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

Cellular ERK Phospho-Form Profiles with Conserved Preference for a Switch-Like Pattern

ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation, and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY, and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC–MS. This method enabled us to determine the abundances of phospho-forms with a relative variability of ≤5% (SD). We observed a switch-like preference of ERK phospho-form abundances toward the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multisite phosphorylation

Cellular ERK Phospho-Form Profiles with Conserved Preference for a Switch-Like Pattern

ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation, and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY, and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC–MS. This method enabled us to determine the abundances of phospho-forms with a relative variability of ≤5% (SD). We observed a switch-like preference of ERK phospho-form abundances toward the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multisite phosphorylation

T160‐phosphorylated CDK

Liver regeneration is a tightly controlled process mainly achieved by proliferation of usually quiescent hepatocytes. The specific molecular mechanisms ensuring cell division only in response to proliferative signals such as hepatocyte growth factor (HGF) are not fully understood. Here, we combined quantitative time-resolved analysis of primary mouse hepatocyte proliferation at the single cell and at the population level with mathematical modeling. We showed that numerous G1/S transition components are activated upon hepatocyte isolation whereas DNA replication only occurs upon additional HGF stimulation. In response to HGF, Cyclin:CDK complex formation was increased, p21 rather than p27 was regulated, and Rb expression was enhanced. Quantification of protein levels at the restriction point showed an excess of CDK2 over CDK4 and limiting amounts of the transcription factor E2F-1. Analysis with our mathematical model revealed that T160 phosphorylation of CDK2 correlated best with growth factor-dependent proliferation, which we validated experimentally on both the population and the single cell level. In conclusion, we identified CDK2 phosphorylation as a gate-keeping mechanism to maintain hepatocyte quiescence in the absence of HGF

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

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

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

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

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

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