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

Recent contributions to Quantitative Linguistics.

Quantitative Linguistics is a rapidly developing discipline covering more and more areas of linguistic and textological research. The book represents an overview of the state of the art in Quantitative Linguistics, its scope and reach. Some of the topics: linguistic laws, frequency analyses, synergetic models of language, networks, part-of-speech systems, authorship attribution, polyfunctionality and polysemy, and opinion target identification

Bispecific PSMA-617 / RM2 Heterodimer for Theranostics Applications in Prostate Cancer

PSMA and GRPR protein receptors are upregulated during prostate cancer (PCa) progression, and thus they have both been used for diagnostic molecular imaging and therapy of the disease. To address tumor heterogeneity, we synthesized and evaluated the bispecific PSMA/GRPR ligand (3) with a 10 atom spacer between PSMA-617 (1) and the GRPR antagonist RM2 (2) generated with click chemistry and coupled with chelator DOTA, to enable radiolabelling. Ligand 3 was radiolabelled with 68Ga, [68Ga]Ga-3 and 177Lu, [177Lu]Lu-3. [68Ga]Ga-3 was tested with PCa cell lines PC-3 and LNCaP for its affinity for GRPR and PSMA receptors, lipophilicity, for its cell-binding specificity, time kinetic binding affinities and cell-internalization. Heterodimer 3 showed specific cell binding, similar affinities for PSMA receptor and GRPR and higher lipophilicity compared to monomers PSMA-617 (1) and RM2 (2), while total internalization rates and cell-binding were superior over monomers. Docking calculations showed that the PSMA-617 (1) /RM2 (2) heterodimer 3 can have binding interactions of PSMA-617 (1) inside the PSMA receptor funnel and of RM2 (2) inside the GRPR. In vivo biodistribution studies for [68Ga]Ga-3 showed dual targeting of PSMA-positive tumors and GRPR-positive tumors and fast pharmacokinetic properties, higher cancer cell-uptake and lower kidney uptake in comparison to the monomers

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

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

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

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

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