Hfe Deficiency Impairs Pulmonary Neutrophil Recruitment in Response to Inflammation

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

Computerized analysis of cytoplasmic Prussian blue (PB) stained AM.

<p>Analysis of iron deposits in AM and cell size as a surrogate parameter for cell activation of AM obtained from female wild-type and <i>Hfe^−/−</i> mice and <i>Hfe^LysMCre</i> mice at 4 h after intratracheal instillation of vehicle or 20 µg LPS. (A) PB-stained iron deposits in AM of female wild-type and <i>Hfe^−/−</i> mice. ^‡<i>P</i><0.05 and ^★<i>P</i>≤0.001 versus WT control mice; ^†<i>P</i><0.005 versus <i>Hfe^−/−</i> control mice. (B) AM size in female wild-type and <i>Hfe^−/−</i> mice. n = 5–7 per group. ^★<i>P</i>≤0.001 versus WT control mice; ^†<i>P</i><0.005 versus <i>Hfe^−/−</i> control mice. (C) PB-stained iron deposits in AM of <i>Hfe^LysMCre</i> mice. ^★<i>P</i>≤0.001 versus <i>Hfe^LysMCre</i> (−) control mice; ^†<i>P</i><0.001 versus <i>Hfe^LysMCre</i> (+) control mice. (D) AM size in <i>Hfe^LysMCre</i> mice. n = 4–15 per group. ^†<i>P</i><0.05 versus <i>Hfe^LysMCre</i> (+) control mice.</p

Attenuated inflammatory cell counts in the BAL of wild-type and <i>Hfe^−/−</i> mice.

<p>BAL obtained 4 h after intratracheal instillation of vehicle or 20 µg LPS. (A) Female wild-type and <i>Hfe^−/−</i> mice. n = 5–7 per group. ^★<i>P</i><0.001 versus WT control mice; ^¶<i>P</i><0.05 and ^†<i>P</i><0.001 versus <i>Hfe^−/−</i> control mice; ^⧫<i>P</i><0.005 versus LPS-treated WT mice. (B) Male wild-type and <i>Hfe^−/−</i> mice. n = 9–11 per group. ^★<i>P</i>≤0.001 versus WT control mice; ^‡<i>P</i><0.05 and ^†<i>P</i><0.005 versus <i>Hfe^−/−</i> control mice; ^⧫<i>P</i><0.005 versus LPS-treated WT mice. Mac.  =  macrophages; PMN  =  polymorphonuclear leukocytes/neutrophils; Eos.  =  eosinophils; Lymph.  =  lymphocytes. (C–F) Representative images of BAL cytospin slides obtained from WT and <i>Hfe^−/−</i> mice (females). MayGrünwald-Giemsa stain, images obtained at 400× magnification. Scale bars, 20 µm.</p

Cytokine protein levels in female wild-type, <i>Hfe^−/−</i> and <i>Hfe^LysMCre</i> mice.

<p>Cytokine protein levels are represented by the fluorescence intensity (FI) as assessed by a Multiplex bead-array based technology assay.</p><p>(A) Female wild-type and <i>Hfe^−/−</i> mice. n = 5–7 per group.</p>★<p><i>P</i><0.05 and ^★★<i>P</i><0.01 versus WT control mice;</p>†<p><i>P</i><0.05 and ^††<i>P</i><0.01 versus <i>Hfe^−/−</i> control mice;</p>⧫<p><i>P</i><0.05 versus LPS-treated WT mice. (B) <i>Hfe^LysMCre</i> mice. Vehicle-treated groups: n = 4–5 per group; LPS-treated groups: n = 9–15 per group.</p>★<p><i>P</i><0.01 versus <i>Hfe^LysMCre</i> (−) control mice;</p>†<p><i>P</i><0.05 versus <i>Hfe^LysMCre</i> (+) control mice.</p

Plasma iron and non-heme tissue iron content in female wild-type, <i>Hfe^−/−</i> and <i>Hfe^LysMCre</i> mice.

<p>Plasma iron in µg/dL, non-heme tissue iron content in µg iron/g dry tissue.</p><p>(A) Female wild-type and <i>Hfe^−/−</i> mice. n = 5–7 per group.</p>‡<p><i>P</i>≤0.005 versus WT control mice;</p>⧫<p><i>P</i>≤0.001 versus LPS-treated WT mice.</p><p>(B) <i>Hfe^LysMCre</i> mice. Vehicle-treated groups: n = 4–5 per group; LPS-treated groups: n = 9–15 per group.</p

Circulating neutrophil levels (in cells/nL) in wild-type and Hfe^−/− mice.

<p>Blood was obtained 4 h after intratracheal instillation of vehicle or 20 µg LPS. (A) Female wild-type and <i>Hfe^−/−</i> mice. n = 5–7 per group. ^‡<i>P</i><0.05 and ^★<i>P</i><0.001 versus WT control mice; ^†<i>P</i><0.05 versus <i>Hfe^−/−</i> control mice. (B) Male wild-type and <i>Hfe^−/−</i> mice. n = 9–11 per group. ^★<i>P</i><0.001 versus WT control mice; ^†<i>P</i><0.001 versus <i>Hfe^−/−</i> control mice.</p

mRNA expression of selected inflammatory mediators in lung samples of female <i>Hfe^LysMCre</i> mice.

<p>qPCR results are shown as relative mRNA expression normalized to GAPDH-expression. n = 4–15 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 <i>Hfe^LysMCre</i> (−) and <i>Hfe^LysMCre</i> (+) mice in either vehicle- or LPS-treated groups are highlighted in grey and bold letters. ^‡<i>P</i><0.05 versus <i>Hfe^LysMCre</i> (−) control mice; ^★<i>P</i><0.05 and ^★★<i>P</i>≤0.005 versus <i>Hfe^LysMCre</i> (−) control mice; ^†<i>P</i><0.05 and ^††<i>P</i>≤0.005 versus <i>Hfe^LysMCre</i> (+) control mice; ^⧫<i>P</i><0.05 versus LPS-treated <i>Hfe^LysMCre</i> (−) mice.</p

mRNA expression of selected inflammatory mediators in lung samples of female wild-type and <i>Hfe^−/−</i> mice.

<p>qPCR results are shown as relative mRNA expression normalized to <i>GAPDH</i> 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 <i>Hfe^−/−</i> mice in either vehicle- or LPS-treated groups are highlighted in grey and bold letters. ^‡<i>P</i><0.05 and ^‡‡<i>P</i>≤0.001 versus WT control mice; ^★<i>P</i><0.05 and ^★★<i>P</i>≤0.005 versus WT control mice; ^†<i>P</i><0.05 and ^††<i>P</i>≤0.005 versus <i>Hfe^−/−</i> control mice; ^⧫<i>P</i><0.05 and ^⧫⧫<i>P</i>≤0.005 versus LPS-treated WT mice.</p

Inflammatory cell counts in the BAL of <i>Hfe^LysMCre</i> mice.

<p>BAL obtained 4 h after intratracheal instillation of vehicle (n = 4–5 per group) or 20 µg LPS (n = 9–15 per group). ^★<i>P</i>≤0.005 versus <i>Hfe^LysMCre</i> (−) control mice; ^†<i>P</i>≤0.005 versus <i>Hfe^LysMCre</i> (+) control mice. Mac.  =  macrophages; PMN  =  polymorphonuclear leukocytes/neutrophils; Eos.  =  eosinophils; Lymph.  =  lymphocytes.</p