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

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI

Comparative Proteomic Phenotyping of Cell Lines and Primary Cells to Assess Preservation of Cell Type-specific Functions *S⃞

Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1–6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1–6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions

Programm zur Beschaeftigung und Qualifizierung von Langzeitarbeitlosen und Sozialhilfeempfaengern

SIGLEIAB-96-150-3 AN 274 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Quenched Substrates for Live-Cell Labeling of SNAP-Tagged Fusion Proteins with Improved Fluorescent Background

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells

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

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

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

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