT control mice; ★P<0.05 and ★★P≤0.005 versus WT control mice; †P<0.05 and ††P≤0.005 versus Hfe−/− control mice; ⧫P<0.05 and ⧫⧫P≤0.005 versus LPS-treated WT mice.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383765&req=5

pone-0039363-g005: mRNA expression of selected inflammatory mediators in lung samples of female wild-type and Hfe−/− mice.qPCR results are shown as relative mRNA expression normalized to GAPDH mRNA expression. n = 5–7 mice per group. The affiliation to functional annotation groups is indicated by brackets. An overlap of bracket indicates the affiliation of the respective inflammatory mediators to more than one functional annotation group. Genes that differed significantly in expression between wild-type and Hfe−/− mice in either vehicle- or LPS-treated groups are highlighted in grey and bold letters. ‡P<0.05 and ‡‡P≤0.001 versus WT control mice; ★P<0.05 and ★★P≤0.005 versus WT control mice; †P<0.05 and ††P≤0.005 versus Hfe−/− control mice; ⧫P<0.05 and ⧫⧫P≤0.005 versus LPS-treated WT mice.
Mentions: Inappropriately low hepatic hepcidin mRNA levels in Hfe−/− mice (Fig. S2) cause iron depletion of macrophages by deregulating ferroportin levels. To assess whether iron levels are depleted in AM we analyzed intracellular ferric iron deposits by computerized analysis of Prussian blue (PB) staining. We show that AM of female Hfe−/− mice contained significantly less PB-stained precipitate in the cytoplasm compared to wild-type control mice (Fig. 4A). Our results extend previous observations that show iron depletion in peritoneal macrophages of Hfe−/− mice [9], [10], [11]. In principle this finding can be explained by low hepatic hepcidin expression (Fig. S2). It is however of interest that pulmonary hepcidin levels are reduced in Hfe−/− compared to wild-type mice (Fig. 5). Thus, the impairment of a pulmonary hepcidin-controlled ferroportin autoregulatory loop in Hfe−/− mice may contribute to increased iron export from AM. By contrast, intracellular PB-stained iron deposits in AM of HfeLysMCre (+) and HfeLysMCre (−) mice did not differ significantly (Fig. 4C), suggesting that macrophage Hfe is not required to control iron levels in AM. Consistent with earlier reports of inflammation-triggered iron sequestration in macrophages [30], [31], LPS-instillation significantly increased the cytoplasmic PB-stained precipitates in AM of wild-type and Hfe−/− mice, HfeLysMCre (−) and HfeLysMCre (+) mice (Fig. 4A, 4C). To exclude that differences in the size of the cell area analyzed account for the iron depletion in Hfe−/− control mice, we additionally measured the cell size of the AM. While the size of the AM of vehicle-treated Hfe−/− was not different from the one of wild-type mice, we observed a significant increase in AM size in all mouse strains studied following pulmonary LPS instillation. Our finding is consistent with previous observations that relate AM size to inflammatory activity (Fig. 4B, 4D) [32]. Taken together, our analyses suggest that the reduced macrophage iron content in AM of Hfe−/− mice may contribute to attenuated neutrophil recruitment (Fig. 1). It is further of note that BAL neutrophils of LPS-instilled Hfe−/− mice showed elevated iron levels compared to LPS-treated wild-type controls (0.54±0.08% vs. 1.10±0.09%; P<0.01). To interpret these data measurements of iron levels in BAL neutrophils of untreated mice would be required. However, due to the low numbers of BAL neutrophils in untreated mice accurate assessment of iron levels was not possible.

Bottom Line: The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types.As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections.In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Hematology and Immunology, University Hospital of Heidelberg, Heidelberg, Germany.

ABSTRACT
Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe(-/-) mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe(-/-) and wild-type mice by intratracheal instillation of 20 µg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe(-/-) mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe(-/-) and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.

Show MeSH
Related in: MedlinePlus